scholarly journals Rapid nanopore-based DNA sequencing protocol of antibiotic-resistant bacteria for use in surveillance and outbreak investigation

2021 ◽  
Vol 7 (4) ◽  
Author(s):  
Fabienne Antunes Ferreira ◽  
Karin Helmersen ◽  
Tina Visnovska ◽  
Silje Bakken Jørgensen ◽  
Hege Vangstein Aamot
Keyword(s):  
Phylogenetic Analysis ◽  
Real Time ◽  
Type Species ◽  
Outbreak Investigation ◽  
Complete Analysis ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Link Type ◽  
Outbreak Investigations

Outbreak investigations are essential to control and prevent the dissemination of pathogens. This study developed and validated a complete analysis protocol for faster and more accurate surveillance and outbreak investigations of antibiotic-resistant microbes based on Oxford Nanopore Technologies (ONT) DNA whole-genome sequencing. The protocol was developed using 42 methicillin-resistant Staphylococcus aureus (MRSA) isolates identified from former well-characterized outbreaks. The validation of the protocol was performed using Illumina technology (MiSeq, Illumina). Additionally, a real-time outbreak investigation of six clinical S. aureus isolates was conducted to test the ONT-based protocol. The suggested protocol includes: (1) a 20 h sequencing run; (2) identification of the sequence type (ST); (3) de novo genome assembly; (4) polishing of the draft genomes; and (5) phylogenetic analysis based on SNPs. After the sequencing run, it was possible to identify the ST in 2 h (20 min per isolate). Assemblies were achieved after 4 h (40 min per isolate) while the polishing was carried out in 7 min per isolate (42 min in total). The phylogenetic analysis took 0.6 h to confirm an outbreak. Overall, the developed protocol was able to at least discard an outbreak in 27 h (mean) after the bacterial identification and less than 33 h to confirm it. All these estimated times were calculated considering the average time for six MRSA isolates per sequencing run. During the real-time S. aureus outbreak investigation, the protocol was able to identify two outbreaks in less than 31 h. The suggested protocol enables identification of outbreaks in early stages using a portable and low-cost device along with a streamlined downstream analysis, therefore having the potential to be incorporated in routine surveillance analysis workflows. In addition, further analysis may include identification of virulence and antibiotic resistance genes for improved pathogen characterization.

scholarly journals Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp.

2020 ◽  
Vol 6 (7) ◽  
Author(s):  
Erin P. Price ◽  
Valentina Soler Arango ◽  
Timothy J. Kidd ◽  
Tamieka A. Fraser ◽  
Thuy-Khanh Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Type Species ◽  
Simultaneous Detection ◽  
Diagnostic Methods ◽  
Achromobacter Xylosoxidans ◽  
Resistant Bacteria ◽  
Pcr Assay ◽  
Content Type ◽  
Link Type

Several members of the Gram-negative environmental bacterial genus Achromobacter are associated with serious infections, with Achromobacter xylosoxidans being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 Achromobacter spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus Achromobacter and for A. xylosoxidans . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans , with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive in silico analysis, and laboratory testing of 34 non- Achromobacter isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

scholarly journals Marinomonas mangrovi sp. nov., isolated from mangrove sediment

2015 ◽  
Vol 65 (Pt_5) ◽  
pp. 1537-1541 ◽  
Author(s):  
De-Chao Zhang ◽  
Rosa Margesin
Keyword(s):  
Phylogenetic Analysis ◽  
Type Species ◽  
Mangrove Sediment ◽  
Rrna Gene ◽  
Phenotypic Characteristics ◽  
Content Type ◽  
Content Sharing ◽  
Link Type ◽  
Curved Rods ◽  
Sequence Similarities

A Gram-stain-negative, Na+-requiring bacterial strain, designated B20-1T, was isolated from soil of the root system of mangrove forest. Cells were curved rods and motile by means of a polar flagellum. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain B20-1T belonged to the genus Marinomonas , sharing highest sequence similarities with Marinomonas rhizomae IVIA-Po-145T (97.6 %), Marinomonas dokdonensis DSW10-10T (97.0 %) and Marinomonas foliarum IVIA-Po-155T (96.9 %). The predominant cellular fatty acids of strain B20-1T were C10 : 0 3-OH, C18 : 1ω7c, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and C16 : 0. Phosphatidylethanolamine and phosphatidylglycerol were identified as the predominant phospholipids. The predominant ubiquinone was Q-8. The genomic DNA G+C content of strain B20-1T was 46.6 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness, a novel species, Marinomonas mangrovi sp. nov., is proposed with B20-1T ( = DSM 28136T = LMG 28077T) as the type strain.

Sphingomonas indica sp. nov., isolated from hexachlorocyclohexane (HCH)-contaminated soil

2012 ◽  
Vol 62 (Pt_12) ◽  
pp. 2997-3002 ◽  
Author(s):  
Neha Niharika ◽  
Swati Jindal ◽  
Jasvinder Kaur ◽  
Rup Lal
Keyword(s):  
Phylogenetic Analysis ◽  
Type Species ◽  
Sequence Similarity ◽  
Rrna Gene ◽  
Respiratory Quinone ◽  
Polar Lipid Profile ◽  
Content Type ◽  
Link Type ◽  
Total Dna ◽  
Physiological Tests

A bacterial strain, designated Dd16T, was isolated from a hexachlorocyclohexane (HCH) dumpsite at Lucknow, India. Cells of strain Dd16T were Gram-stain-negative, non-motile, rod-shaped and yellow-pigmented. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Sphingomonas in the family Sphingomonadaceae , as it showed highest sequence similarity to Sphingomonas asaccharolytica IFO 15499T (95.36 %), Sphingosinicella vermicomposti YC7378T (95.30), ‘Sphingomonas humi’ PB323 (95.20 %), Sphingomonas sanxanigenens NX02T (95.14 %) and Sphingomonas desiccabilis CP1DT (95.00 %). The major fatty acids were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) C14 : 0 2-OH, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c) and C16 : 0. The polar lipid profile of strain Dd16T also corresponded to those reported for species of the genus Sphingomonas (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine, phosphatidylglycerol, and a sphingoglycolipid), again supporting its identification as a member of the genus Sphingomonas . The predominant respiratory quinone was ubiquinone Q10, and sym-homospermidine was the major polyamine observed. The total DNA G+C content of strain Dd16T was 65.8 mol%. The results obtained on the basis of phenotypic characteristics and phylogenetic analysis and after biochemical and physiological tests, clearly distinguished strain Dd16T from closely related members of the genus Sphingomonas . Thus, strain Dd16T represents a novel species of the genus Sphingomonas for which the name Sphingomonas indica sp. nov. is proposed. The type strain is Dd16T ( = DSM 25434T = CCM 7882T).

Paracoccus huijuniae sp. nov., an amide pesticide-degrading bacterium isolated from activated sludge of a wastewater biotreatment system

2013 ◽  
Vol 63 (Pt_3) ◽  
pp. 1132-1137 ◽  
Author(s):  
Li-Na Sun ◽  
Jun Zhang ◽  
Soon-Wo Kwon ◽  
Jian He ◽  
Shun-Gui Zhou ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Fatty Acid ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Activated Sludge ◽  
Type Species ◽  
Amide Bond ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

A facultatively anaerobic, non-spore-forming, non-motile, catalase- and oxidase-positive, Gram-reaction-negative, coccoid to short rod-shaped strain, designated FLN-7T, was isolated from activated sludge of a wastewater biotreatment facility. The strain was able to hydrolyse amide pesticides (e.g. diflubenzuron, propanil, chlorpropham and dimethoate) through amide bond cleavage. Strain FLN-7T grew at 4–42 °C (optimum 28 °C), at pH 5.0–8.0 (optimum pH 7.0) and with 0–5.0 % (w/v) NaCl (optimum 1.0 %). The major respiratory quinone was ubiquinone-10. The major cellular fatty acid was C18 : 1ω7c. The genomic DNA G+C content of strain FLN-7T was 66.4±0.5 mol%. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylcholine and an unidentified glycolipid. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain FLN-7T was a member of the genus Paracoccus and showed highest 16S rRNA gene sequence similarities with Paracoccus aminovorans JCM 7685T (99.2 %), P. denitrificans DSM 413T (97.8 %), P. yeei CDC G1212T (97.3 %) and P. thiocyanatus THI 011T (97.1 %). Strain FLN-7T showed low DNA–DNA relatedness with P. aminovorans KACC 12261T (36.5±3.4 %), P. denitrificans KACC 12251T (30.5±2.6 %), P. yeei CCUG 46822T (26.2±2.4 %) and P. thiocyanatus KACC 13901T (15.5±0.9 %). Based on the phylogenetic analysis, DNA–DNA hybridization, whole-cell fatty acid composition and biochemical characteristics, strain FLN-7T was clearly distinguished from all recognized species of the genus Paracoccus and should be classified in a novel species, for which the name Paracoccus huijuniae sp. nov. is proposed. The type strain is FLN-7T ( = KACC 16242T  = ACCC 05690T).

Luteimonas huabeiensis sp. nov., isolated from stratum water

2013 ◽  
Vol 63 (Pt_9) ◽  
pp. 3352-3357 ◽  
Author(s):  
Gang Wu ◽  
Yang Liu ◽  
Qing Li ◽  
Huijing Du ◽  
Jing You ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Type Species ◽  
Rrna Gene ◽  
Phenotypic Data ◽  
Content Type ◽  
Link Type ◽  
Physiological And Biochemical Characteristics ◽  
Taxonomic Approach ◽  
Type Strains ◽  
Physiological And Biochemical

A yellow-coloured bacterial strain, designated HB2T, isolated from stratum water was investigated using a polyphasic taxonomic approach. Cells were Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was a member of the genus Luteimonas , its three closest neighbours being Luteimonas aquatica BCRC 17731T (97.5 % similarity), Luteimonas marina JCM 12488T (97.3 %) and Luteimonas aestuarii DSM 19680T (96.9 %). Strain HB2T could clearly be distinguished from these type strains based on phylogenetic analysis, DNA–DNA hybridization, fatty acid composition and a range of physiological and biochemical characteristics. It is evident from the genotypic and phenotypic data that strain HB2T represents a novel species of the genus Luteimonas , for which the name Luteimonas huabeiensis sp. nov. is proposed. The type strain is HB2T ( = DSM 26429T = CICC 11005sT).

Sphingobacterium caeni sp. nov., isolated from activated sludge

2013 ◽  
Vol 63 (Pt_6) ◽  
pp. 2260-2264 ◽  
Author(s):  
Li-Na Sun ◽  
Jun Zhang ◽  
Qing Chen ◽  
Jian He ◽  
Shun-Peng Li
Keyword(s):  
Phylogenetic Analysis ◽  
Activated Sludge ◽  
Dna Hybridization ◽  
Type Species ◽  
Taxonomic Status ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
Taxonomic Approach ◽  
Ph Range

The taxonomic status of a bacterium, strain DC-8T, isolated from activated sludge, was determined using a polyphasic taxonomic approach. The cells of strain DC-8T were Gram-negative, non-motile, non-spore-forming and rod-shaped. The isolate grew at temperature range of 10–40 °C (optimum 30–35 °C), pH range of 5.0–10.0 (optimum 6.5–8.0) and NaCl concentrations of 0–5 % (optimum 0–1 %). The predominant menaquinone of strain DC-8T was MK-7 and major fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c; 39.7 %), iso-C15 : 0 (33.7 %) and C16 : 0 (5.2 %). The DNA G+C content was 39.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain DC-8T was a member of the genus Sphingobacterium . Strain DC-8T shared the highest similarity with Sphingobacterium siyangense SY1T (98.4 %), Sphingobacterium multivorum IAM 14316T (98.3 %), Sphingobacterium canadense CR11T (98.0 %) and Sphingobacterium detergens 6.2ST (97.9 %) and shared less than 97 % similarity with other members of the genus Sphingobacterium . DNA–DNA hybridization experiments showed that the DNA–DNA relatedness values between strain DC-8T and its closest phylogenetic neighbours were below 70 %. Based on the phylogenetic analysis, DNA–DNA hybridization, whole-cell fatty acid composition as well as biochemical characteristics, strain DC-8T was clearly distinguished from all recognized species of the genus Sphingobacterium and should be classified as a representative of a novel species of the genus Sphingobacterium , for which the name Sphingobacterium caeni sp. nov. is proposed. The type strain is DC-8T ( = CCTCC AB 2012020T = KACC 16850T).

Mycobacterium parakoreense sp. nov., a slowly growing non-chromogenic species related to Mycobacterium koreense , isolated from a human clinical specimen

2013 ◽  
Vol 63 (Pt_6) ◽  
pp. 2301-2308 ◽  
Author(s):  
Byoung-Jun Kim ◽  
Seok-Hyun Hong ◽  
Hee-Kyung Yu ◽  
Young-Gil Park ◽  
Joseph Jeong ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Sputum Sample ◽  
Clinical Specimen ◽  
Mycolic Acid ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type

A previously undescribed, slowly growing, non-chromogenic Mycobacterium strain (299T) was isolated from the sputum sample of a patient with a symptomatic pulmonary infection. Phenotypically, strain 299T was generally similar to Mycobacterium koreense DSM 45576T and Mycobacterium triviale ATCC 23292T. The 16S rRNA gene sequence of strain 299T was similar to that of M. koreense DSM 45576T (GenBank accession no. AY734996, 99.5 % similarity); however, it differed substantially from that of M. triviale ATCC 23292T (X88924, 98.2 %). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 299T clustered together with M. koreense DSM 45576T and M. triviale ATCC 23292T, supported by high bootstrapping values (99 %). Unique mycolic acid profiles and phylogenetic analysis based on two different chronometer molecules, the hsp65 and rpoB genes, strongly supported the taxonomic status of this strain as representing a distinct species. These data support the conclusion that strain 299T represents a novel mycobacterial species, for which the name Mycobacterium parakoreense sp. nov. is proposed. The type strain is 299T ( = DSM 45575T = KCTC 19818T).

Rhizobium halophytocola sp. nov., isolated from the root of a coastal dune plant

2012 ◽  
Vol 62 (Pt_8) ◽  
pp. 1997-2003 ◽  
Author(s):  
Fehmida Bibi ◽  
Eu Jin Chung ◽  
Ajmal Khan ◽  
Che Ok Jeon ◽  
Young Ryun Chung
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Coastal Dune ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Content Type ◽  
Link Type

During a study of endophytic bacteria from coastal dune plants, a bacterial strain, designated YC6881T, was isolated from the root of Rosa rugosa collected from the coastal dune areas of Namhae Island, Korea. The bacterium was found to be Gram-staining-negative, motile, halophilic and heterotrophic with a single polar flagellum. Strain YC6881T grew at temperatures of 4–37 °C (optimum, 28–32 °C), at pH 6.0–9.0 (optimum, pH 7.0–8.0), and at NaCl concentrations in the range of 0–7.5 % (w/v) (optimum, 4–5 % NaCl). Strain YC6881T was catalase- and oxidase-positive and negative for nitrate reduction. According to phylogenetic analysis using 16S rRNA gene sequences, strain YC6881T belonged to the genus Rhizobium and showed the highest 16S rRNA gene sequence similarity of 96.9 % to Rhizobium rosettiformans , followed by Rhizobium borbori (96.3 %), Rhizobium radiobacter (96.1 %), Rhizobium daejeonense (95.9 %), Rhizobium larrymoorei (95.6 %) and Rhizobium giardinii (95.4 %). Phylogenetic analysis of strain YC6881T by recA, atpD, glnII and 16S–23S intergenic spacer (IGS) sequences all confirmed the phylogenetic arrangements obtained by using 16S rRNA gene sequences. Cross-nodulation tests showed that strain YC6881T was a symbiotic bacterium that nodulated Vigna unguiculata and Pisum sativum. The major components of the cellular fatty acids were C18 : 1ω7c (53.7 %), C19 : 0 cyclo ω8c (12.6 %) and C12 : 0 (8.1 %). The DNA G+C content was 52.8 mol%. Phenotypic and physiological tests with respect to carbon source utilization, antibiotic resistance, growth conditions, phylogenetic analyses of housekeeping genes recA, atpD and glnII, and fatty acid composition could be used to discriminate strain YC6881T from other species of the genus Rhizobium in the same sublineage. Based on the results obtained in this study, strain YC6881T is considered to represent a novel species of the genus Rhizobium , for which the name Rhizobium halophytocola sp. nov. is proposed. The type strain is YC6881T ( = KACC 13775T = DSM 21600T).

scholarly journals Roles for phthiocerol dimycocerosate lipids in Mycobacterium tuberculosis pathogenesis

Microbiology ◽  
2021 ◽  
Author(s):  
Céline Rens ◽  
Joseph D. Chao ◽  
Danielle L. Sexton ◽  
Elitza I. Tocheva ◽  
Yossef Av-Gay
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Cell Envelope ◽  
Infectious Agent ◽  
Resistant Bacteria ◽  
Content Type ◽  
Link Type ◽  
Drug Resistant Bacteria ◽  
Macrophage Phagocytosis ◽  
Key Steps

The success of Mycobacterium tuberculosis as a pathogen is well established: tuberculosis is the leading cause of death by a single infectious agent worldwide. The threat of multi- and extensively drug-resistant bacteria has renewed global concerns about this pathogen and understanding its virulence strategies will be essential in the fight against tuberculosis. The current review will focus on phthiocerol dimycocerosates (PDIMs), a long-known and well-studied group of complex lipids found in the M. tuberculosis cell envelope. Numerous studies show a role for PDIMs in several key steps of M. tuberculosis pathogenesis, with recent studies highlighting its involvement in bacterial virulence, in association with the ESX-1 secretion system. Yet, the mechanisms by which PDIMs help M. tuberculosis to control macrophage phagocytosis, inhibit phagosome acidification and modulate host innate immunity, remain to be fully elucidated.

scholarly journals Pseudomonas viciae sp. nov., isolated from rhizosphere of broad bean

2020 ◽  
Vol 70 (9) ◽  
pp. 5012-5018 ◽  
Author(s):  
Hui Zhao ◽  
Yinan Ma ◽  
Xiaogang Wu ◽  
Liqun Zhang
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Type Species ◽  
Broad Bean ◽  
Phenotypic Characterization ◽  
Rrna Gene ◽  
Aerobic Bacterium ◽  
Gene Sequences ◽  
Content Type ◽  
Link Type

A Gram-stain-negative aerobic bacterium, strain 11K1T, was isolated from a rhizosphere soil of broad bean collected from Qujing, Yunnan, PR China and characterized by using polyphasic taxonomy. The bacterial cells of strain 11K1T were rod-shaped, motile by two polar flagella and positive for oxidase and catalase. Results of phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain had the highest similarities to Pseudomonas thivervalensis DSM 13194T (99.52 %), Pseudomonas lini CFBP 5737T (99.45 %), Pseudomonas chlororaphis subsp. chlororaphi s NBRC 3904T (99.31 %), Pseudomonas kilonensis DSM 13647T (99.25 %) and Pseudomonas brassicacearum JCM11938T (99.24 %). Multilocus sequence analysis using the 16S rRNA, gyrB, rpoB and rpoD gene sequences demonstrated that strain 11K1T was a member of the Pseudomonas corrugata subgroup within the Pseudomonas fluorescens lineage, but was distant from all closely related species. The average nucleotide identity and in silico DNA–DNA hybridization values were lower than recommended thresholds of 95 and 70 %, respectively, for species delineation. The major isoprenoid quinone of strain 11K1T was ubiquinone (Q-9) and the major cellular fatty acids were C16 : 0, summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c), summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c) and C17 : 0 cyclo. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, aminophospholipid and two unidentified lipids. Based on the results of phenotypic characterization, phylogenetic analysis and genome comparison, strain 11K1T represents a novel species of the genus Pseudomonas , for which the name Pseudomonas viciae sp. nov. is proposed. The type strain is 11K1T (=GDMCC 1.1743T=KACC 21650T).

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