scholarly journals Pathogenomic analyses of Mycobacterium microti, an ESX-1-deleted member of the Mycobacterium tuberculosis complex causing disease in various hosts

2021 ◽  
Vol 7 (2) ◽  
Author(s):  
Mickael Orgeur ◽  
Wafa Frigui ◽  
Alexandre Pawlik ◽  
Simon Clark ◽  
Ann Williams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pig ◽  
Type Species ◽  
Clinical Isolates ◽  
Recent Common Ancestor ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type ◽  
Vaccine Potential ◽  
Mycobacterium Microti

Mycobacterium microti is an animal-adapted member of the Mycobacterium tuberculosis complex (MTBC), which was originally isolated from voles, but has more recently also been isolated from other selected mammalian hosts, including occasionally from humans. Here, we have generated and analysed the complete genome sequences of five representative vole and clinical M. microti isolates using PacBio- and Illumina-based technologies, and have tested their virulence and vaccine potential in SCID (severe combined immune deficient) mouse and/or guinea pig infection models. We show that the clinical isolates studied here cluster separately in the phylogenetic tree from vole isolates and other clades from publicly available M. microti genome sequences. These data also confirm that the vole and clinical M. microti isolates were all lacking the specific RD1mic region, which in other tubercle bacilli encodes the ESX-1 type VII secretion system. Biochemical analysis further revealed marked phenotypic differences between isolates in type VII-mediated secretion of selected PE and PPE proteins, which in part were attributed to specific genetic polymorphisms. Infection experiments in the highly susceptible SCID mouse model showed that the clinical isolates were significantly more virulent than the tested vole isolates, but still much less virulent than the M. tuberculosis H37Rv control strain. The strong attenuation of the ATCC 35872 vole isolate in immunocompromised mice, even compared to the attenuated BCG (bacillus Calmette–Guérin) vaccine, and its historic use in human vaccine trials encouraged us to test this strain’s vaccine potential in a guinea pig model, where it demonstrated similar protective efficacy as a BCG control, making it a strong candidate for vaccination of immunocompromised individuals in whom BCG vaccination is contra-indicated. Overall, we provide new insights into the genomic and phenotypic variabilities and particularities of members of an understudied clade of the MTBC, which all share a recent common ancestor that is characterized by the deletion of the RD1mic region.

Rifampin-resistance-associated mutations in the rifampin-resistance-determining region of the rpoB gene of Mycobacterium tuberculosis clinical isolates in Shanghai, PR China

2021 ◽  
Author(s):  
Yinjuan Guo ◽  
Xingwei Cao ◽  
Jinghui Yang ◽  
Xiaocui Wu ◽  
Yin Liu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Mutation Frequency ◽  
Rpob Gene ◽  
Clinical Isolates ◽  
Content Type ◽  
Link Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Rifampin Resistance

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive. Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance. Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis . Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis. Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01). Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Nirbhay Singh ◽  
Anu Chauhan ◽  
Ram Kumar ◽  
Sudheer Kumar Singh
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Ph ◽  
Stress Conditions ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

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 Xanthomonas hydrangeae sp. nov., a novel plant pathogen isolated from Hydrangea arborescens

2021 ◽  
Vol 71 (12) ◽  
Author(s):  
Nay C. Dia ◽  
Johan Van Vaerenbergh ◽  
Cinzia Van Malderghem ◽  
Jochen Blom ◽  
Theo H. M. Smits ◽  
...  
Keyword(s):  
Dna Hybridization ◽  
Type Strain ◽  
Type Species ◽  
Novel Species ◽  
Close Relation ◽  
Genome Sequences ◽  
Content Type ◽  
Phylogenetic Characterization ◽  
Link Type ◽  
Amplification Assay

This paper describes a novel species isolated in 2011 and 2012 from nursery-grown Hydrangea arborescens cultivars in Flanders, Belgium. After 4 days at 28 °C, the strains yielded yellow, round, convex and mucoid colonies. Pathogenicity of the strains was confirmed on its isolation host, as well as on Hydrangea quercifolia. Analysis using MALDI-TOF MS identified the Hydrangea strains as belonging to the genus Xanthomonas but excluded them from the species Xanthomonas hortorum . A phylogenetic tree based on gyrB confirmed the close relation to X. hortorum . Three fatty acids were dominant in the Hydrangea isolates: anteiso-C15 : 0, iso-C15 : 0 and summed feature 3 (C16 : 1  ω7c/C16 : 1  ω6c). Unlike X. hortorum pathovars, the Hydrangea strains were unable to grow in the presence of lithium chloride and could only weakly utilize d-fructose-6-PO4 and glucuronamide. Phylogenetic characterization based on multilocus sequence analysis and phylogenomic characterization revealed that the strains are close to, yet distinct from, X. hortorum . The genome sequences of the strains had average nucleotide identity values ranging from 94.35–95.19 % and in silico DNA–DNA hybridization values ranging from 55.70 to 59.40 % to genomes of the X. hortorum pathovars. A genomics-based loop-mediated isothermal amplification assay was developed which was specific to the Hydrangea strains for its early detection. A novel species, Xanthomonas hydrangeae sp. nov., is proposed with strain LMG 31884T (=CCOS 1956T) as the type strain.

scholarly journals Genome analysis-based reclassification of Pseudomonas fuscovaginae and Pseudomonas shirazica as later heterotypic synonyms of Pseudomonas asplenii and Pseudomonas asiatica, respectively

2020 ◽  
Vol 70 (5) ◽  
pp. 3547-3552 ◽  
Author(s):  
Mari Tohya ◽  
Shin Watanabe ◽  
Tatsuya Tada ◽  
Htay Htay Tin ◽  
Teruo Kirikae
Keyword(s):  
Dna Hybridization ◽  
Type Species ◽  
Bacterial Species ◽  
Taxonomic Status ◽  
Species Delineation ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type ◽  
Type Strains ◽  
Group Species

This study was conducted to clarify the taxonomic status of the species Pseudomonas fuscovaginae and Pseudomonas shirazica . Whole genome sequences for the type strains of P. fuscovaginae and P. shirazica were compared against the closely related type strains of the Pseudomonas putida group and the Pseudomonas fluorescens group species. Average nucleotide identity and digital DNA–DNA hybridization values between P. fuscovaginae LMG 2158T and Pseudomonas asplenii ATCC 23835T were 98.4 and 85.5 %, and between P. shirazica VM14T and Pseudomonas asiatica RYU5T were 99.3 and 95.3 %. These values were greater than recognized thresholds for bacterial species delineation, indicating that they belong to the same genomospecies, respectively. Therefore, P. fuscovaginae and P. shirazica should be reclassified as later heterotypic synonyms of P. asplenii and P. asiatica , respectively.

scholarly journals Genomic epidemiology of tuberculosis in eastern Malaysia: insights for strengthening public health responses

2021 ◽  
Vol 7 (5) ◽  
Author(s):  
Arnold Bainomugisa ◽  
Ella M. Meumann ◽  
Giri Shan Rajahram ◽  
Rick Twee-Hee Ong ◽  
Lachlan Coin ◽  
...  
Keyword(s):  
Public Health ◽  
Type Species ◽  
Multidrug Resistant ◽  
Resistant Isolate ◽  
Recent Common Ancestor ◽  
Resistance Mutations ◽  
Content Type ◽  
Genomic Epidemiology ◽  
Link Type ◽  
Lineage 2

Tuberculosis is a leading public health priority in eastern Malaysia. Knowledge of the genomic epidemiology of tuberculosis can help tailor public health interventions. Our aims were to determine tuberculosis genomic epidemiology and characterize resistance mutations in the ethnically diverse city of Kota Kinabalu, Sabah, located at the nexus of Malaysia, Indonesia, Philippines and Brunei. We used an archive of prospectively collected Mycobacterium tuberculosis samples paired with epidemiological data. We collected sputum and demographic data from consecutive consenting outpatients with pulmonary tuberculosis at the largest tuberculosis clinic from 2012 to 2014, and selected samples from tuberculosis inpatients from the tertiary referral centre during 2012–2014 and 2016–2017. Two hundred and eight M . tuberculosis sequences were available for analysis, representing 8 % of cases notified during the study periods. Whole-genome phylogenetic analysis demonstrated that most strains were lineage 1 (195/208, 93.8 %), with the remainder being lineages 2 (8/208, 3.8 %) or 4 (5/208, 2.4 %). Lineages or sub-lineages were not associated with patient ethnicity. The lineage 1 strains were diverse, with sub-lineage 1.2.1 being dominant (192, 98 %). Lineage 1.2.1.3 isolates were geographically most widely distributed. The greatest diversity occurred in a border town sub-district. The time to the most recent common ancestor for the three major lineage 1.2.1 clades was estimated to be the year 1966 (95 % HPD 1948–1976). An association was found between failure of culture conversion by week 8 of treatment and infection with lineage 2 (4/6, 67 %) compared with lineage 1 strains (4/83, 5 %) (P<0.001), supporting evidence of greater virulence of lineage 2 strains. Eleven potential transmission clusters (SNP difference ≤12) were identified; at least five included people living in different sub-districts. Some linked cases spanned the whole 4-year study period. One cluster involved a multidrug-resistant tuberculosis strain matching a drug-susceptible strain from 3 years earlier. Drug resistance mutations were uncommon, but revealed one phenotype–genotype mismatch in a genotypically multidrug-resistant isolate, and rare nonsense mutations within the katG gene in two isolates. Consistent with the regionally mobile population, M. tuberculosis strains in Kota Kinabalu were diverse, although several lineage 1 strains dominated and were locally well established. Transmission clusters – uncommonly identified, likely attributable to incomplete sampling – showed clustering occurring across the community, not confined to households or sub-districts. The findings indicate that public health priorities should include active case finding and early institution of tuberculosis management in mobile populations, while there is a need to upscale effective contact investigation beyond households to include other contacts within social networks.

scholarly journals Antibacterial activity of high-dose nitric oxide against pulmonary Mycobacterium abscessus disease

2020 ◽  
Vol 2 (9) ◽  
Author(s):  
Kristijan Bogdanovski ◽  
Trisha Chau ◽  
Chevalia J. Robinson ◽  
Sandra D. MacDonald ◽  
Ann M. Peterson ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Antibacterial Activity ◽  
Type Species ◽  
Mycobacterium Abscessus ◽  
Clinical Isolates ◽  
Content Type ◽  
Link Type ◽  
Susceptibility Tests ◽  
Inhaled No

Introduction. Mycobacterium abscessus is an emerging pulmonary pathogen with limited treatment options. Nitric oxide (NO) demonstrates antibacterial activity against various bacterial species, including mycobacteria. In this study, we evaluated the effect of adjunctive inhaled NO therapy, using a novel NO generator, in a CF patient with pulmonary M. abscessus disease, and examined heterogeneity of response to NO in vitro. Methods. In the compassionate-use treatment, a 24-year-old CF patient with pulmonary M. abscessus was treated with two courses of adjunctive intermittent NO, first at 160 p.p.m. for 21 days and subsequently by escalating the dose up to 240 p.p.m. for 8 days. Methemoglobin, pulmonary function, 6 min walk distance (6MWD), qualify of life and sputum microbiology were assessed. In vitro susceptibility tests were performed against patient’s isolate and comparison clinical isolates and quantified by Hill’s slopes calculated from time–kill curves. Results. M. abscessus lung infection eradication was not achieved, but improvements in selected qualify of life domains, lung function and 6MWD were observed during the study. Inhaled NO was well tolerated at 160 p.p.m. Dosing at 240 p.p.m. was stopped due to adverse symptoms, although methemoglobin levels remained within safety thresholds. In vitro susceptibility tests showed a dose-dependent NO effect on M. abscessus susceptibility and significant heterogeneity in response between M. abscessus clinical isolates. The patient’s isolate was found to be the least susceptible strain in vitro. Conclusion. These results demonstrate heterogeneity in M. abscessus susceptibility to NO and suggest that longer treatment regimens could be required to see the reduction or eradication of more resistant pulmonary strains.

scholarly journals Improved molecular characterization of the Klebsiella oxytoca complex reveals the prevalence of the kleboxymycin biosynthetic gene cluster

2021 ◽  
Vol 7 (6) ◽  
Author(s):  
Preetha Shibu ◽  
Frazer McCuaig ◽  
Anne L. McCartney ◽  
Magdalena Kujawska ◽  
Lindsay J. Hall ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Type Species ◽  
Phylogenetic Analyses ◽  
Klebsiella Oxytoca ◽  
Biosynthetic Gene Cluster ◽  
Biosynthetic Gene ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type

As part of the ongoing studies with clinically relevant Klebsiella spp., we characterized the genomes of three clinical GES-5-positive ST138 strains originally identified as Klebsiella oxytoca. bla OXY gene, average nucleotide identity and phylogenetic analyses showed the strains to be Klebsiella michiganensis . Affiliation of the strains to ST138 led us to demonstrate that the current multi-locus sequence typing scheme for K. oxytoca can be used to distinguish members of this genetically diverse complex of bacteria. The strains encoded the kleboxymycin biosynthetic gene cluster (BGC), previously only found in K. oxytoca strains and one strain of Klebsiella grimontii . The finding of this BGC, associated with antibiotic-associated haemorrhagic colitis, in K. michiganensis led us to carry out a wide-ranging study to determine the prevalence of this BGC in Klebsiella spp. Of 7170 publicly available Klebsiella genome sequences screened, 88 encoded the kleboxymycin BGC. All BGC-positive strains belonged to the K. oxytoca complex, with strains of four ( K. oxytoca , K. pasteurii , K. grimontii , K. michiganensis ) of the six species of complex found to encode the complete BGC. In addition to being found in K. grimontii strains isolated from preterm infants, the BGC was found in K. oxytoca and K. michiganensis metagenome-assembled genomes recovered from neonates. Detection of the kleboxymycin BGC across the K. oxytoca complex may be of clinical relevance and this cluster should be included in databases characterizing virulence factors, in addition to those characterizing BGCs.

scholarly journals Highly multidrug-resistant Morganella morganii clinical isolates from Nepal co-producing NDM-type metallo-β-lactamases and the 16S rRNA methylase ArmA

2020 ◽  
Vol 69 (4) ◽  
pp. 572-575
Author(s):  
Shovita Shrestha ◽  
Tatsuya Tada ◽  
Jatan B. Sherchan ◽  
Hiroki Uchida ◽  
Tomomi Hishinuma ◽  
...  
Keyword(s):  
16S Rrna ◽  
Control Systems ◽  
Type Species ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Morganella Morganii ◽  
Content Type ◽  
Link Type ◽  
16S Rrna Methylase ◽  
Multiple Antibiotics

Morganella morganii can harbour extended-spectrum β-lactamases and carbapenemases, resulting in increased resistance to multiple antibiotics and a high mortality rate. This study describes the emergence of highly multidrug-resistant clinical isolates of M. morganii from Nepal co-producing NDM-type metallo-β-lactamases, including NDM-1 and NDM-5, and the 16S rRNA methylase ArmA. This is the first report of M. morganii clinical isolates from Nepal co-producing NDM-1/-5 and ArmA. It is important to establish infection control systems and effective treatments against multidrug-resistant M. morganii .

scholarly journals Re-identification of strains deposited as Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida in GenBank based on whole genome sequences

2020 ◽  
Vol 70 (11) ◽  
pp. 5958-5963
Author(s):  
Yuh Morimoto ◽  
Mari Tohya ◽  
Zulipiya Aibibula ◽  
Tadashi Baba ◽  
Hiroyuki Daida ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genome Sequencing ◽  
Dna Hybridization ◽  
Type Species ◽  
Whole Genome ◽  
Average Nucleotide Identity ◽  
Genome Sequences ◽  
Content Type ◽  
Link Type

The taxonomic classification of Pseudomonas species has been revised and updated several times. This study utilized average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) cutoff values of 95 and 70 %, respectively, to re-identify the species of strains deposited in GenBank as P. aeruginosa , P. fluorescens and P. putida . Of the 264 deposited P. aeruginosa strains, 259 were correctly identified as P. aeruginosa , but the remaining five were not. All 28 deposited P. fluorescens strains had been incorrectly identified as P. fluorescens . Four of these strains were re-identified, including two as P. kilonensis and one each as P. aeruginosa and P. brassicacearum , but the remaining 24 could not be re-identified. Similarly, all 35 deposited P. putida strains had been incorrectly identified as P. putida . Nineteen of these strains were re-identified, including 12 as P. alloputida , four as P. asiatica and one each as P. juntendi , P. monteilii and P. mosselii . These results strongly suggest that Pseudomonas bacteria should be identified using ANI and dDDH analyses based on whole genome sequencing when Pseudomonas species are initially deposited in GenBank/DDBJ/EMBL databases.

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