Menu

Auto Tag: Virulence

July 7, 2019

Elucidation of quantitative structural diversity of remarkable rearrangement regions, shufflons, in IncI2 plasmids.

A multiple DNA inversion system, the shufflon, exists in incompatibility (Inc) I1 and I2 plasmids. The shufflon generates variants of the PilV protein, a minor component of the thin pilus. The shufflon is one of the most difficult regions for de novo genome assembly because of its structural diversity even in an isolated bacterial clone. We determined complete genome sequences, including those of IncI2 plasmids carrying mcr-1, of three Escherichia coli strains using single-molecule, real-time (SMRT) sequencing and Illumina sequencing. The sequences assembled using only SMRT sequencing contained misassembled regions in the shufflon. A hybrid analysis using SMRT and Illumina sequencing resolved the misassembled region and revealed that the three IncI2 plasmids, excluding the shufflon region, were highly conserved. Moreover, the abundance ratio of whole-shufflon structures could be determined by quantitative structural variation analysis of the SMRT data, suggesting that a remarkable heterogeneity of whole-shufflon structural variations exists in IncI2 plasmids. These findings indicate that remarkable rearrangement regions should be validated using both long-read and short-read sequencing data and that the structural variation of PilV in the shufflon might be closely related to phenotypic heterogeneity of plasmid-mediated transconjugation involved in horizontal gene transfer even in bacterial clonal populations.

Read more
July 7, 2019

Extremely low genomic diversity of Rickettsia japonica distributed in Japan.

Rickettsiae are obligate intracellular bacteria that have small genomes as a result of reductive evolution. Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as “spotted fevers”. The life cycle of SFG rickettsiae is closely associated with that of the tick, which is generally thought to act as a bacterial vector and reservoir that maintains the bacterium through transstadial and transovarial transmission. Each SFG member is thought to have adapted to a specific tick species, thus restricting the bacterial distribution to a relatively limited geographic region. These unique features of SFG rickettsiae allow investigation of how the genomes of such biologically and ecologically specialized bacteria evolve after genome reduction and the types of population structures that are generated. Here, we performed a nationwide, high-resolution phylogenetic analysis of Rickettsia japonica, an etiological agent of Japanese spotted fever that is distributed in Japan and Korea. The comparison of complete or nearly complete sequences obtained from 31 R. japonica strains isolated from various sources in Japan over the past 30 years demonstrated an extremely low level of genomic diversity. In particular, only 34 single nucleotide polymorphisms were identified among the 27 strains of the major lineage containing all clinical isolates and tick isolates from the three tick species. Our data provide novel insights into the biology and genome evolution of R. japonica, including the possibilities of recent clonal expansion and a long generation time in nature due to the long dormant phase associated with tick life cycles.© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Read more
July 7, 2019

Characterization of Class IIa bacteriocin resistance in Enterococcus faecium.

Vancomycin-resistant enterococci, particularly resistant Enterococcus faecium, pose an escalating threat in nosocomial environments because of their innate resistance to many antibiotics, including vancomycin, a treatment of last resort. Many class IIa bacteriocins strongly target these enterococci and may offer a potential alternative for the management of this pathogen. However, E. faecium’s resistance to these peptides remains relatively uncharacterized. Here, we explored the development of resistance of E. faecium to a cocktail of three class IIa bacteriocins: enterocin A, enterocin P, and hiracin JM79. We started by quantifying the frequency of resistance to these peptides in four clinical isolates of E. faecium We then investigated the levels of resistance of E. faecium 6E6 mutants as well as their fitness in different carbon sources. In order to elucidate the mechanism of resistance of E. faecium to class IIa bacteriocins, we completed whole-genome sequencing of resistant mutants and performed reverse transcription-quantitative PCR (qRT-PCR) of a suspected target mannose phosphotransferase (ManPTS). We then verified this ManPTS’s role in bacteriocin susceptibility by showing that expression of the ManPTS in Lactococcus lactis results in susceptibility to the peptide cocktail. Based on the evidence found from these studies, we conclude that, in accord with other studies in E. faecalis and Listeria monocytogenes, resistance to class IIa bacteriocins in E. faecium 6E6 is likely caused by the disruption of a particular ManPTS, which we believe we have identified. Copyright © 2017 American Society for Microbiology.

Read more
July 7, 2019

Regulation of PI-2b pilus expression in hypervirulent Streptococcus agalactiae ST-17 BM110.

The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) “hypervirulent” ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5′ promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110?covR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

Read more
July 7, 2019

Complete genome sequence of Streptococcus iniae 89353, a virulent strain isolated from diseased tilapia in Taiwan.

Streptococcus iniae 89353 is a virulent strain isolated from diseased tilapia in Taiwan. The full-genome sequence of S. iniae 89353 is 2,098,647 bp. The revealed genome information will be beneficial for identification and understanding of potential virulence genes of Streptococcus iniae and possible immunogens for vaccine development against streptococcosis. Copyright © 2017 Gong et al.

Read more
July 7, 2019

Complete genome sequence of a Paenalcaligenes hominis strain isolated from a paraplegic patient with neurogenic bladder using single-molecule real-time sequencing technology.

The genome of Paenalcaligenes hominis, isolated from a paraplegic patient with neurogenic bladder, was sequenced with the Pacific Biosciences RSII platform. The genome size is 2.68 Mb and includes 3,096 annotated coding sequences, including genes associated with quinone cofactors, which play crucial roles in the virulence of Gram-negative bacteria. Copyright © 2017 Mukhopadhyay et al.

Read more
July 7, 2019

Complete genome sequence of the gamma-aminobutyric acid-producing strain Streptococcus thermophilus APC151.

Here is presented the whole-genome sequence of Streptococcus thermophilus APC151, isolated from a marine fish. This bacterium produces gamma-aminobutyric acid (GABA) in high yields and is biotechnologically suitable to produce naturally GABA-enriched biofunctional yogurt. Its complete genome comprises 2,097 genes and 1,839,134 nucleotides, with an average G+C content of 39.1%. Copyright © 2017 Linares et al.

Read more
July 7, 2019

Evolutionary dynamics and genomic features of the Elizabethkingia anophelis 2015 to 2016 Wisconsin outbreak strain.

An atypically large outbreak of Elizabethkingia anophelis infections occurred in Wisconsin. Here we show that it was caused by a single strain with thirteen characteristic genomic regions. Strikingly, the outbreak isolates show an accelerated evolutionary rate and an atypical mutational spectrum. Six phylogenetic sub-clusters with distinctive temporal and geographic dynamics are revealed, and their last common ancestor existed approximately one year before the first recognized human infection. Unlike other E. anophelis, the outbreak strain had a disrupted DNA repair mutY gene caused by insertion of an integrative and conjugative element. This genomic change probably contributed to the high evolutionary rate of the outbreak strain and may have increased its adaptability, as many mutations in protein-coding genes occurred during the outbreak. This unique discovery of an outbreak caused by a naturally occurring mutator bacterial pathogen provides a dramatic example of the potential impact of pathogen evolutionary dynamics on infectious disease epidemiology.

Read more
July 7, 2019

Population genomic analysis of 1,777 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates, Houston, Texas: unexpected abundance of clonal group 307.

Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae, it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K. pneumoniae strains cultured from patients in the 2,000-bed Houston Methodist Hospital system between September 2011 and May 2015, representing a comprehensive, population-based strain sample. Strains of largely uncharacterized clonal group 307 (CG307) caused more infections than those of well-studied epidemic CG258. Strains varied markedly in gene content and had an extensive array of small and very large plasmids, often containing antimicrobial resistance genes. Some patients with multiple strains cultured over time were infected with genetically distinct clones. We identified 15 strains expressing the New Delhi metallo-beta-lactamase 1 (NDM-1) enzyme that confers broad resistance to nearly all beta-lactam antibiotics. Transcriptome sequencing analysis of 10 phylogenetically diverse strains showed that the global transcriptome of each strain was unique and highly variable. Experimental mouse infection provided new information about immunological parameters of host-pathogen interaction. We exploited the large data set to develop whole-genome sequence-based classifiers that accurately predict clinical antimicrobial resistance for 12 of the 16 antibiotics tested. We conclude that analysis of large, comprehensive, population-based strain samples can assist understanding of the molecular diversity of these organisms and contribute to enhanced translational research. IMPORTANCEKlebsiella pneumoniae causes human infections that are increasingly difficult to treat because many strains are resistant to multiple antibiotics. Clonal group 258 (CG258) organisms have caused outbreaks in health care settings worldwide. Using a comprehensive population-based sample of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae strains, we show that a relatively uncommon clonal type, CG307, caused the plurality of ESBL-producing K. pneumoniae infections in our patients. We discovered that CG307 strains have been abundant in Houston for many years. As assessed by experimental mouse infection, CG307 strains were as virulent as pandemic CG258 strains. Our results may portend the emergence of an especially successful clonal group of antibiotic-resistant K. pneumoniae. Copyright © 2017 Long et al.

Read more
July 7, 2019

Acquisition of virulence factors in livestock-associated MRSA: Lysogenic conversion of CC398 strains by virulence gene-containing phages.

Staphylococcus aureus MRSA strains belonging to the clonal complex 398 (CC398) are highly prevalent in livestock and companion animals but may also cause serious infections in humans. CC398 strains in livestock usually do not possess well-known virulence factors that can be frequently found in other MRSA sequence types (ST). Since many staphylococcal virulence genes are residing on the genomes of temperate phages, the question arises why livestock-associated (LA-) CC398 strains are only rarely infected by those phages. We isolated and characterized four temperate phages (P240, P282, P630 and P1105) containing genes of the immune evasion cluster (IEC) and/or for the Panton-Valentine leucocidin (PVL). Sequence analysis of the phage genomes showed that they are closely related to known phages and that the DNA region encoding lysis proteins, virulence factors and the integrase exhibits numerous DNA repeats which may facilitate genomic rearrangements. All phages lysed and lysogenized LA-CC398 strains. Integration of IEC phage P282 was detected at ten sites of the hosts’ chromosome. The prophages were stably inherited in LA-CC398 and enterotoxin A, staphylokinase and PVL toxin were produced. The data demonstrate that lysogenic conversion of LA-CC398 strains by virulence-associated phages may occur and that new pathotypes may emerge by this mechanism.

Read more
July 7, 2019

Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi.

Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology.Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids.Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.