Menu

Auto Tag: Infectious disease

July 7, 2019

Genomic analysis of ST88 community-acquired methicillin resistant Staphylococcus aureus in Ghana.

The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an “African” CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide.We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages.We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the other S. aureus lineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics including ß-lactams, tetracycline and chloramphenicol.This study reveals that S. aureus ST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.

Read more
July 7, 2019

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

Read more
July 7, 2019

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs.

Chronic bacterial infections of the lung are the leading cause of morbidity and mortality in cystic fibrosis patients. Tracking bacterial evolution during chronic infections can provide insights into how host selection pressures-including immune responses and therapeutic interventions-shape bacterial genomes. We carried out genomic and phenotypic analyses of 215 serially collected Burkholderia cenocepacia isolates from 16 cystic fibrosis patients, spanning a period of 2-20 yr and a broad range of epidemic lineages. Systematic phenotypic tests identified longitudinal bacterial series that manifested progressive changes in liquid media growth, motility, biofilm formation, and acute insect virulence, but not in mucoidy. The results suggest that distinct lineages follow distinct evolutionary trajectories during lung infection. Pan-genome analysis identified 10,110 homologous gene clusters present only in a subset of strains, including genes restricted to different molecular types. Our phylogenetic analysis based on 2148 orthologous gene clusters from all isolates is consistent with patient-specific clades. This suggests that initial colonization of patients was likely by individual strains, followed by subsequent diversification. Evidence of clonal lineages shared by some patients was observed, suggesting inter-patient transmission. We observed recurrent gene losses in multiple independent longitudinal series, including complete loss of Chromosome III and deletions on other chromosomes. Recurrently observed loss-of-function mutations were associated with decreases in motility and biofilm formation. Together, our study provides the first comprehensive genome-phenome analyses of B. cenocepacia infection in cystic fibrosis lungs and serves as a valuable resource for understanding the genomic and phenotypic underpinnings of bacterial evolution.© 2017 Lee et al.; Published by Cold Spring Harbor Laboratory Press.

Read more
July 7, 2019

The recent emergence in hospitals of multidrug-resistant community-associated sequence type 1 and spa type t127 methicillin-resistant Staphylococcus aureus investigated by whole-genome sequencing: Implications for screening.

Community-associated spa type t127/t922 methicillin-resistant Staphylococcus aureus (MRSA) prevalence increased from 1%-7% in Ireland between 2010-2015. This study tracked the spread of 89 such isolates from June 2013-June 2016. These included 78 healthcare-associated and 11 community associated-MRSA isolates from a prolonged hospital outbreak (H1) (n = 46), 16 other hospitals (n = 28), four other healthcare facilities (n = 4) and community-associated sources (n = 11). Isolates underwent antimicrobial susceptibility testing, DNA microarray profiling and whole-genome sequencing. Minimum spanning trees were generated following core-genome multilocus sequence typing and pairwise single nucleotide variation (SNV) analysis was performed. All isolates were sequence type 1 MRSA staphylococcal cassette chromosome mec type IV (ST1-MRSA-IV) and 76/89 were multidrug-resistant. Fifty isolates, including 40/46 from H1, were high-level mupirocin-resistant, carrying a conjugative 39 kb iles2-encoding plasmid. Two closely related ST1-MRSA-IV strains (I and II) and multiple sporadic strains were identified. Strain I isolates (57/89), including 43/46 H1 and all high-level mupirocin-resistant isolates, exhibited =80 SNVs. Two strain I isolates from separate H1 healthcare workers differed from other H1/strain I isolates by 7-47 and 12-53 SNVs, respectively, indicating healthcare worker involvement in this outbreak. Strain II isolates (19/89), including the remaining H1 isolates, exhibited =127 SNVs. For each strain, the pairwise SNVs exhibited by healthcare-associated and community-associated isolates indicated recent transmission of ST1-MRSA-IV within and between multiple hospitals, healthcare facilities and communities in Ireland. Given the interchange between healthcare-associated and community-associated isolates in hospitals, the risk factors that inform screening for MRSA require revision.

Read more
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

Complex routes of nosocomial vancomycin-resistant Enterococcus faecium transmission revealed by genome sequencing.

Vancomycin-resistant Enterococcus faecium (VREfm) is a leading cause of nosocomial infection. Here, we describe the utility of whole-genome sequencing in defining nosocomial VREfm transmission.A retrospective study at a single hospital in the United Kingdom identified 342 patients with E. faecium bloodstream infection over 7 years. Of these, 293 patients had a stored isolate and formed the basis for the study. The first stored isolate from each case was sequenced (200 VREfm [197 vanA, 2 vanB, and 1 isolate containing both vanA and vanB], 93 vancomycin-susceptible E. faecium) and epidemiological data were collected. Genomes were also available for E. faecium associated with bloodstream infections in 15 patients in neighboring hospitals, and 456 patients across the United Kingdom and Ireland.The majority of infections in the 293 patients were hospital-acquired (n = 249) or healthcare-associated (n = 42). Phylogenetic analysis showed that 291 of 293 isolates resided in a hospital-associated clade that contained numerous discrete clusters of closely related isolates, indicative of multiple introductions into the hospital followed by clonal expansion associated with transmission. Fine-scale analysis of 6 exemplar phylogenetic clusters containing isolates from 93 patients (32%) identified complex transmission routes that spanned numerous wards and years, extending beyond the detection of conventional infection control. These contained both vancomycin-resistant and -susceptible isolates. We also identified closely related isolates from patients at Cambridge University Hospitals NHS Foundation Trust and regional and national hospitals, suggesting interhospital transmission.These findings provide important insights for infection control practice and signpost areas for interventions. We conclude that sequencing represents a powerful tool for the enhanced surveillance and control of nosocomial E. faecium transmission and infection.

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

Antibodyomics: bioinformatics technologies for understanding B-cell immunity to HIV-1.

Numerous antibodies have been identified from HIV-1-infected donors that neutralize diverse strains of HIV-1. These antibodies may provide the basis for a B cell-mediated HIV-1 vaccine. However, it has been unclear how to elicit similar antibodies by vaccination. To address this issue, we have undertaken an informatics-based approach to understand the genetic and immunologic processes controlling the development of HIV-1-neutralizing antibodies. As DNA sequencing comprises the fastest growing database of biological information, we focused on incorporating next-generation sequencing of B-cell transcripts to determine the origin, maturation pathway, and prevalence of broadly neutralizing antibody lineages (Antibodyomics1, 2, 4, and 6). We also incorporated large-scale robotic analyses of serum neutralization to identify and quantify neutralizing antibodies in donor cohorts (Antibodyomics3). Statistical analyses furnish another layer of insight (Antibodyomics5), with physical characteristics of antibodies and their targets through molecular dynamics simulations (Antibodyomics7) and free energy perturbation analyses (Antibodyomics8) providing information-rich output. Functional interrogation of individual antibodies (Antibodyomics9) and synthetic antibody libraries (Antibodyomics10) also yields multi-dimensional data by which to understand and improve antibodies. Antibodyomics, described here, thus comprise resolution-enhancing tools, which collectively embody an information-driven discovery engine aimed toward the development of effective B cell-based vaccines.© 2017 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.

Read more
July 7, 2019

Whole-genome sequences of Burkholderia pseudomallei isolates exhibiting decreased meropenem susceptibility.

We report here paired isogenic Burkholderia pseudomallei genomes obtained from three patients receiving intravenous meropenem for melioidosis treatment, with post-meropenem isolates developing decreased susceptibility. Two genomes were finished, and four were drafted to improved high-quality standard. These genomes will be used to identify meropenem resistance mechanisms in B. pseudomallei. Copyright © 2017 Price et al.

Read more
July 7, 2019

Complete genome of Brachybacterium sp. P6-10-X1 isolated from deep-sea sediments of the Southern Ocean

Brachybacterium sp. P6-10-X1 is a rare actinobacterium isolated from deep-sea sediments in the Southern Ocean. To explore the potential of natural product biosynthesis, the genome was completely sequenced. It contained a circular chromosome of 4,385,603 bp with an average GC content of 70.9%. Genome mining revealed four biosynthetic gene clusters potentially producing new natural products.

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.