Genomanalyse und Identifikation von Biomarkern von Leptospira spp.

Leptospirosis is the most common zoonotic disease worldwide. In this work, a pathogenomics approach was used to associates distinctive phenotypic features with genomic and transcriptomic characteristics in clinical strains of Leptospira interrogans (L. interrogans). In addition, ORFeome phage display was used to identify seroreactive peptides. Six L. interrogans isolated from Malaysia were studied and compared clinically and tested for virulence in the guinea pig model. SMRT sequencing was carried out on the PacBio RSII and complemented using an Illumina MiSeq. Two L. interrogans strains from both extremes of the virulence spectrum were subjected in triplicates to RNA-seq using Illumina HiSeq2500. All six L. interrogans strains were typed with multilocus sequence typing (MLST), core genome MLST and single nucleotide polymorphism (SNP) genotyping. Two ORFeome phage display libraries of Leptospira spp. genomes from Malaysian strains and WHO reference strains were constructed. Subsequently, five seroreactive synthetic peptides were constructed and validated for immunogenicity with 16 control and 16 patients sera by titration ELISA. Clinically, the patient infected with strain Langkawi presented with the most severe clinical course, while strain 1530d had mild leptospirosis. These findings correlated well with those of the animal study. Both cgMLST and SNP genotyping are comparable to ST clustering in MLST, however, SNP genotyping resulted in a higher coverage of the core genome i.e. 77.5% compared to 57.7% in cgMLST. There is remarkable genome plasticity in nearly all genomes, which is mainly driven by plasmids and insertion sequences elements. At logarithmic phase culture, genes associated with chemotaxis were expressed significantly more highly in strain Langkawi compared to strain 1530d, most likely explains the greater virulence of strain Langkawi compared to 1530d. In the phage display approach, two peptides (SIR16-D1 and SIR16-H1) were identified to have good accuracy for acute leptospirosis detection (area under the ROC curve: 0.86 and 0.78 respectively). Genome plasticity were gained through horizontal gene transfer and homologous recombination as an adaptive strategy in a variety of ecological niches. Differences in pathogenicity of all the strains appeared to be largely determined by genomic make-up. ORFeome phage display proved to be a powerful technique for the identification of leptospiral immunogenic peptides.Leptospirose ist die weltweit häufigste Zoonose-Erkrankung. In dieser Arbeit wurde ein pathogenomischer Ansatz verwendet, um phänotypische Merkmale mit Genom und Transkriptom Daten von klinischen Stämmen von Leptospira interrogans (L. interrogans) zu verknüpfen. Zusätzlich wurde ORFeome-Phagendisplay verwendet, um seroreaktive Peptide zu identifizieren. Sechs aus Malaysia isolierte L. interrogans Stämme wurden untersucht,klinisch verglichen und auf Virulenz im Meerschweinchenmodell getestet. Die SMRT-Sequenzierung wurde am PacBio RSII durchgeführt und mit einem Illumina MiSeq ergänzt. Zwei L. interrogans-Stämme aus beiden Extremen des Virulenzspektrums wurden mittels RNA-seq untersucht. Alle sechs L. interrogans-Stämme wurden mittels MLST Core-Genom-MLST und (SNP) typisiert. Zwei ORFeome-Phagendisplaybibliotheken von Leptospira spp. Genomen vMalaysischer und WHO Referenzstämmen wurden konstruiert. Anschließend wurden fünf seroreaktive synthetische Peptide konstruiert und mit 16 Kontrollseren und 16 Patientenseren durch Titrations-ELISA auf Immunogenität validiert. Klinisch zeigte sich der mit dem Stamm Langkawi infizierte Patient dieschwerwiegendsten Symptome, während der Stamm 1530d eine leichte Leptospirose aufwies. Diese Befunde korrelierten gut mit denen der Tierstudie. Sowohl cgMLST als auch SNP-Genotypisierung sind vergleichbar mit ST-Clustering in MLST, jedoch führte die SNP-Genotypisierung zu einer höheren Abdeckung des Kerngenoms, d. H. 77,5%, verglichen mit 57,7% in cgMLST. In fast allen Genomen gibt es eine bemerkenswerte Genom-Plastizität, die hauptsächlich von Plasmiden und Insertionssequenzelementen bestimmt wird. In der Kultivierung wurden Gene, die mit Chemotaxis in Verbindung stehen, im Stamm Langkawi im Vergleich zum Stamm 1530d signifikant höher exprimiert. Dies erklärt höchstwahrscheinlich die höhere Virulenz des Stammes Langkawi im Vergleich zu 1530d. Beim Phagendisplay-Ansatz wurden zwei Peptide (SIR16-D1 und SIR16-H1) mit hoher Genauigkeit für den Nachweis akuter Leptospirosen identifiziert (AUC: 0.86 bzw. 0.78). Die Genomplastizität wurde durch horizontalen Gentransfer und homologe Rekombination als adaptive Strategie in verschiedenen ökologischen Nischen erlangt. Die Unterschiede in der Pathogenität aller Stämme schienen weitgehend durch die Genomzusammensetzung bestimmt zu sein. Das ORFeome-Phagendisplay erwies sich als leistungsfähige Methode zur Identifizierung von immunogenen Leptospiral-Peptiden

Screening and detection of heterogenous vancomycin intermediate Staphylococcus aureus in Hospital Kuala Lumpur Malaysia, using the glycopeptide resistance detection etest and population analysis profiling

In a 3-month study done in Hospital Kuala Lumpur (HKL), 7 out of 320 methicillin resistant Staphylococcus aureus isolates were confirmed as heterogeneous vancomycin intermediate S. aureus (hVISA) using the glycopeptide resistance detection e-test and population analysis, giving a prevalence rate of 2.19%. This is the first report of hVISA in Malaysia

A Severe Case of Arthrographis kalrae Keratomycosis

A 52-year-old man with diabetes developed a unilateral central corneal ulcer after accidental foreign body inoculation. He complained of pain and loss of visual acuity in the injured eye, which displayed redness and edema and eventually discharged pus. A corneal scraping from the left eye orbit revealed fungal elements, and cultures of the material grew a fungus. The isolate was identified as Arthrographis kalrae based on gross and microscopic morphologies. The patient received amphotericin B intravenously and itraconazole orally. The wound healed following surgical intervention, but the patient lost the use of his left eye

Complete Genome Sequencing Leptospira interrogans Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes

The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence

Complete Genome Sequencing of Isolates from Malaysia Reveals Massive Genome Rearrangement but High Conservation of Virulence-Associated Genes.

The ability of Leptospirae to persist in environments and animal hosts but to cause clinically highly variable disease in humans has made leptospirosis the most common zoonotic disease. Considering the paucity of data on variation in complete genomes of human pathogenic Leptospirae, we have used a combination of Single Molecule Real-Time (SMRT) and Illumina sequencing to obtain complete genome sequences of six human clinical L. interrogans isolates from Malaysia. All six contained the larger (4.28-4.56 Mb) and smaller (0.34-0.395 Mb) chromosome typical of human pathogenic Leptospirae and 0-7 plasmids. Only 24% of the plasmid sequences could be matched to databases. We identified a chromosomal core genome of 3318 coding sequences and strain-specific accessory genomes of 49-179 coding sequences. These sequences enabled detailed genomic strain typing (Genome BLAST Distance Phylogeny, DNA-DNA hybridization, and multi locus sequence typing) and phylogenetic classification (whole-genome SNP genotyping). Even though there was some shared synteny and collinearity across the six genomes, there was evidence of major genome rearrangement, likely driven by horizontal gene transfer and homologous recombination. Mobile genetic elements were identified in all strains in highly varying numbers, including in the rfb locus, which defines serogroups and contributes to immune escape and pathogenesis. On the other hand, there was high conservation of virulence-associated genes including those relating to sialic acid, alginate, and lipid A biosynthesis. These findings suggest (i) that the antigenic variation, adaption to various host environments, and broad spectrum of virulence of L. interrogans are in part due to a high degree of genomic plasticity and (ii) that human pathogenic strains maintain a core set of genes required for virulence

Discovery of Leptospira spp. seroreactive peptides using ORFeome phage display.

BACKGROUND:Leptospirosis is the most common zoonotic disease worldwide. The diagnostic performance of a serological test for human leptospirosis is mainly influenced by the antigen used in the test assay. An ideal serological test should cover all serovars of pathogenic leptospires with high sensitivity and specificity and use reagents that are relatively inexpensive to produce and can be used in tropical climates. Peptide-based tests fulfil at least the latter two requirements, and ORFeome phage display has been successfully used to identify immunogenic peptides from other pathogens. METHODOLOGY/PRINCIPAL FINDINGS:Two ORFeome phage display libraries of the entire Leptospira spp. genomes from five local strains isolated in Malaysia and seven WHO reference strains were constructed. Subsequently, 18 unique Leptospira peptides were identified in a screen using a pool of sera from patients with acute leptospirosis. Five of these were validated by titration ELISA using different pools of patient or control sera. The diagnostic performance of these five peptides was then assessed against 16 individual sera from patients with acute leptospirosis and 16 healthy donors and was compared to that of two recombinant reference proteins from L. interrogans. This analysis revealed two peptides (SIR16-D1 and SIR16-H1) from the local isolates with good accuracy for the detection of acute leptospirosis (area under the ROC curve: 0.86 and 0.78, respectively; sensitivity: 0.88 and 0.94; specificity: 0.81 and 0.69), which was close to that of the reference proteins LipL32 and Loa22 (area under the ROC curve: 0.91 and 0.80; sensitivity: 0.94 and 0.81; specificity: 0.75 and 0.75). CONCLUSIONS/SIGNIFICANCE:This analysis lends further support for using ORFeome phage display to identify pathogen-associated immunogenic peptides, and it suggests that this technique holds promise for the development of peptide-based diagnostics for leptospirosis and, possibly, of vaccines against this pathogen