Prevalence and molecular characterization of Rickettsia spp. from wild small mammals in public parks and urban areas of Bangkok Metropolitan, Thailand

Rural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia

Blood transcriptomics to characterize key biological pathways and identify biomarkers for predicting mortality in melioidosis.

Melioidosis is an often lethal tropical disease caused by the Gram-negative bacillus, Burkholderia pseudomallei. The study objective was to characterize transcriptomes in melioidosis patients and identify genes associated with outcome. Whole blood RNA-seq was performed in a discovery set of 29 melioidosis patients and 3 healthy controls. Transcriptomic profiles of patients who did not survive to 28 days were compared with patients who survived and healthy controls, showing 65 genes were significantly up-regulated and 218 were down-regulated in non-survivors compared to survivors. Up-regulated genes were involved in myeloid leukocyte activation, Toll-like receptor cascades and reactive oxygen species metabolic processes. Down-regulated genes were hematopoietic cell lineage, adaptive immune system and lymphocyte activation pathways. RT-qPCR was performed for 28 genes in a validation set of 60 melioidosis patients and 20 healthy controls, confirming differential expression. IL1R2, GAS7, S100A9, IRAK3, and NFKBIA were significantly higher in non-survivors compared with survivors (P < 0.005) and healthy controls (P < 0.0001). The AUROCC of these genes for mortality discrimination ranged from 0.80-0.88. In survivors, expression of IL1R2, S100A9 and IRAK3 genes decreased significantly over 28 days (P < 0.05). These findings augment our understanding of this severe infection, showing expression levels of specific genes are potential biomarkers to predict melioidosis outcomes

HOUSEKEEPING GENES ANALYSIS OF FOODBORNE PATHOGENIC BACTERIA Vibrio parahaemolyticus ISOLATED FROM AQUATIC BIRDS IN THAILAND

Background: Vibrio parahaemolyticus is one of the leading causative agent of foodborne disease. Infection is caused by consumption of undercooked contaminated seafood. V. parahaemolyticus is commonly found in crustacean species and marine environment. Presence of this organism in avian host has been previously reported, however genetic analysis of avian V. parahaemolyticus is required for molecular epidemiological study of this organism. The aim of this study was to determine genetic profile of V. parahaemolyticus isolated from fecal aquatic bird samples by modified Multilocus Sequence Typing (MLST) method. Methods: Three housekeeping genes fragments (dnaE, gyrB and pntA) of total 18 V. parahaemolyticus isolates from fecal aquatic bird samples at Bangpoo resort, Samut Prakarn province, Thailand, during 2016-2017, were amplified by using conventional PCR for nucleotide sequencing. Nucleotide sequences were analyzed and phylogenetic tree were constructed by MEGA 7.0 software. Comparative genetic analysis of avian isolates from Thailand and worldwide isolates were performed by using information from MLST database of V. parahaemolyticus ( https://pubmlst.org /vparahaemolyticus/). Results: Three housekeeping genes of 18 isolates were successfully amplified and purified for nucleotide sequencing. Phylogenetic tree analysis of concatenated nucleotide sequences indicated that 18 Thai avian isolates were genetically diverse. Five isolates (MUVP 9, 11, 22, 23 and 24) represented identical genetic profile with clinical isolates from China, India, Japan and Peru. Other examined isolates were closely related to environmental isolates from China and United Kingdom. These resultsshowed that aquatic birds are natural reservoir of V. parahaemolyticus strains with multiple genetic background. Conclusion: This study indicated that aquatic birds possessed potentially pathogenic V. parahaemolyticus and may play a role in transmission of this organism across the countries. &nbsp

Prevalence and Molecular Characterization of Rickettsia spp. from Wild Small Mammals in Public Parks and Urban Areas of Bangkok Metropolitan, Thailand

International audienceRural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia

Inflammasome signaling pathways exert antiviral effect against Chikungunya virus in human dermal fibroblasts.

International audienceArboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bite of infected arthropods. Early transmission to vertebrates is initiated by skin puncture and deposition of virus in this organ. However, events at the bite site remain largely unknown. Here, we report that Chikungunya virus (CHIKV) and West Nile virus (WNV), despite belonging to distinct viral families, elicit a common antiviral signature in primary human dermal fibroblasts, attesting for the up regulation of interferon signaling pathways and leading to an increased expression of IFN-β, interleukins and chemokines. Remarkably, CHIKV and WNV enhance IL-1β expression and induce maturation of caspase-1, indicating the capacity of these pathogens to elicit activation of the inflammasome program in resident skin cells. CHIKV and WNV also induce the expression of the inflammasome sensor AIM2 in dermal fibroblasts, whereas inhibition of caspase-1 and AIM2 with siRNA interferes with both CHIKV- and WNV-induced IL-1β production by these cells. Finally, inhibition of the inflammasome via caspase-1 silencing was found to enhance CHIKV replication in dermal fibroblasts. Together, these results indicate that the skin contributes to the pro-inflammatory and anti-viral microenvironment via the activation of the inflammasome in the early stages following infection with arboviruses

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of Burkholderia pseudomallei from Asia and Australia and differentiation between Burkholderia species.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is increasingly used for rapid bacterial identification. Studies of Burkholderia pseudomallei identification have involved small isolate numbers drawn from a restricted geographic region. There is a need to expand the reference database and evaluate B. pseudomallei from a wider geographic distribution that more fully captures the extensive genetic diversity of this species. Here, we describe the evaluation of over 650 isolates. Main spectral profiles (MSP) for 26 isolates of B. pseudomallei (N = 5) and other Burkholderia species (N = 21) were added to the Biotyper database. MALDI-TOF MS was then performed on 581 B. pseudomallei, 19 B. mallei, 6 B. thailandensis and 23 isolates representing a range of other bacterial species. B. pseudomallei originated from northeast and east Thailand (N = 524), Laos (N = 12), Cambodia (N = 14), Hong Kong (N = 4) and Australia (N = 27). All 581 B. pseudomallei were correctly identified, with 100% sensitivity and specificity. Accurate identification required a minimum inoculum of 5 x 107 CFU/ml, and identification could be performed on spiked blood cultures after 24 hours of incubation. Comparison between a dendrogram constructed from MALDI-TOF MS main spectrum profiles and a phylogenetic tree based on recA gene sequencing demonstrated that MALDI-TOF MS distinguished between B. pseudomallei and B. mallei, while the recA tree did not. MALDI-TOF MS is an accurate method for the identification of B. pseudomallei, and discriminates between this and other related Burkholderia species

SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells

Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking

International audienceChikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment