Impact of AbaI mutation on virulence, biofilm development, and antibiotic susceptibility in Acinetobacter baumannii.

The quorum sensing (QS) system mediated by the abaI gene in Acinetobacter baumannii is crucial for various physiological and pathogenic processes. In this study, we constructed a stable markerless abaI knockout mutant (ΔabaI) strain using a pEXKm5-based allele replacement method to investigate the impact of abaI on A. baumannii. Proteomic analysis revealed significant alterations in protein expression between the wild type (WT) and ΔabaI mutant strains, particularly in proteins associated with membrane structure, antibiotic resistance, and virulence. Notably, the downregulation of key outer membrane proteins such as SurA, OmpA, OmpW, and BamA suggests potential vulnerabilities in outer membrane integrity, which correlate with structural abnormalities in the ΔabaI mutant strain, including irregular cell shapes and compromised membrane integrity, observed by scanning and transmission electron microscopy. Furthermore, diminished expression of regulatory proteins such as OmpR and GacA-GacS highlights the broader regulatory networks affected by abaI deletion. Functional assays revealed impaired biofilm formation and surface-associated motility in the mutant strain, indicative of altered colonization capabilities. Interestingly, the mutant showed a complex antibiotic susceptibility profile. While it demonstrated increased susceptibility to membrane-targeting antibiotics, its response to beta-lactams was more nuanced. Despite increased expression of metallo-beta-lactamase (MBL) superfamily proteins and DcaP-like protein, the mutant unexpectedly showed lower MICs for carbapenems (imipenem and meropenem) compared to the wild-type strain. This suggests that abaI deletion affects antibiotic susceptibility through multiple, potentially competing mechanisms. Further investigation is needed to fully elucidate the interplay between quorum sensing, antibiotic resistance genes, and overall antibiotic susceptibility in A. baumannii. Our findings underscore the multifaceted role of the abaI gene in modulating various cellular processes and highlight its significance in A. baumannii physiology, pathogenesis, and antibiotic resistance. Targeting the abaI QS system may offer novel therapeutic strategies for this clinically significant pathogen.

Case Report: Soft tissue infection with Burkholderia thailandensis capsular variant: case report from the Lao PDR.

Background: Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandednisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case: A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods: A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion: This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.

> Burkholderia thailandensis is a bacteria that is found in the environment. Rarely, this bacteria can cause infection in humans. Here we report a B. thailandensis infection in a 44 year old male in Laos. The patient sustained a puncture wound in his left foot and when presenting at a district hospital was prescribed cloxacillin. The wound did not improve and on day three of admission, a pus sample was sent to Mahosot Hospital Microbiology Laboratory for investigation. A preliminary diagnosis of melioidosis, caused by the bacteria Burkholderia pseudomallei, was made and antibiotic treatment was changed. Additional laboratory investigation determined that the isolate was actually B. thailandensis and antibiotic treatment was further changed. Due to the inconclusive results of the initial laboratory tests, the isolate was sent for sequencing and was identified as a strain which expresses a B. pseudomallei-like capsular polysaccharide. This is the first report of infection with B. thailandensis in Laos and the first report of infection with a B. thailandensis capsular variant.

Genetic diversity, determinants, and dissemination of Burkholderia pseudomallei lineages implicated in melioidosis in Northeast Thailand.

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conduct a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identifies three dominant lineages, each with unique gene sets potentially enhancing bacterial fitness in the environment. We find that recombination drives lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage reveal increased expression of lineage-specific genes under environmental conditions in two out of three lineages. This underscores the potential importance of environmental persistence for these dominant lineages. The study also highlights the influence of environmental factors such as terrain slope, altitude, and river direction on the geographical dispersal of B. pseudomallei. Collectively, our findings suggest that environmental persistence may play a role in facilitating the spread of B. pseudomallei, and as a prerequisite for exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.

Genetic diversity, determinants, and dissemination of Burkholderia pseudomallei lineages implicated in melioidosis in northeast Thailand.

Melioidosis is an often-fatal neglected tropical disease caused by an environmental bacterium Burkholderia pseudomallei. However, our understanding of the disease-causing bacterial lineages, their dissemination, and adaptive mechanisms remains limited. To address this, we conducted a comprehensive genomic analysis of 1,391 B. pseudomallei isolates collected from nine hospitals in northeast Thailand between 2015 and 2018, and contemporaneous isolates from neighbouring countries, representing the most densely sampled collection to date. Our study identified three dominant lineages with unique gene sets enhancing bacterial fitness, indicating lineage-specific adaptation strategies. Crucially, recombination was found to drive lineage-specific gene flow. Transcriptome analyses of representative clinical isolates from each dominant lineage revealed heightened expression of lineage-specific genes in environmental versus infection conditions, notably under nutrient depletion, highlighting environmental persistence as a key factor in the success of dominant lineages. The study also revealed the role of environmental factors - slope of terrain, altitude, direction of rivers, and the northeast monsoons - in shaping B. pseudomallei geographical dispersal. Collectively, our findings highlight persistence in the environment as a pivotal element facilitating B. pseudomallei spread, and as a prelude to exposure and infection, thereby providing useful insights for informing melioidosis prevention and control strategies.

Type VI Secretion System Accessory Protein TagAB-5 Promotes Burkholderia pseudomallei Pathogenicity in Human Microglia.

Central nervous system (CNS) melioidosis caused by Burkholderia pseudomallei is being increasingly reported. Because of the high mortality associated with CNS melioidosis, understanding the underlying mechanism of B. pseudomallei pathogenesis in the CNS needs to be intensively investigated to develop better therapeutic strategies against this deadly disease. The type VI secretion system (T6SS) is a multiprotein machine that uses a spring-like mechanism to inject effectors into target cells to benefit the infection process. In this study, the role of the T6SS accessory protein TagAB-5 in B. pseudomallei pathogenicity was examined using the human microglial cell line HCM3, a unique resident immune cell of the CNS acting as a primary mediator of inflammation. We constructed B. pseudomallei tagAB-5 mutant and complementary strains by the markerless allele replacement method. The effects of tagAB-5 deletion on the pathogenicity of B. pseudomallei were studied by bacterial infection assays of HCM3 cells. Compared with the wild type, the tagAB-5 mutant exhibited defective pathogenic abilities in intracellular replication, multinucleated giant cell formation, and induction of cell damage. Additionally, infection by the tagAB-5 mutant elicited a decreased production of interleukin 8 (IL-8) in HCM3, suggesting that efficient pathogenicity of B. pseudomallei is required for IL-8 production in microglia. However, no significant differences in virulence in the Galleria mellonella model were observed between the tagAB-5 mutant and the wild type. Taken together, this study indicated that microglia might be an important intracellular niche for B. pseudomallei, particularly in CNS infection, and TagAB-5 confers B. pseudomallei pathogenicity in these cells.

Optimization and prospective evaluation of sensitive real-time PCR assays with an internal control for the diagnosis of melioidosis in Thailand.

IMPORTANCE: Melioidosis is a serious infectious disease caused by Burkholderia pseudomallei, an environmental Gram-negative bacterium. Early detection of B. pseudomallei infection is crucial for successful antibiotic treatment and reducing mortality rates associated with melioidosis. Bacteria culture is currently used to identify B. pseudomallei in clinical samples, but the method is slow. Therefore, there is a need for more accurate and sensitive molecular-based diagnostic methods that can detect B. pseudomallei in all sample types, including samples from blood. We developed an optimal DNA extraction method for B. pseudomallei from plasma samples and used an internal control for real-time PCR. We evaluated six PCR target genes and identified the most effective target for the early detection of B. pseudomallei infection in patients. To prevent delays in the treatment of melioidosis that can lead to fatal outcomes, we recommend implementing this new approach for routine early detection of B. pseudomallei in clinical settings.

Abundance of ACVR1B transcript is elevated during septic conditions: Perspectives obtained from a hands-on reductionist investigation.

Sepsis is a complex heterogeneous condition, and the current lack of effective risk and outcome predictors hinders the improvement of its management. Using a reductionist approach leveraging publicly available transcriptomic data, we describe a knowledge gap for the role of ACVR1B (activin A receptor type 1B) in sepsis. ACVR1B, a member of the transforming growth factor-beta (TGF-beta) superfamily, was selected based on the following: 1) induction upon in vitro exposure of neutrophils from healthy subjects with the serum of septic patients (GSE49755), and 2) absence or minimal overlap between ACVR1B, sepsis, inflammation, or neutrophil in published literature. Moreover, ACVR1B expression is upregulated in septic melioidosis, a widespread cause of fatal sepsis in the tropics. Key biological concepts extracted from a series of PubMed queries established indirect links between ACVR1B and "cancer", "TGF-beta superfamily", "cell proliferation", "inhibitors of activin", and "apoptosis". We confirmed our observations by measuring ACVR1B transcript abundance in buffy coat samples obtained from healthy individuals (n=3) exposed to septic plasma (n = 26 melioidosis sepsis cases)ex vivo. Based on our re-investigation of publicly available transcriptomic data and newly generated ex vivo data, we provide perspective on the role of ACVR1B during sepsis. Additional experiments for addressing this knowledge gap are discussed.

Entomopathogenic nematodes isolated from agricultural areas of Thailand and their activity against the larvae of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus (Diptera: Culicidae).

Entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis have been considered to be effective biological control agents for several insects. In this study, we isolated and identified EPNs from soil samples in agricultural areas of northern Thailand and evaluated their efficacy for controlling larvae of three mosquito vector species, Aedes aegypti, Ae. albopictus and Culex quinquefasciatus. A total of 51 of 1,000 soil samples were positive (5.1% prevalence) for EPNs, which were identified through sequencing of the rDNA and ITS to 37 Steinernema isolates (3.7%) and 14 Heterorhabditis isolates (1.4%). For the bioassay, the larvae of mosquitoes were exposed to Steinernema surkhetense (eALN6.3_TH), Steinernema lamjungense (eALN11.5_TH), Heterorhabditis indica (eACM14.2_TH) and Heterorhabditis bacteriophora (eALN18.2_TH). Heterorhabditis bacteriophora showed the highest efficacy against Ae. aegypti and Cx. quinquefasciatus. At 96 h after exposure, the mortality rates were 60.0 and 91.7%, respectively. The EPNs were observed in the dead mosquito larvae, which were mostly found in the thorax followed by the head and abdomen. Some EPNs were dead with melanization, and some were able to survive in the cavity of mosquito larvae. Our results show the low prevalence of EPN in agricultural areas of Thailand. Moreover, H. bacteriophora may be considered an alternative biocontrol agent for managing and controlling these vector mosquitoes.

Antibiotic susceptibility of clinical Burkholderia pseudomallei isolates in northeast Thailand during 2015-2018 and the genomic characterization of ß-lactam-resistant isolates.

Melioidosis is an often fatal infection in tropical regions caused by an environmental bacterium, Burkholderia pseudomallei Current recommended melioidosis treatment requires intravenous ß-lactam antibiotics such as ceftazidime (CAZ), meropenem (MEM) or amoxicillin-clavulanic acid (AMC) and oral trimethoprim-sulfamethoxazole. Emerging antibiotic resistance could lead to therapy failure and high mortality. We performed a prospective multicentre study in northeast Thailand during 2015-2018 to evaluate antibiotic susceptibility and characterize ß-lactam resistance in clinical B. pseudomallei isolates. Collection of 1,317 B. pseudomallei isolates from patients with primary and relapse infections were evaluated for susceptibility to CAZ, imipenem (IPM), MEM and AMC. ß-lactam resistant isolates were confirmed by broth microdilution method and characterized by whole genome sequence analysis, penA expression and ß-lactamase activity. The resistant phenotype was verified via penA mutagenesis. All primary isolates were IPM-susceptible but we observed two CAZ-resistant and one CAZ-intermediate resistant isolates, two MEM-less susceptible isolates, one AMC-resistant and two AMC-intermediate resistant isolates. One of 13 relapse isolates was resistant to both CAZ and AMC. Two isolates were MEM-less susceptible. Strains DR10212A (primary) and DR50054E (relapse) were multi-drug resistant. Genomic and mutagenesis analyses supplemented with gene expression and ß-lactamase analyses demonstrated that CAZ-resistant phenotype was caused by PenA variants: P167S (N=2) and penA amplification (N=1). Despite the high mortality rate in melioidosis, our study revealed that B. pseudomallei isolates had a low frequency of ß-lactam resistance caused by penA alterations. Clinical data suggest that resistant variants may emerge in patients during antibiotic therapy and be associated with poor response to treatment.

Entomopathogenic Nematodes and Their Symbiotic Bacteria from the National Parks of Thailand and Larvicidal Property of Symbiotic Bacteria against Aedes aegypti and Culex quinquefasciatus.

Entomopathogenic nematodes (EPNs) are insect parasitic nematodes of the genera Het-erorhabditis and Steinernema. These nematodes are symbiotically associated with the bacteria, Photorhabdus and Xenorhabdus, respectively. National parks in Thailand are a potentially rich resource for recovering native EPNs and their symbiotic bacteria. The objectives of this study are to isolate and identify EPNs and their bacterial flora from soil samples in four national parks in Thailand and to evaluate their efficacy for controlling mosquito larvae. Using a baiting method with a Galleria mellonella moth larvae and a White trap technique, 80 out of 840 soil samples (9.5%) from 168 field sites were positive for EPNs. Sequencing of an internal transcribed spacer resulted in the molecular identification of Heterorhabditis nematode isolates as H. indica, H. baujardi and Heterorhabditis SGmg3, while using 28S rDNA sequencing, Steinernema nematode species were identified as S. guang-dongense, S. surkhetense, S. minutum, S. longicaudum and one closely related to S. yirgalemense. For the symbiotic bacterial isolates, based on recA sequencing, the Photorhabdus spp. were identified as P. luminescens subsp. akhurstii, P. luminescens subsp. hainanensis and P. luminescens subsp. australis. Xenorhabdus isolates were identified as X. stockiae, X. indica, X. griffiniae, X. japonica and X. hominickii. Results of bioassays demonstrate that Photorhabdus isolates were effective on both Aedes aegypti and Culex quinquefasciatus. Therefore, we conclude that soil from Thailand's national parks contain a high diversity of entomopathogenic nematodes and their symbiotic bacteria. Photorhabdus bacteria are larvicidal against culicine mosquitoes and may serve as effective biocontrol agents.

Cycle-Inhibiting Factor Is Associated with Burkholderia pseudomallei Invasion in Human Neuronal Cells.

Burkholderia pseudomallei is a pathogenic bacterium that causes human melioidosis, which is associated with a high mortality rate. However, the underlying mechanisms of B. pseudomallei pathogenesis are largely unknown. In this study, we examined the infection of human neuronal SH-Sy5y cells by several clinically relevant B. pseudomallei strains. We found that all tested B. pseudomallei strains can invade SH-Sy5y cells, undergo intracellular replication, cause actin-tail formation, and form multinucleated giant cells. Additionally, a deletion mutant of B. pseudomallei cycle-inhibiting factor (cif) was constructed that exhibited reduced invasion in SH-Sy5y cells. Complementation of cif restored invasion of the B. pseudomallei cif-deleted mutant. Our findings enhance understanding of B. pseudomallei pathogenicity in terms of the virulence factor Cif and demonstrate the function of Cif in neurological melioidosis. This may eventually lead to the discovery of novel targets for treatment and a strategy to control the disease.

Genome analysis of secondary metabolite­biosynthetic gene clusters of Photorhabdus akhurstii subsp. akhurstii and its antibacterial activity against antibiotic-resistant bacteria.

Xenorhabdus and Photorhabdus can produce a variety of secondary metabolites with broad spectrum bioactivity against microorganisms. We investigated the antibacterial activity of Xenorhabdus and Photorhabdus against 15 antibiotic-resistant bacteria strains. Photorhabdus extracts had strong inhibitory the growth of Methicillin-resistant Staphylococcus aureus (MRSA) by disk diffusion. The P. akhurstii s subsp. akhurstii (bNN168.5_TH) extract showed lower minimum inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC). The interaction between either P. akhurstii subsp. akhurstii (bNN141.3_TH) or P. akhurstii subsp. akhurstii (bNN168.5_TH) or P. hainanensis (bNN163.3_TH) extract in combination with oxacillin determined by checkerboard assay exhibited partially synergistic interaction with fractional inhibitory concentration index (FICI) of 0.53. Time-killing assay for P. akhurstii subsp. akhurstii (bNN168.5_TH) extract against S. aureus strain PB36 significantly decreased cell viability from 105 CFU/ml to 103 CFU/ml within 30 min (P < 0.001, t-test). Transmission electron microscopic investigation elucidated that the bNN168.5_TH extract caused treated S. aureus strain PB36 (MRSA) cell membrane damage. The biosynthetic gene clusters of the bNN168.5_TH contained non-ribosomal peptide synthetase cluster (NRPS), hybrid NRPS-type l polyketide synthase (PKS) and siderophore, which identified potentially interesting bioactive products: xenematide, luminmide, xenortide A-D, luminmycin A, putrebactin/avaroferrin and rhizomide A-C. This study demonstrates that bNN168.5_TH showed antibacterial activity by disrupting bacterial cytoplasmic membrane and the draft genome provided insights into the classes of bioactive products. This also provides a potential approach in developing a novel antibacterial agent.

Larvicidal activity of Photorhabdus and Xenorhabdus bacteria isolated from insect parasitic nematodes against Aedes aegypti and Aedes albopictus.

Aedes aegypti and Aedes albopictus are important vectors for several arboviruses such as the dengue virus. The chemical control of Aedes spp., which is usually implemented, affects both humans and the environment. The biological control of Aedes spp. with entomopathogenic bacteria such as Photorhabdus and Xenorhabdus may be an alternative method that can overcome such issues. This study aimed to isolate and identify Photorhabdus and Xenorhabdus bacteria from entomopathogenic nematodes (EPNs) collected in Thailand and evaluate their larvicidal properties in controlling A. aegypti and A. albopictus. Colony morphology and recA sequencing of the 118 symbiotic isolated bacteria indicated that most were P. luminescens subsp. akhurstii and X. stockiae with minor prevalence of P. luminescens subsp. hainanensis, P. asymbiotica subsp. australis, X. indica, X. griffiniae, X. japonica, X. thuongxuanensis, and X. eapokensis. The larvicidal bioassay with the third- and fourth-instar mosquito larvae suggested that a whole-cell suspension of X. griffiniae (bMSN3.3_TH) had the highest efficiency in eradicating A. aegypti and A. albopictus, with 90 ± 3.71% and 81 ± 2.13% mortality, respectively, after 96 h exposure. In contrast, 1% of ethyl acetate extracted from X. indica (bSNK8.5_TH) showed reduced mortality for A. aegypti of only 50 ± 3.66% after 96 h exposure. The results indicate that both X. griffiniae (bMSN3.3_TH) and X. indica (bSNK8.5_TH) could be used as biocontrol agents against Aedes larvae.

False Positivity of Anti-SARS-CoV-2 Antibodies in Patients with Acute Tropical Diseases in Thailand.

Serology remains a useful indirect method of diagnosing tropical diseases, especially in dengue infection. However, the current literature regarding cross-reactivity between SARS-CoV-2 and dengue serology is limited and revealed conflicting results. As a means to uncover relevant serological insight involving antibody classes against SARS-CoV-2 and cross-reactivity, anti-SARS-CoV-2 IgA, IgM, and IgG ELISA, based on spike and nucleocapsid proteins, were selected for a fever-presenting tropical disease patient investigation. The study was conducted at the Faculty of Tropical Medicine during March to December 2021. The study data source comprised (i) 170 non-COVID-19 sera from 140 adults and children presenting with acute undifferentiated febrile illness and 30 healthy volunteers, and (ii) 31 COVID-19 sera from 17 RT-PCR-confirmed COVID-19 patients. Among 170 non-COVID-19 samples, 27 were false positives (15.9%), of which IgA, IgM, and IgG cross-reactive antibody classes were detected in 18 (10.6%), 9 (5.3%), and 3 (1.8%) cases, respectively. Interestingly, one case exhibited both IgA and IgM false positivity, while two cases exhibited both IgA and IgG false positivity. The false positivity rate in anti-SARS-CoV-2 IgA and IgM was reported in adults with dengue infection (11.3% and 5%) and adults with other tropical diseases (16.7% and 13.3%). The urea dissociation method applied to mitigate false positivity resulted in significantly decreased ELISA-based false and true positives. In conclusion, the analysis of antibody against SARS-CoV-2 in sera of patients with different tropical diseases showed that high IgA and IgM false positivity thus potentially limits serological assay utility in fever-presenting patients in tropical areas.

Molecular identification and phylogeny of Steinernema and Heterorhabditis nematodes and their efficacy in controlling the larvae of Aedes aegypti, a major vector of the dengue virus.

Aedes aegypti is the mosquito vector of several arboviruses, especially the dengue virus. Aedes aegypti strain resistant to chemical insecticides have been reported worldwide. To tackle this, an entomopathogenic nematode (EPN) may be an alternative bio-control agent. To this end, this study aims to isolate, identify, and analyze the phylogeny of EPNs in Thailand and evaluate their efficacy for controlling the Ae. aegypti larvae. From 12 provinces in Thailand, soil samples were randomly collected, with 118 out of 1,100 them being positive for EPNs (10.73% prevalence) in genera Steinernema (4.46%) and Heterorhabditis (6.27%). Then, molecular discrimination of these two genus was performed based on the sequencing and phylogenetic analysis of the 28S rDNA and internal transcribed spacer regions. The most abundant species of EPN were Heterorhabditis indica, with minor species of Heterorhabditis sp. SGmg3, H. baujardi, S. surkhetense, S. kushidai, S. siamkayai, Steinernema sp. YNd80, Steinernema sp. YNc215, S. guangdongense, and S. huense. The larvicidal activity of five selected EPN isolates were tested against Ae. aegypti. Ten larvae of Ae. aegypti were incubated with different concentration (80, 160, 320, and 640 IJs/larva) of the infective juveniles of EPN in a 24-well and 6-well plates for 4 days. The mortality rates of the larvae were observed daily. Steinernema surkhetense (ePYO8.5_TH) showed the potential to kill mosquito larvae, with the highest mortality rate of 92 ± 9.37% and 89 ± 9.91% after it was treated with 640 IJs/larva in a 24-well plate and 1600 IJs/larva in a 6-well plate, respectively. There is an abundant distribution of EPNs across the country, and S. surkhetense ePYO8.5_TH may be used as a biocontrol agent against Ae. aegypti larvae.

Longitudinal analysis to characterize classes and subclasses of antibody responses to recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients in Thailand.

Serological assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might contribute to confirming the suspected coronavirus disease 2019 (COVID-19) in patients not detected with molecular assays. Human antibodies that target the host angiotensin-converting enzyme 2-binding domain of the viral spike protein are a target for serodiagnosis and therapeutics. This study aimed to characterize the classes and subclasses of antibody responses to a recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients and investigated the reactivity of these antibodies in patients with other tropical infections and healthy individuals in Thailand. ELISAs for IgM, IgA, IgG and IgG subclasses based on RBD antigen were developed and tested with time series of 27 serum samples from 15 patients with COVID-19 and 60 samples from pre-COVID-19 outbreaks including acute dengue fever, murine typhus, influenza, leptospirosis and healthy individuals. Both RBD-specific IgA and IgG were detected in only 21% of the COVID-19 patients in the acute phase. The median IgA and IgG levels were significantly higher in the convalescent serum sample compared to the acute serum sample (P < 0.05). We observed the highest correlation between levels of IgG and IgA (rho = 0. 92). IgG1 and IgG3 were the major IgG subclasses detected in SARS-CoV-2 infection. Only acute IgG3 level was negatively associated with viral detection based on RT-PCR of ORF1ab gene (rho = -0.57). The median IgA and IgG levels in convalescence sera of COVID-19 patients were significantly higher than healthy individuals and convalescent sera of other febrile infectious patients. The analyses of antibody classes and subclasses provide insights into human immune responses against SARS-CoV-2 during natural infection and interpretation of antibody assays.

Genomic loss in environmental and isogenic morphotype isolates of Burkholderia pseudomallei is associated with intracellular survival and plaque-forming efficiency.

BACKGROUND: Burkholderia pseudomallei is an environmental bacterium that causes melioidosis. A facultative intracellular pathogen, B. pseudomallei can induce multinucleated giant cells (MNGCs) leading to plaque formation in vitro. B. pseudomallei can switch colony morphotypes under stress conditions. In addition, different isolates have been reported to have varying virulence in vivo, but genomic evolution and the relationship with plaque formation is poorly understood. METHODOLOGY/PRINCIPLE FINDINGS: To gain insights into genetic underpinnings of virulence of B. pseudomallei, we screened plaque formation of 52 clinical isolates and 11 environmental isolates as well as 4 isogenic morphotype isolates of B. pseudomallei strains K96243 (types II and III) and 153 (types II and III) from Thailand in A549 and HeLa cells. All isolates except one environmental strain (A4) and K96243 morphotype II were able to induce plaque formation in both cell lines. Intracellular growth assay and confocal microscopy analyses demonstrated that the two plaque-forming-defective isolates were also impaired in intracellular replication, actin polymerization and MNGC formation in infected cells. Whole genome sequencing analysis and PCR revealed that both isolates had a large genomic loss on the same region in chromosome 2, which included Bim cluster, T3SS-3 and T6SS-5 genes. CONCLUSIONS/SIGNIFICANCE: Our plaque screening and genomic studies revealed evidence of impairment in plaque formation in environmental isolates of B. pseudomallei that is associated with large genomic loss of genes important for intracellular multiplication and MNGC formation. These findings suggest that the genomic and phenotypic differences of environmental isolates may be associated with clinical infection.

Lactoferrin is a dynamic protein in human melioidosis and is a TLR4-dependent driver of TNF-α release in Burkholderia thailandensis infection in vitro.

Melioidosis is an often-severe tropical infection caused by Burkholderia pseudomallei (Bp) with high associated morbidity and mortality. Burkholderia thailandensis (Bt) is a closely related surrogate that does not require BSL-3 conditions for study. Lactoferrin is an iron-binding glycoprotein that can modulate the innate inflammatory response. Here we investigated the impact of lactoferrin on the host immune response in melioidosis. Lactoferrin concentrations were measured in plasma from patients with melioidosis and following ex vivo stimulation of blood from healthy individuals. Bt growth was quantified in liquid media in the presence of purified and recombinant human lactoferrin. Differentiated THP-1 cells and human blood monocytes were infected with Bt in the presence of purified and recombinant human lactoferrin, and bacterial intracellular replication and cytokine responses (tumor necrosis factor-α (TNF-α), interleukin-1ß and interferon-γ) were measured. In a cohort of 49 melioidosis patients, non-survivors to 28 days had significantly higher plasma lactoferrin concentrations compared to survivors (median (interquartile range (IQR)): 326 ng/ml (230-748) vs 144 ng/ml (99-277), p<0.001). In blood stimulated with heat-killed Bp, plasma lactoferrin concentration significantly increased compared to unstimulated blood (median (IQR): 424 ng/ml (349-479) vs 130 ng/ml (91-214), respectively; p<0.001). Neither purified nor recombinant human lactoferrin impaired growth of Bt in media. Lactoferrin significantly increased TNF-α production by differentiated THP-1 cells and blood monocytes after Bt infection. This phenotype was largely abrogated when Toll-like receptor 4 (TLR4) was blocked with a monoclonal antibody. In sum, lactoferrin is produced by blood cells after exposure to Bp and lactoferrin concentrations are higher in 28-day survivors in melioidosis. Lactoferrin induces proinflammatory cytokine production after Bt infection that may be TLR4 dependent.

Advances on Vibrio parahaemolyticus research in the postgenomic era.

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.

Predictive Validity of the qSOFA Score for Sepsis in Adults with Community-Onset Staphylococcal Infection in Thailand.

The quick sequential organ failure assessment (qSOFA) score has had limited validation in lower resource settings and was developed using data from high-income countries. We sought to evaluate the predictive validity of the qSOFA score for sepsis within a low- and middle-income country (LMIC) population with culture-proven staphylococcal infection. This was a secondary analysis of a prospective multicenter cohort in Thailand with culture-positive infection due to Staphylococcus aureus or S. argenteus within 24 h of admission and positive (≥2/4) systemic inflammatory response syndrome (SIRS) criteria. Primary exposure was maximum qSOFA score within 48 h of culture collection and primary outcome was mortality at 28 days. Baseline risk of mortality was determined using a multivariable logistic regression model with age, gender, and co-morbidities significantly associated with the outcome. Predictive validity was assessed by discrimination of mortality using area under the receiver operating characteristic (AUROC) curve compared to a model using baseline risk factors alone. Of 253 patients (mean age 54 years (SD 16)) included in the analysis, 23 (9.1%) died by 28 days after enrollment. Of those who died, 0 (0%) had a qSOFA score of 0, 8 (35%) had a score of 1, and 15 (65%) had a score ≥2. The AUROC of qSOFA plus baseline risk was significantly greater than for the baseline risk model alone (AUROCqSOFA = 0.80 (95% CI, 0.70-0.89), AUROCbaseline = 0.62 (95% CI, 0.49-0.75); p < 0.001). Among adults admitted to four Thai hospitals with community-onset coagulase-positive staphylococcal infection and SIRS, the qSOFA score had good predictive validity for sepsis.