Search

Background: Due to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication., Methods: A novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area., Results: BiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area., Conclusions: Due to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis., (© 2024. The Author(s).)

Chronic rhinosinusitis (CRS) is long-term inflammation of the sinuses that can be caused by infection due to antibiotic-resistant bacteria. Biofilm developed by microbes is postulated to cause antibiotic treatment failure. Thus, the anti-biofilm activities of seven Thai herbal essential oils (EOs) against antibiotic-resistant bacteria isolated from CRS patients was investigated. Lemongrass ( Cymbopogon citratus L.) EO showed the most effective antibiofilm activity against Klebsiella pneumoniae , Pseudomonas aeruginosa and Staphylococcus epidermidis grown as biofilm. GC-MS analysis found that myrcene was the major bioactive compound. Pretreatment with lemongrass EO significantly inhibited biofilm formation of all bacterial strains in more than 50% of cases. Furthermore, confocal microscopy analysis revealed the biofilm-disrupting activity of lemongrass EO against the biofilm matrix of all these bacterial species and also increased P. aeruginosa swarming motility with no toxicity to human cells. These results suggest that lemongrass EO has promising clinical applications as an anti-biofilm agent for CRS patients.

Several vaccine programs were introduced during the COVID-19 pandemic, which included inactivated virus, DNA viral vectors and mRNA vaccines. Booster programs are recommended, especially for those in high-risk groups. However, many of these booster programs involve heterologous vaccines. This study enrolled volunteers who first received two full-dose CoronaVac vaccinations before receiving heterologous boosters with DNA- and/or mRNA-vaccines for an additional 2 doses (n = 40) or an additional 3 doses (n = 16). Our results showed no difference in side effects, neutralizing antibodies, or T-cell responses for any of the heterologous vaccination programs. However, the neutralizing capacity and IFN-γ responses against the Omicron variant in volunteers who received 4 or 5 doses were improved. Polarization of peripheral memory T cells after stimulation in all booster groups with Omicron peptide showed an increased trend of naïve and central memory phenotypes of both CD4+ and CD8+ T cells, suggesting that exposure to Omicron antigens will drive T cells into a lymphoid resident T cell phenotype. Our data support a continuous vaccination program to maximize the effectiveness of immunity, especially in people at high risk. Furthermore, the number of boosting doses is important for maintaining immunity., (© 2023. The Author(s).)

Melioidosis is an infectious disease with high mortality rates in human, caused by the bacterium Burkholderia pseudomallei. As an intracellular pathogen, B. pseudomallei can escape from the phagosome and induce multinucleated giant cells (MNGCs) formation resulting in antibiotic resistance and immune evasion. A novel strategy to modulate host response against B. pseudomallei pathogenesis is required. In this study, an active metabolite of vitamin D3 (1α,25-dihydroxyvitamin D3 or 1α,25(OH)2D3) was selected to interrupt pathogenesis of B. pseudomallei in a human lung epithelium cell line, A549. The results demonstrated that pretreatment with 10-6 M 1α,25(OH)2D3 could reduce B. pseudomallei internalization to A549 cells at 4 h post infection (P < 0.05). Interestingly, the presence of 1α,25(OH)2D3 gradually reduced MNGC formation at 8, 10 and 12 h compared to that of the untreated cells (P < 0.05). Furthermore, pretreatment with 10-6 M 1α,25(OH)2D3 considerably increased hCAP-18/LL-37 mRNA expression (P < 0.001). Additionally, pro-inflammatory cytokines, including MIF, PAI-1, IL-18, CXCL1, CXCL12 and IL-8, were statistically decreased (P < 0.05) in 10-6 M 1α,25(OH)2D3-pretreated A549 cells by 12 h post-infection. Taken together, this study indicates that pretreatment with 10-6 M 1α,25(OH)2D3 has the potential to reduce the internalization of B. pseudomallei into host cells, decrease MNGC formation and modulate host response during B. pseudomallei infection by minimizing the excessive inflammatory response. Therefore, 1α,25(OH)2D3 supplement may provide an effective supportive treatment for melioidosis patients to combat B. pseudomallei infection and reduce inflammation in these patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mattrasongkram et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The pandemic coronavirus disease 2019 (COVID-19) is a major global public health concern and several protective vaccines, or preventive/therapeutic approaches have been developed. Sinovac-CoronaVac, an inactivated whole virus vaccine, can protect against severe COVID-19 disease and hospitalization, but less is known whether it elicits long-term T cell responses and provides prolonged protection., Methods: This is a longitudinal surveillance study of SARS-CoV-2 receptor binding domain (RBD)-specific IgG levels, neutralizing antibody levels (NAb), T cell subsets and activation, and memory B cells of 335 participants who received two doses of CoronaVac. SARS-CoV-2 RBD-specific IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), while NAb were measured against two strains of SARS-CoV-2, the Wuhan and Delta variants. Activated T cells and subsets were identified by flow cytometry. Memory B and T cells were evaluated by enzyme-linked immune absorbent spot (ELISpot)., Findings: Two doses of CoronaVac elicited serum anti-RBD antibody response, elevated B cells with NAb capacity and CD4 + T cell-, but not CD8 + T cell-responses. Among the CD4 + T cells, CoronaVac activated mainly Th2 (CD4 + T) cells. Serum antibody levels significantly declined three months after the second dose., Interpretation: CoronaVac mainly activated B cells but T cells, especially Th1 cells, were poorly activated. Activated T cells were mainly Th2 biased, demonstrating development of effector B cells but not long-lasting memory plasma cells. Taken together, these results suggest that protection with CoronaVac is short-lived and that a third booster dose of vaccine may improve protection., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.

Staphylococcus aureus is deemed to be one of the major causes of hospital and community-acquired infections, especially in methicillin-resistant S. aureus (MRSA) strains. Because antimicrobial peptides have captured attention as novel drug candidates due to their rapid and broad-spectrum antimicrobial activity, anti-MRSA peptides have emerged as potential therapeutics for the treatment of bacterial infections. Although experimental approaches can precisely identify anti-MRSA peptides, they are usually cost-ineffective and labor-intensive. Therefore, computational approaches that are able to identify and characterize anti-MRSA peptides by using sequence information are highly desirable. In this study, we present the first computational approach (termed SCMRSA) for identifying and characterizing anti-MRSA peptides by using sequence information without the use of 3D structural information. In SCMRSA, we employed an interpretable scoring card method (SCM) coupled with the estimated propensity scores of 400 dipeptides. Comparative experiments indicated that SCMRSA was more effective and could outperform several machine learning-based classifiers with an accuracy of 0.960 and Matthews correlation coefficient of 0.848 on the independent test data set. In addition, we employed the SCMRSA-derived propensity scores to provide a more in-depth explanation regarding the functional mechanisms of anti-MRSA peptides. Finally, in order to serve community-wide use of the proposed SCMRSA, we established a user-friendly webserver which can be accessed online at http://pmlabstack.pythonanywhere.com/SCMRSA. SCMRSA is anticipated to be an open-source and useful tool for screening and identifying novel anti-MRSA peptides for follow-up experimental studies., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Phage virion proteins (PVPs) are effective at recognizing and binding to host cell receptors while having no deleterious effects on human or animal cells. Understanding their functional mechanisms is regarded as a critical goal that will aid in rational antibacterial drug discovery and development. Although high-throughput experimental methods for identifying PVPs are considered the gold standard for exploring crucial PVP features, these procedures are frequently time-consuming and labor-intensive. Thusfar, more than ten sequence-based predictors have been established for the in silico identification of PVPs in conjunction with traditional experimental approaches. As a result, a revised and more thorough assessment is extremely desirable. With this purpose in mind, we first conduct a thorough survey and evaluation of a vast array of 13 state-of-the-art PVP predictors. Among these PVP predictors, they can be classified into three groups according to the types of machine learning (ML) algorithms employed (i.e. traditional ML-based methods, ensemble-based methods and deep learning-based methods). Subsequently, we explored which factors are important for building more accurate and stable predictors and this included training/independent datasets, feature encoding algorithms, feature selection methods, core algorithms, performance evaluation metrics/strategies and web servers. Finally, we provide insights and future perspectives for the design and development of new and more effective computational approaches for the detection and characterization of PVPs., (Copyright © 2022 Kabir et al.)

Mycobacterium abscessus (Mab) is one of the most drug resistant bacteria with a high treatment failure rate. Antimicrobial peptides (AMPs) are alternative therapeutic agents against this infection. This study was aimed to assess the in vitro activities of thirteen AMPs (S5, S52, S6, S61, S62, S63, KLK, KLK1, KLK2, Pug-1, Pug-2, Pug-3 and Pug-4) that have never been investigated against drug resistant Mab isolates. Only four novel modified AMPs (S61, S62, S63 and KLK1) provided the lowest minimum inhibitory concentration (MIC) values ranging from 200-400 μg/ml against the Mab ATCC19977 strain. These four potential AMPs were further tested with 16 clinical isolates of clarithromycin resistant Mab. The majority of the tested strains (10/16 isolates, 62.5%) showed ~99% kill by all four AMPs within 24 hours with an MIC <50 μg/ml. Only two isolates (12.5%) with acquired clarithromycin resistance, however, exhibited values <50 μg/ml of four potential AMPs, S61, S62, S63 and KLK1 after 3-days-incubation. At the MICs level, S63 showed the lowest toxicity with 1.50% hemolysis and 100% PBMC viability whereas KLK1 showed the highest hemolysis (10.21%) and lowest PBMC viability (93.52%). S61, S62 and S63 were further tested with clarithromycin-AMP interaction assays and found that 5/10 (50%) of selected isolates exhibited a synergistic interaction with 0.02-0.41 FICI values. This present study demonstrated the potential application of novel AMPs as an adjunctive treatment with clarithromycin against drug resistant Mab infection., Competing Interests: The authors have declared that no competing interests exist.

A large-scale surveillance is an important measure to monitor the regional spread of antimicrobial resistance. We prospectively studied the prevalence and molecular characteristics of clinically important Gram-negative bacilli, including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii complex (ABC), and Pseudomonas aeruginosa, from blood, respiratory tract, urine, and sterile sites at 47 hospitals across Thailand. Among 187,619 isolates, 93,810 isolates (50.0%) were critically drug resistant, of which 12,915 isolates (13.8%) were randomly selected for molecular characterization. E. coli was most commonly isolated from all specimens, except the respiratory tract, in which ABC was predominant. Prevalence of extended-spectrum cephalosporin resistance (ESCR) was higher in E. coli (42.5%) than K. pneumoniae (32.0%), but carbapenem-resistant (CR)-K. pneumoniae (17.2%) was 4.5-fold higher than CR- E. coli (3.8%). The majority of ESCR/CR- E. coli and K. pneumoniae isolates carried bla CTX-M (64.6% to 82.1%). bla NDM and bla OXA-48-like were the most prevalent carbapenemase genes in CR- E. coli /CR-K. pneumoniae (74.9%/52.9% and 22.4%/54.1%, respectively). In addition, 12.9%/23.0% of CR- E. coli /CR-K. pneumoniae cocarried bla NDM and bla OXA-48-like. Among ABC isolates, 41.9% were extensively drug resistant (XDR) and 35.7% were multidrug resistant (MDR), while P. aeruginosa showed XDR/MDR at 6.3%/16.5%. A. baumannii was the most common species among ABC isolates. The major carbapenemase gene in MDR-A. baumannii/XDR-A. baumannii was bla OXA-23-like (85.8%/93.0%), which had much higher rates than other ABC species. bla IMP , bla VIM , bla OXA-40-like , and bla OXA-58-like were also detected in ABC at lower rates. The most common carbapenemase gene in MDR/XDR-P. aeruginosa was bla IMP (29.0%/30.6%), followed by bla VIM (9.5%/25.3%). The findings reiterate an alarming situation of drug resistance that requires serious control measures.

Despite the synergistic effect of Opisthorchis viverrini and Helicobacter pylori co-infection on pathogenesis of severe hepatobiliary abnormalities (HBA) including advanced periductal fibrosis and replace with cholangiocarcinoma (CCA) have been established, the immune response to H. pylori in O. viverrini infected population has never been explored. Hence, this study aimed to investigate the antibody responses to 2 immunogenic H. pylori proteins in O. viverrini-infected patients with HBA and CCA. The risk analysis by multinomial logistic regression revealed that GroEL seropositivity was associated with higher risks of hepatobiliary abnormalities and CCA with adjusted odds ratios (95% confidence intervals) of 2.11 (95% CI=1.20-3.71, P=0.008) and 2.13 (95% CI=1.21-3.75, P=0.009), respectively. These findings indicate that GroEL seropositivity might be a biomarker for early detection of O. viverrini associated HBA and CCA.

Fast, accurate identification and characterization of amyloid proteins at a large-scale is essential for understating their role in therapeutic intervention strategies. As a matter of fact, there exist only one in silico model for amyloid protein identification using the random forest (RF) model in conjunction with various feature types namely the RFAmy. However, it suffers from low interpretability for biologists. Thus, it is highly desirable to develop a simple and easily interpretable prediction method with robust accuracy as compared to the existing complicated model. In this study, we propose iAMY-SCM, the first scoring card method-based predictor for predicting and analyzing amyloid proteins. Herein, the iAMY-SCM made use of a simple weighted-sum function in conjunction with the propensity scores of dipeptides for the amyloid protein identification. Cross-validation results indicated that iAMY-SCM provided an accuracy of 0.895 that corresponded to 10-22% higher performance than that of widely used machine learning models. Furthermore, iAMY-SCM achieving an accuracy of 0.827 as evaluated by an independent test, which was found to be comparable to that of RFAmy and was approximately 9-13% higher than widely used machine learning models. Furthermore, the analysis of estimated propensity scores of amino acids and dipeptides were performed to provide insights into the biophysical and biochemical properties of amyloid proteins. As such, this demonstrates that the proposed iAMY-SCM is efficient and reliable in terms of simplicity, interpretability and implementation. To facilitate ease of use of the proposed iAMY-SCM, a user-friendly and publicly accessible web server at http://camt.pythonanywhere.com/iAMY-SCM has been established. We anticipate that that iAMY-SCM will be an important tool for facilitating the large-scale prediction and characterization of amyloid protein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chronic rhinosinusitis (CRS) is a chronic disease that involves long-term inflammation of the nasal cavity and paranasal sinuses. Bacterial biofilms present on the sinus mucosa of certain patients reportedly exhibit resistance against traditional antibiotics, as evidenced by relapse, resulting in severe disease. The aim of this study was to determine the killing activity of human cathelicidin antimicrobial peptides (LL-37, LL-31) and their D-enantiomers (D-LL-37, D-LL-31), alone and in combination with conventional antibiotics (amoxicillin; AMX and tobramycin; TOB), against bacteria grown as biofilm, and to investigate the biological activities of the peptides on human lung epithelial cells. D-LL-31 was the most effective peptide against bacteria under biofilm-stimulating conditions based on IC50 values. The synergistic effect of D-LL-31 with AMX and TOB decreased the IC50 values of antibiotics by 16-fold and could eliminate the biofilm matrix in all tested bacterial strains. D-LL-31 did not cause cytotoxic effects in A549 cells at 25 μM after 24 h of incubation. Moreover, a cytokine array indicated that there was no significant induction of the cytokines involving in immunopathogenesis of CRS in the presence of D-LL-31. However, a tissue-remodeling-associated protein was observed that may prevent the progression of nasal polyposis in CRS patients. Therefore, a combination of D-LL-31 with AMX or TOB may improve the efficacy of currently used antibiotics to kill biofilm-embedded bacteria and eliminate the biofilm matrix. This combination might be clinically applicable for treatment of patients with biofilm-associated CRS., Competing Interests: The authors have declared that no competing interests exist.

The inhibition of dipeptidyl peptidase IV (DPP-IV, E.C.3.4.14.5) is well recognized as a new avenue for the treatment of Type 2 diabetes (T2D). Until now, peptide-like DDP-IV inhibitors have been shown to normalize the blood glucose concentration in T2D subjects. To the best of our knowledge, there is yet no computational model for predicting and analyzing DPP-IV inhibitory peptides using sequence information. In this study, we present for the first time a simple and easily interpretable sequence-based predictor using the scoring card method (SCM) for modeling the bioactivity of DPP-IV inhibitory peptides (iDPPIV-SCM). Particularly, the iDPPIV-SCM was developed by employing the SCM method together with the propensity scores of amino acids. Rigorous independent test results demonstrated that the proposed iDPPIV-SCM was found to be superior to those of well-known machine learning (ML) classifiers (e.g., k-nearest neighbor, logistic regression, and decision tree) with demonstrated improvements of 2-11, 4-22, and 7-10% for accuracy, MCC, and AUC, respectively, while also achieving comparable results to that of the support vector machine. Furthermore, the analysis of estimated propensity scores of amino acids as derived from the iDPPIV-SCM was performed so as to provide a more in-depth understanding on the molecular basis for enhancing the DPP-IV inhibitory potency. Taken together, these results revealed that iDPPIV-SCM was superior to those of other well-known ML classifiers owing to its simplicity, interpretability, and validity. For the convenience of biologists, the predictive model is deployed as a publicly accessible web server at http://camt.pythonanywhere.com/iDPPIV-SCM. It is anticipated that iDPPIV-SCM can serve as an important tool for the rapid screening of promising DPP-IV inhibitory peptides prior to their synthesis.

Although, existing methods have been successful in predicting phage (or bacteriophage) virion proteins (PVPs) using various types of protein features and complex classifiers, such as support vector machine and naïve Bayes, these two methods do not allow interpretability. However, the characterization and analysis of PVPs might be of great significance to understanding the molecular mechanisms of bacteriophage genetics and the development of antibacterial drugs. Hence, we herein proposed a novel method (PVPred-SCM) based on the scoring card method (SCM) in conjunction with dipeptide composition to identify and characterize PVPs. In PVPred-SCM, the propensity scores of 400 dipeptides were calculated using the statistical discrimination approach. Rigorous independent validation test showed that PVPred-SCM utilizing only dipeptide composition yielded an accuracy of 77.56%, indicating that PVPred-SCM performed well relative to the state-of-the-art method utilizing a number of protein features. Furthermore, the propensity scores of dipeptides were used to provide insights into the biochemical and biophysical properties of PVPs. Upon comparison, it was found that PVPred-SCM was superior to the existing methods considering its simplicity, interpretability, and implementation. Finally, in an effort to facilitate high-throughput prediction of PVPs, we provided a user-friendly web-server for identifying the likelihood of whether or not these sequences are PVPs. It is anticipated that PVPred-SCM will become a useful tool or at least a complementary existing method for predicting and analyzing PVPs., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Melioidosis is a severe disease caused by Burkholderia pseudomallei . The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC 50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC 50 values of the peptide and antibiotic and caused also disruption of biofilms of B . pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

The biofilm-forming ability of Burkholderia pseudomallei is crucial for its survival in unsuitable environments and is correlated with antibiotic resistance and relapsing cases of melioidosis. Extracellular DNA (eDNA) is an essential component for biofilm development and maturation in many bacteria. The aim of this study was to investigate the eDNA released by B. pseudomallei during biofilm formation using DNase treatment. The extent of biofilm formation and quantity of eDNA were assessed by crystal-violet staining and fluorescent dye-based quantification, respectively, and visualized by confocal laser scanning microscopy (CLSM). Variation in B. pseudomallei biofilm formation and eDNA quantity was demonstrated among isolates. CLSM images of biofilms stained with FITC-ConA (biofilm) and TOTO-3 (eDNA) revealed the localization of eDNA in the biofilm matrix. A positive correlation of biofilm biomass with quantity of eDNA during the 2-day biofilm-formation observation period was found. The increasing eDNA quantity over time, despite constant living/dead ratios of bacterial cells during the experiment suggests that eDNA is delivered from living bacterial cells. CLSM images demonstrated that depletion of eDNA by DNase I significantly lessened bacterial attachment (if DNase added at 0 h) and biofilm developing stages (if added at 24 h) but had no effect on mature biofilm (if added at 45 h). Collectively, our results reveal that eDNA is released from living B. pseudomallei and is correlated with biofilm formation. It was also apparent that eDNA is essential during bacterial cell attachment and biofilm-forming steps. The depletion of eDNA by DNase may provide an option for the prevention or dispersal of B. pseudomallei biofilm., Competing Interests: The authors have declared that no competing interests exist.

Cholangiocarcinoma is a malignant tumor with high metastatic and mortality rates. We investigated the effects of rhinacanthin-C on cell proliferation, migration, invasion and the expression of proteins regulating cancer cell invasion-regulated proteins in a cholangiocarcinoma (KKU-M156) cell line. Cytotoxicity of rhinacanthin-C was determined by the SRB assay. Using wound-migration, chamber-migration and chamber-invasion assays, we assessed the effects of rhinacanthin-C against KKU-M156 cells. The activities of matrix metalloproteinases 2 and 9 (MMP-2, MMP-9) and urokinase-type plasminogen activator (uPA) were determined using gelatinase and uPA zymography assays. The expression of invasion-regulated proteins was investigated using western-blot analysis. After treatment with rhinacanthin-C, KKU-M156 cells exhibited antiproliferative effects in a dose-dependent manner with greater efficacy than in Vero cells: IC50 values were 1.50 and 2.37 μM, respectively. Rhinacanthin-C significantly inhibited cell migration and invasion of KKU-M156 cells in a dose-dependent manner. Consistent with this observation, treatment with rhinacanthin-C was associated with a decrease in the expression levels of FAK, p-FAK, MMP-2, and a decrease in the levels of p38-, JNK1/2- and ERK1/2-MAPK pathways as well as inhibiting NF-κB/p65 expression and translocation of NF-κB/p65 to the nucleus. We have shown for the first time that the anti-metastatic effects of rhinacanthin-C on KKU-M156 cells are mediated via inhibition of the expression of invasion-regulated proteins. Rhinacanthin-C may deserve consideration as a potential agent for the treatment of cholangiocarcinoma., (Creative Commons Attribution License)

Burkholderia pseudomallei is the causative agent of melioidosis and regarded as a bioterrorism threat. It can adapt to the nutrient-limited environment as the bacteria can survive in triple distilled water for 16 years. Moreover, B. pseudomallei exhibits intrinsic resistance to diverse groups of antibiotics in particular while growing in biofilms. Recently, nutrient-limited condition influenced both biofilm formation and ceftazidime (CAZ) tolerance of B. pseudomallei were found. However, there is no information about how nutrient-limitation together with antibiotics used in melioidosis treatment affects the structure of the biofilm produced by B. pseudomallei. Moreover, no comparative study to investigate the biofilm architectures of B. pseudomallei and the related B. thailandensis under different nutrient concentrations has been reported. Therefore, this study aims to provide new information on the effects of four antibiotics used in melioidosis treatment, viz. ceftazidime (CAZ), imipenem (IMI), meropenem (MEM) and doxycycline (DOX) on biofilm architecture of B. pseudomallei and B. thailandensis with different nutrient concentrations under static and flow conditions using confocal laser scanning microscopy. Impact of nutritional stress on drug susceptibility of B. pseudomallei and B. thailandensis grown planktonically or as biofilm was also evaluated. The findings of this study indicate that nutrient-limited environment enhanced survival of B. pseudomallei in biofilm after exposure to the tested antibiotics. The shedding planktonic B. pseudomallei and B. thailandensis were also found to have increased CAZ tolerance in nutrient-limited environment. However, killing activities of MEM and IMI were stronger than CAZ and DOX on B. pseudomallei and B. thailandensis both in planktonic cells and in 2-day old biofilm. In addition, MEM and IMI were able to inhibit B. pseudomallei and B. thailandensis biofilm formation to a larger extend compared to CAZ and DOX. Differences in biofilm architecture were observed for biofilms grown under static and flow conditions. Under static conditions, biofilms grown in full strength modified Vogel and Bonner's medium (MVBM) showed honeycomb-like architecture while a knitted-like structure was observed under limited nutrient condition (0.1×MVBM). Under flow conditions, biofilms grown in MVBM showed a multilayer structure while merely dispersed bacteria were found when grown in 0.1×MVBM. Altogether, this study provides more insight on the effect of four antibiotics against B. pseudomallei and B. thailandensis in biofilm under different nutrient and flow conditions. Since biofilm formation is believed to be involved in disease relapse, MEM and IMI may be better therapeutic options than CAZ for melioidosis treatment.

The aims of the study were to develop nested-PCR (targeting vacA and cagA), SYBR green quantitative PCR (targeting 16S rDNA) tests and compared them with indirect fluorescent-monoclonal antibody (IFA) method for determination of the prevalence of Helicobacter pylori in 118 saliva samples from asymptomatic individuals in Khon Kaen, Thailand. Detection limit of both PCR-based assays was one cell. Prevalence of H. pylori in saliva samples was 55% based on the criterion of positivity of IFA test and one of the PCR-based methods or positivity of both PCR assays. Forty-nine percent of H. pylori detected carried cagA, encoding a cytotoxin associated with severe clinical outcomes. These results imply that the mouth may be an important reservoir for H. pylori, with nearly 50% of the virulent type that could possibly lead to gastroduodenal disease.

Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis. Inhalational exposure leading to pulmonary melioidosis is the most common clinical manifestation with significant mortality. However, the role of B. pseudomallei biofilm phenotype during bacterial-host interaction remains unclear. We hypothesize that biofilm phenotype may play a role in such interactions. In this study, B. pseudomallei H777 (biofilm wild type), B. pseudomallei M10 (biofilm mutant) and B. pseudomallei C17 (biofilm-complemented) strains were used to assess the contribution of biofilm to adhesion to human lung epithelial cells (A549), intracellular interactions, apoptosis/necrosis and impact on proinflammatory responses. Confocal laser scanning microscopy demonstrated that B. pseudomallei H777 and C17 produced biofilm, whereas M10 did not. To determine the role of biofilm in host interaction, we assessed the ability of each of the three strains to interact with the A549 cells at MOI 10. Strain H777 exhibited higher levels of attachment and invasion compared to strain M10 (p < 0.05). In addition, the biofilm-complemented strain, C17 exhibited restored bacterial invasion ability. Flow cytometry combined with a double-staining assay using annexin V and propidium iodide revealed significantly higher numbers of early apoptotic and late apoptotic A549 cells when these were infected with strain H777 (1.52%) and C17 (1.43%) compared to strain M10 (0.85%) (p < 0.05). Strains H777 and C17 were able to stimulate significant secretion of IL-6 and IL-8 compared with the biofilm mutant (p < 0.05). Together, these findings demonstrated the role of biofilm-associated phenotypes of B. pseudomallei in cellular pathogenesis of human lung epithelial cells with respect to initial attachment and invasion, apoptosis and proinflammatory responses.

Lactoferrin chimera (LFchimera), a hybrid peptide containing the two antimicrobial stretches of the innate immunity factor bovine lactoferrin, viz. LFampin265-284 and LFcin17-30, has strikingly high antimicrobial activity against the category B pathogen Burkholderia pseudomallei. The action mechanisms of LFchimera against B. pseudomallei is not fully understood. The aim of this study was to further investigate the effect of treated B. pseudomallei with LFchimera using (immune) electron microscopy. The effects of LFchimera on biofilm formation and against preformed biofilm of B. pseudomallei were also determined. After exposure to LFchimera, transmission electron microscopy revealed swelling of the periplasmic space of B. pseudomallei and a highly inhomogeneous electron density in the intracellular DNA region. Localization of LFchimera in B. pseudomallei using immunoelectron microscopy showed gold particles in intracellular structures without accumulation on the membranes. LFchimera also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Moreover, limited exposure of B. pseudomallei to LFchimera at subcidal concentration could reduce biofilm formation. Altogether, the results indicate that LFchimera possesses antibacterial and antibiofilm activities and can modulate B. pseudomallei colonization. Therefore, the efficacy of LFchimera merits further development of this agent for the therapy of melioidosis.

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous studies, isomorellin and forbesione, caged xanthones isolated from Garcinia hanburyi, were found to induce cell cycle arrest and apoptosis in CCA cell lines. The subject of our inquiry is the synergistic effect(s) of these caged xanthones with doxorubicin on growth inhibition and apoptosis induction in human CCA cell lines., Methods: KKU-100, KKU-M139 and KKU-M156 cell lines and Chang cells were treated with either isomorellin or forbesione alone or in combination with doxorubicin. Cell viability was determined using the sulforhodamine B assay. The combined effects of plant compounds with doxorubicin were analyzed using the isobologram and combination index method of Chou-Talalay. Apoptosis was determined by ethidium bromide/acridine orange staining. Protein expressions were determined by Western blot analysis., Results: Isomorellin or forbesione alone inhibited the growth of these CCA cell lines in a dose-dependent manner and showed selective cytotoxicity against CCA cells but not against Chang cells. Isomorellin/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-M139 and KKU-M156 cells, while the forbesione/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-100 and KKU-M139 cells. The percentages of apoptotic cells were significantly higher in the combined treatments than in the respective single drug treatments. The combined treatments strongly enhanced the expression of Bax/Bcl-2, activated caspase-9 and caspase-3, while suppressing the expression of survivin, procaspase-9 and procaspase-3, compared with single drug treatments. The degree of suppression of NF-κB activation mediated by a decrease in the expression of NF-κB/p65, a reduction of the pIκB-α level and an increase in the IκB-α protein level, was significantly higher in the combined treatment groups than in the single drug treatment groups. The degree of suppression of MRP1 protein expression was also significantly higher in the combined treatment than in the single drug treatment groups., Conclusion: The combinations of isomorellin/doxorubicin and forbesione/doxorubicin showed significant synergistic effects on the growth inhibition and apoptosis induction in KKU-M156 and KKU-100 cells. Caged xanthones may be useful adjunct treatments with chemotherapy for Opisthorchis viverrini (OV)-associated CCA.

LFchimera, a construct combining two antimicrobial domains of bovine lactoferrin, lactoferrampin265-284 and lactoferricin17-30, possesses strong bactericidal activity. As yet, no experimental evidence was presented to evaluate the mechanisms of LFchimera against Burkholderia isolates. In this study we analyzed the killing activity of LFchimera on the category B pathogen Burkholderia pseudomallei in comparison to the lesser virulent Burkholderia thailandensis often used as a model for the highly virulent B. pseudomallei. Killing kinetics showed that B. thailandensis E264 was more susceptible for LFchimera than B. pseudomallei 1026b. Interestingly the bactericidal activity of LFchimera appeared highly pH dependent; B. thailandensis killing was completely abolished at and below pH 6.4. FITC-labeled LFchimera caused a rapid accumulation within 15 min in the cytoplasm of both bacterial species. Moreover, freeze-fracture electron microscopy demonstrated extreme effects on the membrane morphology of both bacterial species within 1 h of incubation, accompanied by altered membrane permeability monitored as leakage of nucleotides. These data indicate that the mechanism of action of LFchimera is similar for both species and encompasses disruption of the plasma membrane and subsequently leakage of intracellular nucleotides leading to cell dead.

Melioidosis is a severe infectious disease that is endemic in Southeast Asia and Northern Australia. Burkholderia pseudomallei, the causative agent of this disease, has developed resistance to an increasing list of antibiotics, demanding a search for novel agents. Lactoferricin and lactoferrampin are two antimicrobial domains of lactoferrin with a broad spectrum of antimicrobial activity. A hybrid peptide (LFchimera) containing lactoferrampin (LFampin265-284) and a part of lactoferricin (LFcin17-30) has strikingly higher antimicrobial activities compared to the individual peptides. In this study, the antimicrobial activities of this chimeric construct (LFchimera1), as well as of another one containing LFcin17-30 and LFampin268-284, a shorter fragment of LFampin265-284 (LFchimera2), and the constituent peptides were tested against 7 isolates of B. pseudomallei and compared to the preferential antibiotic ceftazidime (CAZ). All isolates including B. pseudomallei 979b shown to be resistant to CAZ, at a density of 10(5) CFU/ml, could be killed by 5-10 μM of LFchimera1 within 2 h, while the other peptides as well as the antibiotic CAZ only inhibited the B. pseudomallei strains resulting in an overgrowth in 24 h. These data indicate that LFchimera1 could be considered for development of therapeutic agents against B. pseudomallei.

The Gram-negative bacterium Burkholderia pseudomallei is the aetiological agent of melioidosis, which is an endemic disease in tropical areas of Southeast Asia and Northern Australia. Burkholderia pseudomallei has intrinsic resistance to a number of commonly used antibiotics and has also been reported to develop a biofilm. Resistance to killing by antimicrobial agents is one of the hallmarks of bacteria grown in biofilm. The aim of this study was to determine the antimicrobial activity and mechanisms of action of LL-37 and its truncated variants against B. pseudomallei both in planktonic and biofilm form, as LL-37 is an antimicrobial peptide that possessed strong killing activity against several pathogens. Antimicrobial assays revealed that LL-31, a truncated variant of LL-37 lacking the six C-terminus residues, exhibited the strongest killing effect. Time-kill experiments showed that 20 μM LL-31 can reach the bactericidal endpoint within 2h. Freeze-fracture electron microscopy of bacterial cells demonstrated that these peptides disrupt the membrane and cause leakage of intracellular molecules leading to cell death. Moreover, LL-31 also possessed stronger bactericidal activity than ceftazidime against B. pseudomallei grown in biofilm. Thus, LL-31 should be considered as a potent antimicrobial agent against B. pseudomallei both in planktonic and biofilm form., (Copyright © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Antimicrobial peptides (AMPs) are essential host defence molecules found in a wide variety of species and are promising antibacterial therapeutic candidates. Focusing on the human cathelicidin peptide LL-37, the aim of the present study was to explore the mechanisms of action and antimicrobial activities of a library of LL-37 fragments using Burkholderia thailandensis E264 as a model. The results revealed that IG-19 was the shortest fragment within LL-37 that exhibited antibacterial activity. LL-31, missing six residues at the C-terminus of LL-37, exhibited the strongest killing effect. Freeze-fracture electron microscopy of bacterial cells treated with either LL-37 or LL-31 revealed irregular bacterial surfaces with bleb projections, indicating that these peptides disrupted the integrity of the membrane. In addition, these peptides induced leakage of cell components, including nucleotides and even proteins. Altogether, the results obtained indicate the potential of using LL-31 as a new AMP to combat Burkholderia spp., (Copyright © 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Burkholderia pseudomallei, the causative agent of melioidosis, is intrinsically resistant to many antibiotics, resulting in high mortality rates of 19% in Australia and even 50% in Thailand. Antimicrobial peptides (AMPs) possess potent broad-spectrum bactericidal activities and are regarded as promising therapeutic alternatives in the fight against resistant microorganisms. Moreover, these peptides may also affect inflammation, immune activation and wound healing. In this study, the in vitro activities of 10 AMPs, including histatin 5 and histatin variants, human cathelicidin peptide LL-37 and lactoferrin peptides, against 24 isolates of B. pseudomallei were investigated. The results showed that the antibacterial activities of the individual peptides depended on peptide dose and bacterial isolate. Among the 10 peptides tested, LL-37 exhibited the most effective killing activity. The smooth type A lipopolysaccharide (LPS) phenotype B. pseudomallei appeared to be more susceptible than those expressing the smooth type B LPS and the rough type LPS. Four isolates of B. pseudomallei shown to be resistant to ceftazidime and trimethoprim/sulfamethoxazole were also highly susceptible to LL-37. These data indicate that LL-37 possesses antimicrobial activity against all isolates independent of the LPS phenotype and is therefore a promising peptide to combat B. pseudomallei infections.