BURKHOLDERIA PSEUDOMALLEI BIOFILM PLAYS A KEY ROLE IN CHRONIC INFLAMMATION IN C57BL/6 MICE.

Burkholderia pseudomallei is a causative agent of melioidosis. Clinical signs of melioidosis vary from acute septicemia to chronic inflammation or subclinical infection. This study investigated the role of B. pseudomallei biofilm in chronic inflammation in lungs of infected C57BL/6 mice. Low doses of B. pseudomallei H777 and its biofilm defective M10 mutant were fed intra-gastrically to C57BL/6 mice and inflammatory responses were investigated by histopathological techniques. Two hundred colony forming units (CFUs) of B. pseudomallei H777 induced chronic inflammatory responses in mice on day 20 post-infection, with discrete interstitial infiltration by mononuclear inflammatory cells. On day 40 postinfection, there were marked thickening of alveolar septa and congested capillaries, which increased in severity by day 60. On the other hand, mice infected with B. pseudomallei M10 showed less mononuclear infiltration. The results indicate that B. pseudomallei defective in biofilm production gave rise to less severe pathology, resulting a higher rate of survival in infected mice; and pulmonary melioidosis could be developed in C57BL/6 mice by intra-gastric feeding makes it a possible animal model of chronic human melioidosis.

Relapsed Melioidosis Model in C57BL/6 Mice.

BACKGROUND: Burkholderia pseudomallei are the causative agents of melioidosis, a disease that has a high relapse rate in endemic areas. The mechanism of relapse is unclear OBJECTIVE: This study aimed to establish relapsed melioidosis in C57BL/6 mice by induction with B. pseudomallei. MATERIAL AND METHOD: Low doses of B. pseudomallei H777 and its biofilm defective mutant (M10) were intra-gastric fed to C57BL/6 mice. All the infected mice had suppressed immune status by intra-peritoneal injection of hydrocortisone at 2.5 mg per mouse at day 60 post-infection. Inflammatory response to the infection was investigated by histo-pathological studies and monitoring bacterial counts in the blood and organs. RESULTS: All the infected mice were found to have a high infiltration of mononuclear cells at day 60 post-infection. The results showed high bacterial counts in the blood in both strains post-suppressed immune status after two days. The biofilm mutant and wild type strains produced relapse in C57BL/6 mice but the latter was responsible for significantly more severe inflammation than the biofilm mutant. CONCLUSION: Low immune status may cause relapsed melioidosis in hosts with chronic inflammation.

Antibacterial Activity of Excretions-Secretions from Chrysomya megacephala Against Escherichia coli.

BACKGROUND: The blowfly, Chrysomya megacephala, is distributed worldwide. Previous studies found maggot excretions-secretions from other blowfly species inhibited pro-inflammatory response and antimicrobial activity. OBJECTIVE: This study aimed to test the bactericidal activity of excretions-secretions from C. megacephala larvae. MATERIAL AND METHOD: A total of 1,500 3-day-old larvae were used to collect excretions-secretions (ES) modified by the Barnes method. The bactericidal activity ofthe excretions-secretions was test by Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli using suitable liquid culture assay. Scanning electron microscope (SEM) was used to investigate the morphological change ofthe bacteria. RESULTS: E. coli were significantly inhibited by excretions-secretions from C. megacephala larvae. P. aeruginosa and S. aureus were not found to inhibit growth. CONCLUSION: The excretions-secretions from C. megacephala larvae may have a medical property for the inhibition of bacterial growth.

Prevalence of bla(PenA) and bla(OXA) in Burkholderia pseudomallei Isolated from Patients at Sappasitthiprasong Hospital and Their Susceptibility to Ceftazidime and Carbapenems.

BACKGROUND: Burkholderia pseudomallei is a causative agent of melioidosis. Ceftazidime is the preferred drug of choice for treatment. However, the motility rate is high in endemic areas. OBJECTIVE: This study aimed to determine the susceptibility tofour different antimicrobial agents and to detect the ß-lactamase genes in B. pseudomallei isolates from patients admitted to Sappasitthiprasong Hospital. MATERIAL AND METHOD: 85 B. pseudomallei isolates from patients admitted to Sappasitthiprasong Hospital between November 2010 and May 2011 were determined for antimicrobial susceptibility by standard disk diffusion and minimum inhibitory concentration (MIC). Real-time polymerase chain reaction (PCR) was used for the detection of bla(penA) and bla(OXA) in ß-lactamase genes. RESULTS: Almost all of the clinical isolates ofB. pseudomallei were susceptible to ceftazidime and imipenem. Cefatzidime MIC was ≤ 1-16 µg/ml and imipenem MIC was ≤ 1-4 µg/ml. The real-time PCR revealed that more than 90% of B. pseudomallei isolates carried bla(PenA) and bla(OXA). CONCLUSION: Although the clinical isolates of B. pseudomallei were susceptible to ceftazidime and imipenem, this study showed B. pseudomallei had a gene that produced beta-lactamase enzyme and may be poorly effective in the use of beta-lactam drugs.

Comparison of Automated and Conventional IHC Visual Scoring Analysis for MHC Class I and Tapasin Expression in Cervical Carcinoma.

BACKGROUND: Cervical cancer (CXCA) is the second most common cancer among women in Thailand and worldwide. Immune evasion caused by down-regulation of host immune responsive genes, such as MHC class I and loss of antigen processing machinery (APM), presents a capability leading to cancer development. Immunohistochemical staining (HC) is regarded as a common technique for protein marker detection in clinical laboratories. At present, IHC automation has been launched to facilitate the speed and feasibility to replace conventional IHC. However, evaluation of its use is still limited. OBJECTIVE: This study aimed to evaluate IHC scoring by automated visual analysis compared to conventional IHC analysis. MATERIAL AND METHOD: The paraffin-embedded tissues of 96 invasive CXCA were processed using a tissue microarray (TMA) platform followed by automated IHC staining of the anti-MHC class I (heavy chain, ß2M) and an APM-Tapasin expression. Conventional IHC and automated slide scanning with scoring visual analysis were compared. RESULTS: The results showed significant association between conventional and automated IHC evaluation (p-value > 0.05, Chi-square) for MHC class I and Tapasin stated in percentage of positive cancer cells, whereas intensity was found (p-value < 0.05, Chi-square) with moderate agreement (p-value < 0.001, kappa) 0.434-0.615 and 0.353-0.554, respectively. After calculated values, the results showed significant association between conventional and automated IHC evaluation (p-value > 0.05, Chi-square) for MHC class I and Tapasin with the highest agreement level (p-value < 0.001, kappa) of summation 0.595-0.755 and multiply scoring 0.633-0.689, respectively. CONCLUSION AND DISCUSSION: The automation softwarefor IHC scoring and interpretation can be used for the determination of MHC class I and Tapasin in CXCA. In addition, an antigen presentation pattern must be included to allow an accurate result for MHC class I in clinical use. An appropriate sample size and design of staging coverage as well as clinical prognosis outcomes of progression should be used infurther investigation.

Antibiotic resistance patterns of Enterococcus spp. isolated from Musca domestica and Chrysomya megacephala in ubon Ratchathani.

BACKGROUND: The housefly Musca domestica and the blowfly Chrysomya megacephala are found worldwide and are medically significant as mechanical vectors of various pathogens from unsanitary locations to food, resulting in diseases in humans. OBJECTIVES: This study aimed to test the antimicrobial activity against Enterococcus spp. isolated from M. domestica and C. megacephala by standard disk diffusion and minimal inhibitory concentration (MIC), and to study the potential of M. domestica and C. megacephala to transfer multi-drug resistant enterococcus to humans. MATERIAL AND METHOD: Seven hundred adult flies were collected from fresh-food markets, garbage piles, restaurants, school cafeterias, and rice paddy fields in Muang Ubon Ratchathani and Warinchamrap in Ubon Ratchathani Province. Antimicrobial susceptibility for Enterococcus spp. isolated from adult flies was performed by disk diffusion test and minimum inhibitory concentration (MIC) determination. RESULTS: One hundred and twenty isolates of Enterococcus spp. were taken from 67 M. domestica and 53 C. megacephala. Standard disk diffusion showed the Enterococcus spp. isolates exhibited susceptibility to ampilcillin (99.2%), chloramphenicol (74.20%), tetracycline (75.0%), vancomycin (50.8%), and erythromycin (42.5%). The MICs of antimicrobial agents for all isolates were < or = 0.25-8 microg/mL for vancomycin, 1- > 16 microg/mL for tetracycline, 4- > 16 microg/mL for chloramphenicol, and 0.5-8 microg/mL for ciprofloxacin. CONCLUSION: The study demonstrated the potential of M. domestica and C. megacephala to carry Enterococcus spp. Nine antimicrobial susceptibility patterns were obtained among the 120 enterococci isolates.

Methicillin-resistant Staphylococcus aureus with reduced susceptibility to vancomycin in Sanprasitthiprasong Hospital.

BACKGROUND: Staphylococcus aureus is a species of bacteria that causes a number of diseases and more than 60% of it is presently resistant to methicillin. Vancomycin is the drug of choice for the eradication of methicillin-resistant S. aureus (MRSA). OBJECTIVE: This study aimed to investigate the susceptibility of heterogeneous vancomycin intermediate S. aureus (hVISA) and vancomycin intermediate S. aureus (VISA) to vancomycin by standard disk diffusion, microbroth dilution, a one-point population assay, and a population analysis profile. MATERIAL AND METHOD: Sixty-eight MRSA isolates from patients admitted to Sanprasitthiprasong Hospital between November 2010 and November 2011 were tested. RESULTS: Standard disk diffusion showed that all the MRSA isolates were susceptible to vancomycin. Vancomycin MICs for all isolates were 1-2 microg/mL. Only two MRSA isolates (2.9%) were able to grow on brain heart infusion agar supplemented with vancomycin 4 microg/mL and were confirmed by a population analysis as hVISA. CONCLUSION: This study showed the effect of vancomycin on MRSA and the need for early detection and controlled planning.

Prognostic factors of human papillomavirus genotypes of invasive cervical carcinoma: an analytical cross-sectional study in lower north-east Thailand.

BACKGROUND: Cervical cancer (CXCA) caused by persistent infections by high-risk human papillomavirus (HR-HPV) can lead to multi-step carcinogenesis. The best management strategy and significant prognosis for cervical cancer patients remain unclear. OBJECTIVE: To investigate the associations of the two most common HR-HPVs with clinical outcomes of progression and recurrence status as well as prognosis outcomes of patients. MATERIAL AND METHOD: An analytical cross-sectional study of patients registered at Ubon Ratchathani Cancer Hospital was conducted from 2007 to 2010. Clinical data, histopathological features, and clinical outcomes of progression and recurrence status were recorded. HPV type-specific E6/E7 nested multiplex polymerase chain reaction (NMPCR) was performed to identify HR-HPV16 and 18 using extracted deoxyribonucleic acid (DNA) from embedded paraffin. Clinical findings and HPV genotypes were analyzed using Fisher's exact test. Association studies of crucial factors and HR-HPV genotypes were performed using logistic regression analysis (odds ratio [OR]) and 95% confidence interval [CI]). A p-value of less than 0.05 was considered statistically significant. RESULTS: The study found single HPV16 infection in 57.3%, single HPV18 in 17.3%, mixed HR-HPV16/18 in 13.1%, and non-HPV16, 18, or 16/18 in 12.3%. The findings showed significant association among their genotypes and histopathological types and grading (p < 0.0001 and p = 0.014). Clinical outcomes of progression and recurrence status with increased severity of clinical staging were associated significantly (p = 0.001 and p = 0.002). HPV18 type-specific was shown as a poor prognostic type with its relevance to the severity of disease higher than that of HPV16. CONCLUSION AND DISCUSSION: HPV16 and 18 remain the major type-specifics especially in relation to invasive CXCA, requiring further therapeutic vaccination study and proper prognosis. HR-HPV type-specific is very important during cervical carcinogenesis but other crucial contributing factors for prognostic outcomes should be further elucidated.

Activated charcoal enhanced the antigen-expression and dendritic cell maturation of the vaccine using Listeria-platform.

BACKGROUND: Listeria monocytogenes (LM) has been used as a vaccine vector based upon its ability to induce a strong cell-mediated immune response. LM inactivated with γ-irradiation retains immunogenic properties and is an attractive platform for clinical use since it would have improved safety concerns compared to live vectors. Activated charcoal has been shown to enhance expression of LM proteins such as PrfA. AIM: To investigate the effect of various growth conditions supplemented with activated charcoal on recombinant antigen expression. METHODS: We prepared γ-irradiated ovalbumin-expressing LM (LM-OVA) after growth under various culture conditions. We cultured LM-OVA at various temperatures including 25°C, 37°C and 37°C with activated charcoal and compared OVA expression by western blot analysis, dendritic cells maturation and OVA-specific T cells. RESULTS: The OVA expression was highest in γ-irradiated LM-OVA grown with activated charcoal at 37°C. Compared to other growth conditions, γ-irradiated LM-OVA grown with activated charcoal at 37°C induce better DC maturation as well as production of the highest number of antigen-specific IFN γ-secreting T cells. CONCLUSION: The further study should be demonstrated the potential to alter growth conditions to enhance OVA expression resulting for vaccine vectors, thereby improving their safety and efficacy.

Immune response to Burkholderia pseudomallei.

Burkholderia pseudomallei are the causative agents of melioidosis, a disease found mostly in people with underlying risk factors. Fifty percent of cases are community-acquired septicemias and manifestation can vary from acute septicemia (with or without shock), to chronic, to subclinical infections. There is no vaccine to prevent the condition. It is difficult to eradicate the bacteria. Prolonged antibiotic therapy is required. Finally, there is a high rate of relapse when therapy is not completed The bacteria can activate both innate and adaptive immune responses but B. pseudomallei employ numerous tactics to evade these immune responses. The pathogenesis of melioidosis is poorly understood, especially the interaction between the host and the pathogen that results in acute and chronic infections. The objective of this review was to summarize the current understanding of the immunology of melioidosis. This review presents an overview of host immune response to B. pseudomallei and benefits the development of research into immunology and promotes an understanding of the mechanism and pathology of B. pseudomallei infection.

TBK1 does not play a role in the control of in vitro Burkholderia pseudomallei growth.

Burkholderia pseudomallei, the causative agent of melioidosis, is an important intracellular pathogen in tropical regions. TANK-binding kinase (TBK1), part of the pathway that induces transcription of Type I interferon genes, has been demonstrated to play an important role in controlling intracellular bacterial infections. To investigate the role of tbk1 in protecting against B. pseudomallei we developed tbk1-deficient cell lines by using shRNA for transient knockdown of the tbk1 gene in HeLa and RAW 264.7 cells. In tbk1-deficient RAW cells, the replication of invasive and non-invasive Escherichia coli was significantly increased at 48 h after infection compared with wild-type cells. The result was confirmed using Brucella melitensis in tbk1-deficient HeLa cells, which demonstrated a >1.5-2.0 log higher bacterial count at 6-48 h after infection compared to wild-type cells. By contrast, the growth of Burkholderia pseudomallei expressing either typical (A2) or atypical (G207) lipopolysaccharide was not significantly different between the tbk1-deficient and control cells. These results suggest that the tbk1 gene and its activation may be able to control invasive E. coli, non-invasive E. coli and B. melitensis growth but may not be able to control B. pseudomallei infection. The role of the tbk1 gene in proinflammatory cytokine induction and bacterial intracellular infection needs further investigation to identify mechanistic differences among the life cycles of various intracellular bacteria.