Distribution and characterization of Stenotrophomonas maltophilia isolates from environmental and clinical samples in Thailand.

BACKGROUND: Stenotrophomonas maltophilia has emerged as an important opportunistic pathogen, especially in patients who are immunocompromised, suffering from malignancy or have been hospitalized for a prolonged period. Information of this bacterium in Thailand has not been elucidated. AIMS: To investigate the phenotype and genotype of environmental and clinical isolates of S. maltophilia in Songklanagarind Hospital, southern Thailand. METHODS: Isolates of S. maltophilia were collected from various environmental sources on three hospital wards and clinical samples from seven wards. Antibiotic susceptibility and minimum inhibitory concentration (MIC) testing were performed using disk diffusion and E-test, respectively. Isolates were genotyped by pulsed-field gel electrophoresis. FINDINGS: The majority of S. maltophilia environmental isolates were from sink drains (67.5%), followed by drinking water (18.7%) and tap water (7.5%). Clinical isolates of the bacterium mainly originated from sputum samples (56.2% of all isolates). Antibiotic resistance was more common in clinical isolates than in environmental isolates; resistance to co-trimoxazole was associated with the presence of the sul1 gene. The MIC values for ciprofloxacin and co-trimoxazole correlated closely with the results obtained from disk diffusion assay. DNA profile analysis revealed seven clusters with high diversity among the isolates. CONCLUSION: No genotypic relationship was detected between isolates from environmental and clinical samples. As such, acquisition of this bacterium may occur outside the hospital.

Isolation of Bdellovibrio and like organisms and potential to reduce acute hepatopancreatic necrosis disease caused by Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease, a severe disease of shrimp, is caused by Vibrio parahaemolyticus (AHPND Vp), a halophilic bacterium harboring a plasmid that contains toxin genes homologous to Photorhabdus insect-related toxins. We obtained 9 isolates of Bdellovibrio and like organisms (BALOs) from water and sediment samples in Thailand. Using 16S rRNA sequencing, all of the organisms were identified as Bacteriovorax spp. and were able to attack all tested AHPND Vp isolates. In addition, their various susceptible hosts, including Gram-positive and Gram-negative bacteria, were observed. The optimal ratio for interaction between the Bacteriovorax isolate BV-A and AHPND Vp was determined to be 1:10. The suitable conditions applied for co-culture between BV-A and AHPND Vp were 30°C, 2% NaCl, and pH 7.6. The capability of BV-A to reduce numbers of AHPND Vp in vitro was observed in co-culture after incubation for 2 d and continued until the end of the incubation period. In vivo, BV-A was able to reduce mortality of shrimp post-larvae infected with AHPND Vp. In addition, BV-A significantly decreased the formation of biofilm by AHPND Vp. These findings provide evidence for using Bacteriovorax as a biocontrol of AHPND Vp in shrimp aquaculture.

Characterization of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease in southern Thailand.

Vibrio parahaemolyticus was isolated from shrimp of five farms located in the Pattani and Songkhla provinces of southern Thailand. Using a PCR method targeted to the unique DNA sequences derived from the plasmid (AP2 primers) and the toxin gene (AP3 primers) of V. parahaemolyticus that caused acute hepatopancreatic necrosis disease (AHPND), a total of 33 of 108 isolates were positive. In contrast, all 63 and 66 isolates of clinical and environmental V. parahaemolyticus, respectively, obtained previously from 2008 to 2014 from the same area were negative. This implied that these strains were likely to be the cause of the outbreak of AHPND in this area. Intestinal samples proved to be a better source for the isolation of V. parahaemolyticus AHPND than the hepatopancreas. All isolates were investigated for haemolytic activity, virulence genes, serotypes, genotypes and antibiotic susceptibility. All the AHPND isolates had a unique O antigen, but small variations of the K antigens were detected from different farms. In addition, the DNA profiles of V. parahaemolyticus AHPND isolates were similar, but distinct from those clinical and environmental isolates. It is postulated that the causative agent of AHPND might have originated from one clone and then slightly different serotypes subsequently developed.

A randomized clinical trial of chlorhexidine in the maintenance of oral candidiasis-free period in HIV infection.

OBJECTIVE: To determine if chlorhexidine can be used as an intervention to prolong the time to relapse of oral candidiasis. SUBJECTS AND METHODS: A double-blinded randomized clinical trial was performed in 75 HIV/AIDS subjects with oral candidiasis. Clotrimazole troche was prescribed, and the subjects were re-examined every 2 weeks until the lesions were completely eradicated. The subjects were then randomly divided into two groups; 0.12% chlorhexidine (n = 37, aged 22-52 years, mean 34 years) and 0.9% normal saline (n = 38, aged 22-55 years, mean 38 years). They were re-examined every 2 weeks until the next episode was observed. RESULTS: The time to recurrence of oral candidiasis between the chlorhexidine and the saline group was not statistically significant (P > 0.05). The following variables were significantly associated with the time of recurrence; frequency of antifungal therapy (P = 0.011), total lymphocyte (P = 0.017), alcohol consumption (P = 0.043), and candidiasis on gingiva (P = 0.048). The subjects with lower lymphocyte showed shorter oral candidiasis-free periods (P = 0.034). CONCLUSIONS: Chlorhexidine showed a small but not statistically significant effect in maintenance of oral candidiasis-free period. This lack of significance may be due to the small sample size. Further study should be performed to better assess the size of the effect, or to confirm our findings.

Tuberculosis in Thai prisons: magnitude, transmission and drug susceptibility.

BACKGROUND: Because of the human immunodeficiency virus (HIV) epidemic, tuberculosis has reemerged as a major public health problem in Thailand. Prison inmates are at high risk for developing tuberculosis because of the high prevalence of HIV infection. OBJECTIVES: To determine the magnitude, transmission, and drug susceptibility of tuberculosis in Thai prisons. SETTINGS: Four provincial prisons in Southern Thailand. DESIGN: Cross-sectional, descriptive, clinical and molecular study. RESULTS: Miniature chest roentgenograms were performed on 304 (6.4%) of 4751 inmates screened for a > or = 2 week history of chronic cough and fever. At least 17 (35%) of 49 inmates who had a miniature chest roentgenogram compatible with tuberculosis were HIV-positive. The prevalence of smear-positive pulmonary tuberculosis was 568 per 100,000 inmates, which was eight times higher than that in the general population. Eight (38%) of 21 culture-positive Mycobacterium tuberculosis isolates had DNA fingerprints matching those of another inmate who was housed in the same room or in the same dormitory unit; 39% of the M. tuberculosis isolates were resistant to isoniazid; three of these isolates were also borderline resistant to rifampicin. CONCLUSION: The prevalence of pulmonary tuberculosis in these prisons was high. A substantial proportion were acquired in the prisons. Isoniazid (INH) resistance was common, and theoretically precludes the use of INH-preventive therapy for contacts of these cases. Active case finding should be done and directly observed therapy implemented to prevent the spread of tuberculosis into the community.

Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V. hollisae from the environment and its identification.

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.

Isolation of a pandemic O3:K6 clone of a Vibrio parahaemolyticus strain from environmental and clinical sources in Thailand.

Application of an immunomagnetic enrichment method selective for Vibrio parahaemolyticus serovar K6 allowed isolation of a strain belonging to the pandemic O3:K6 clone of V. parahaemolyticus from fresh shellfish not implicated in a clinical case in southern Thailand. Arbitrarily primed PCR profiles of this strain, clinical O3:K6 strains isolated from sporadic diarrhea cases in the same area, and a standard pandemic O3:K6 strain were indistinguishable.

Isolation and characterization of Escherichia coli O157 from retail beef and bovine feces in Thailand.

Antibody to Escherichia coli O157 lipopolysaccharide was detected in the sera of healthy individuals more frequently in Southern Thailand than in Japan. The result suggested possible exposure of Thai people to E. coli O157. E. coli O157:H7 or O157:H(-) was isolated from four of 95 retail beef and one of 55 bovine feces samples collected in Southern Thailand by enrichment culture followed by immunomagnetic bead separation. Four of the five strains carried the stx(2) gene alone or in combination with the stx(1) gene. The strains were shown to be genetically distinct by an arbitrarily primed PCR method.

Epidemiology of Burkholderia pseudomallei in Thailand.

The distribution of Burkholderia pseudomallei in soil collected from four regions of Thailand and the frequency of B. pseudomallei infections in patients attending government hospitals throughout Thailand in 1997 were surveyed. A total of 3,585 soil samples collected from 896 sites in four regions of Thailand were cultured for B. pseudomallei using selective enrichment broth and modified Ashdown's agar. The organism was recovered in 4.4%, 6.1%, 20.4%, and 5.9% of the soil samples collected from the northern, central, northeastern, and southern regions, respectively, of Thailand (P < 0.0001). Burkholderia pseudomallei was cultured from 50.1% of the sites in the northeastern region compared with 13.8%, 24.5%, and 18.4% in the northern, central, and southern regions, respectively (P < 0.0001). The infection rate in patients attending government hospitals in the northeastern region (137.9 per 100,000 inpatients) was significantly higher than those in the northern (18 per 100,000 inpatients), central (13.4 per 100,000 inpatients), and southern (14.4 per 100,000 inpatients) regions, respectively (P < 0.0001). It is suggested that melioidosis, which is endemic in Thailand, is associated with the presence of B. pseudomallei in soil.

Epidemiology of arabinose assimilation in burkholderia pseudomallei isolated from patients and soil in Thailand.

Burkholderia pseudomallei is an environmental saprophyte that has been isolated widely from soil in Southeast Asia and the relationship between environmental contamination and clinical melioidosis has been established. It has been shown that the arabinose assimilation property of B. pseudonrallei is probably one of the determinants indicating virulence of this organism. Therefore, the distribution of arabinose assimilation biotypes of B. pseudomallei collected from four geographic regions of Thailand was studied in order to determine an association between arabinose assimilation of B. pseudomallei and the uneven distribution of melioidosis found among these four areas. A total of 830 isolates of B. pseudomallei (412 patient isolates and 418 soil isolates) collected from the patients and soil in four regions of Thailand in 1997 were tested for an ability to grow on a minimal agar medium supplemented with L-arabinose. All patient isolates except one could not utilise arabinose (Ara-). For 418 soil isolates, 232 (55.5%) isolates were identified as Ara type. They comprised 180 (62.5%), 36 (46.8%), 6 (35.3%) and 10 (27.8%) isolates derived from northeastern, southern, northern and central regions respectively. The ratios of Ara- to Ara, were 1.7, 0.9. 0.5 and 0.4 among isolates collected from northeastern, southern, northern and central regions respectively. The prevalence of Ara- in soil isolates in northeast is significantly higher than those in other regions. This observation suggests that in addition to the presence of B. pseudomallei in soil which is one of the factors contributing to a burden of melioidosis in northeastern Thailand, the distribution of more virulent biotype (Ara-) soil isolates is a factor contributing to a high prevalence of melioidosis in northeastern Thailand as well.

Interaction of respiratory syncytial virus with human cord blood mononuclear cells.

This study on the interaction between respiratory syncytial virus (RSV) and human cord blood mononuclear cells shows that RSV replication can occur in neonatal macrophages. Although neonatal lymphocytes were not supportive of RSV replication, exposure to RSV resulted in significant inhibition of mitogen-induced transformation. Both adult and neonatal NK cell cytotoxicity were unaffected by exposure to RSV. These results suggest that RSV has preferential effects on human cord blood mononuclear cell subpopulations.

Suppression of neutrophil and lymphoproliferative responses in vitro by itraconazole but not fluconazole.

Itraconazole and fluconazole are triazole compounds recently licensed for the therapy of systemic fungal infections. At 10 micrograms/ml concentrations, itraconazole was found to suppress neutrophil chemotaxis, random movement, deoxyglucose uptake and hexose-monophosphate shunt activity to the same extent as ketoconazole, an older generation azole antifungal. Itraconazole was also found to suppress mitogen-induced lymphocyte transformation to the same extent as ketoconazole at concentrations as low as 1 microgram/ml. By contrast, significant inhibition of both neutrophil and lymphocyte functions was not observed with fluconazole at concentrations as high as 50 micrograms/ml. These results suggest that fluconazole may be less immunotoxic than itraconazole, and may be more suitable for use in immunocompromised patients.

Inhibitory effects of human neutrophil granules and oxygen radicals on adherence of Candida albicans.

The adherence of Candida albicans to dacron fibre microcolumns was significantly suppressed after interaction with human neutrophils. The adherence-inhibiting properties of neutrophils were shown to reside in their cytoplasmic granules and granular enzymes. Oxygen-derived free radicals produced by the respiratory burst may also be responsible, as shown by experiments in which oxygen radicals were generated by the cell-free hypoxanthine-xanthine oxidase system. Dose-response studies with H2O2 and beta-glucoronidase demonstrated that lower concentrations of these agents inhibited adherence without affecting viability of C. albicans. These results suggest that interference with adherence mechanisms may be an effective means of host defence by neutrophils against the colonisation of mucosal surfaces by C. albicans.

Direct modulation of human neutrophil behaviour by Candida albicans.

We examined the direct effect of unopsonized yeast particles of Candida albicans on two aspects of neutrophil behaviour, namely adherence and 3H-deoxyglucose uptake. The data show that brief exposure of C. albicans to human neutrophils resulted in decreased ability of the neutrophils to adhere to Dacron fibre and take up deoxyglucose. This inhibitory effect was further shown to be dependent on yeast concentration and on the integrity of the yeast cell wall. Additional experiments indicate that this effect was direct rather than indirect through soluble mediators in the supernatant. Interference experiments with glucan and mannan, the two major polysaccharide components of the yeast cell wall, suggest that the ligand in direct interaction between C. albicans and neutrophil contains glucan. Finally, it was shown that nonpathogenic species of Candida such as C. krusei, C. parapsilosis and C. guilliermondii did not display neutrophil-modulatory properties while the occasionally pathogenic C. tropicalis did. These results indicate that C. albicans has the ability to circumvent neutrophil defence mechanisms, and may in part explain the propensity of this fungus to cause infection.

Effects of the newer antifungal agents (bifonazole, ICI 195, 739 and amorolfin) on in vitro phagocytic, lymphocytic and natural-killer cell responses.

Amphotericin B and some of the imidazole drugs have been shown to suppress certain neutrophil and lymphocyte functions both in vitro and in vivo. We present here the in vitro effects of: amorolfin, a morpholine derivative; the imidazoles clotrimazole and ketoconazole; the N-substituted imidazole bifonazole and a triazole (ICE 195, 739), on neutrophil and lymphocyte function. All of these drugs inhibited neutrophil random migration, chemotaxis and hexose monophosphate shunt activity. The effects of the drugs on neutrophil adherence, deoxyglucose transport and beta-glucuronidase release were variable while lysozyme release was unaffected. Natural Killer cell cytoxicity was depressed by all drugs tested except for amorolfin. Mitogen-induced lymphocyte blastogenesis was suppressed by all the antifungal drugs tested. Similar results were obtained using the mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen. The mechanism of action of these drugs on these cell functions remains unknown, there may be a correlation between their effects on fungi and their effects on leukocytes. Clearance of systemic fungal infection is heavily dependent on integrity of the cellular immune system and it is clearly undesirable that antifungal drugs have immunosuppressive properties. Further studies are required to determine the in vivo and clinical relevance of our observations.

Inhibition of neutrophil locomotion, natural killer cell cytotoxicity and lymphocyte transformation by 1-methyl-3-phenyl-1,2,4-triazinium-5-olate, a novel triazinium zwitterion.

We studied a novel triazinium zwitterion compound for its effects on neutrophil locomotion and deoxyglucose uptake, Natural Killer (NK) cell cytotoxicity and mitogen-induced lymphocyte transformation. The results show significant inhibition of neutrophil locomotion at concentrations of 10 micrograms/ml or greater; by contrast, there was no significant effect on neutrophil deoxyglucose uptake. Significant suppression of NK cell cytotoxicity occurred at similar concentrations in a dose-dependent fashion. Marked suppression of mitogen-induced lymphocyte transformation was also observed for all three mitogens used in the assays. This effect was dose-dependent, reversible by washing and still evident even when it was added 37 h after the initiation of cultures. These results suggest that 1-methyl-3-phenyl-1,2,4-triazinium-5-olate may have application as an anti-inflammatory and immunosuppressive agent.

1-Methyl-3-phenyl-1,2,4-triazinium-5-olate: a new zwitterion with cytotoxic activity against human cancer cell-lines.

One of 6 newly synthesized triazinium zwitterions (JR-1--JR-6) was shown to induce 51Cr-release from leukemic (HL60 and CEM) and solid tumor (MM170 and HeLa) cell-lines. Leukemic cells were more sensitive to this compound than solid tumors as demonstrated by dose-response and time-course studies. Other experiments showed that JR-6 (1-methyl-3-phenyl-1,2,4-triazinium-5-olate) significantly suppressed protein, RNA and DNA synthesis at tumoricidal concentrations.

Inhibition of adherence of Candida albicans by conventional and experimental antifungal drugs.

We tested the effects of antifungal drugs on adherence of Candida albicans in vitro. Significant reduction of adherence occurred after 2 h incubation with amphotericin B, nystatin, miconazole, econazole, ketoconazole, chlorohexidine and ICI 195,739. Significant inhibition of candida adherence by 5-fluorocytosine and amorolfin required 18 h incubation. Combinations of amphotericin B with 5-fluorocytosine, miconazole, ICI 195,739 and amorolfin resulted in synergistic inhibition of adherence. Adherence is an important pathogenic mechanism in candida infections and interference with this process may represent a major component of the mode of action of antifungal drugs.