Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42°C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.Burkholderia pseudomallei is a biothreat agent and the cause of melioidosis (29). This gram-negative motile bacillus is present in soil and water over a wide area of the Far East, where infection is acquired by inoculation or inhalation (29). B. pseudomallei causes 20% of community-acquired septicemias in northeast Thailand (7) and is the most common cause of fatal community-acquired pneumonia in Darwin, Australia (10, 14). Overall, mortality is around 50% in northeast Thailand (35% in children) and 20% in Australia (10, 11, 29).A major feature of melioidosis is that bacterial eradication is difficult to achieve. The clinical response to intravenous antibiotics is slow (median fever clearance time, 8 days), and recurrent disease is common (6% in the first year in Thailand), despite appropriate antibiotic therapy for 12 to 20 weeks (6, 9). A prolonged period of dormancy may also occur between exposure to B. pseudomallei and clinical manifestations of infection, with the maximum recorded time being 62 years (8,20,21). It is clear that B. pseudomallei can become adapted for survival in vivo, but the mechanisms by which this occurs in humans have not been demonstrated.In the 1930s, it was observed that colony morphology could change in vitro between rough and smooth colonies (22). We have observed over a period of 20...

Seventy-five clinical isolates of Pseudomonas aeruginosa collected in a tertiary teaching hospital in Thailand were investigated for susceptibility to antimicrobials including imipenem. Metallo-β-lactamase (MBL) enzymes were detected by E-test MBL assay and PCR; class 1 integron genes were also detected by PCR. Strains positive for bla(IMP) and bla(VIM) genes were further characterized by DNA sequencing and examined for clonality by pulsed-field gel electrophoresis. High rates of resistance to anti-pseudomonal agents were found. MBL enzymes were found in 13 (17·3%) strains and 24 (32%) carried class 1 integron genes. Twelve of the latter strains harboured the bla(IMP-14) gene and one strain the bla(VIM-2) gene. All of the IMP-14 strains were identical or closely related suggesting clonal dissemination of these genes.

Pseudomonas aeruginosa is one of the most important causes of nosocomial infection and it has increasing resistance to many antimicrobial agents. β-lactamase production is the most frequent mechanism for β-lactam resistance in P. aeruginosa. We evaluated the prevalence of β-lactamase genes in P. aeruginosa for classes A, C, and D by polymerase chain reaction, and investigated clonal diversity by pulsed-field gel electrophoresis (PFGE). We used the disk diffusion method to test 118 non-duplicate clinical isolates of P. aeruginosa for antimicrobial susceptibility. We identified 51 isolates (43.22%) as multidrug-resistant P. aeruginosa, approximately 44.91% of which were resistant to ceftazidime. β-lactamase genes were found in 80 isolates of P. aeruginosa (67.80%). The genes that encode VEB-1, AmpC, and OXA-10 were detected in 9 (7.62%), 75 (63.56%), and 18 (15.25%) of these isolates, respectively. The genes that encode PER-1, CTX-M, TEM-1 and derivatives, and SHV-1 were not found in any of the P. aeruginosa isolates. We identified 29 different pulsotypes by PFGE. Two predominant pulsotypes were found. In pulsotype 1, OXA- 10, which was co-produced with the AmpC gene, was predominant. Moreover, VEB-1-producing strains were found to be scattered in many pulsotypes, and AmpC-producing strains showed high pulsotype diversity. The prevalence of β-lactamase genes in P. aeruginosa was represented by the genetic heterogeneity of OXA-10, AmpC, and VEB-1. The predominant clone of P. aeruginosa clinical isolates was OXA-10. This raises concern about oxacillinases among P. aeruginosa clinical isolates.

Candidiasis caused by the fluconazole-resistant opportunistic pathogen Candida albicans is an intractable clinical problem that threatens immunocompromised or normal individuals. The most common mechanism of fluconazole resistance in C. albicans is the failure of cells to accumulate the drug due to increased expression of the efflux proteins encoded by the CDR1, CDR2, and MDR1 genes. Because the number of current antifungal drugs is limited, it is necessary to develop new therapeutic strategies. This study aimed to evaluate the antifungal activity of Thai Cajuput oil, its synergism with fluconazole, and its effect on efflux-pump gene expression in fluconazole-resistant C. albicans clinical isolates. Thus, we first detected the efflux-pump genes in fourteen resistant strains by PCR. The frequencies of the CDR1, CDR2, and MDR1 genes were 68.75%, 62.5%, and 87.5%, respectively, and these efflux-pump genes were distributed in three distinct patterns. Subsequently, the antifungal activity of Thai Cajuput oil was assessed by broth macrodilution and its synergism with fluconazole was evaluated by the checkerboard assay. The changes in the expression levels of CDR1, CDR2, and MDR1 after treatment with Thai Cajuput oil were analyzed by qRT-PCR. The MICs and MFCs of Thai Cajuput oil ranged from 0.31 to 1.25 μl/ml and 0.63 to 1.25 μl/ml, respectively, and its activity was defined as fungicidal activity. The MICs of the combination of Thai Cajuput oil and fluconazole were much lower than the MICs of the individual drugs. Interestingly, sub-MICs of Thai Cajuput oil significantly reduced the MDR1 expression level in resistant strains P < 0.05 . Our study suggests that Thai Cajuput oil can be used to create new potential combination therapies to combat the antifungal resistance of C. albicans.

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