Abstract. Respiratory infections are initiated by the attachment of bacteria to pharyngeal epithelial cells. We studied the attachment of Burkholderia pseudomallei to pharyngeal epithelial cells. After one, two, three, and four washes, there were 22.6 Ϯ 8.9, 15.7 Ϯ 7.0, 6.8 Ϯ 3.1, and 4.6 Ϯ 1.1 (mean Ϯ SD) attached bacteria/cell, respectively. If the bacterial concentration was maintained at 1 ϫ 10 8 colony-forming units (cfu)/ml and three washes were done, at concentrations of 2.5 ϫ 10 4 , 5 ϫ 10 4 , and 1 ϫ 10 5 cells/ml there were 9.9 Ϯ 3.6, 3.3 Ϯ 0.8, and 2.5 Ϯ 1.1 attached bacteria/cell, respectively. If the cell concentration was kept at 2.5 ϫ 10 4 cells/ml and three washes were done, at bacterial concentrations of 1 ϫ 10 5 , 1 ϫ10 6 , 1 ϫ 10 7 , 1 ϫ 10 8 , and 1 ϫ 10 9 cfu/ml, there were 0.3 Ϯ 0.3, 0.6 Ϯ 0.6, 1.0 Ϯ 0.2, 5.1 Ϯ 2.3, and 9.6 Ϯ 1.9 attached bacteria/cell, respectively. There were 4.8 Ϯ 1.9, 5.5 Ϯ 2.5, 5.6 Ϯ 1.9, and 6.4 Ϯ 2.6 attached bacteria/cell at 0, 30, 120, and 240 min of incubation, respectively. Pharyngeal cells from 10 persons (seven men and three women, mean Ϯ SD age ϭ 30.7 Ϯ 8.1 years, 12 experiments with a single isolate) showed that there were 7.8 Ϯ 4.3 attached bacteria/cell. It was found that the efficiency of attachment of this bacteria was very low (7.0 Ϯ 3.3 bacteria/cell). Electron microscopy revealed that there were no fimbriae but a thin capsular polysaccharide layer on the surface of B. pseudomallei. Attachment to pharyngeal epithelial cells appeared to be mediated by this structure.Burkholderia pseudomallei, a gram-negative bacilli, is a natural saprophyte that can be isolated from soil, stagnant streams, ponds, and rice paddies in areas endemic for melioidosis. This bacteria is usually transmitted by cutaneous and respiratory routes; however cutaneous transmission is significantly more prevalent than the respiratory route. 1-3 The most common form of this disease is a pulmonary infections that ranges from acute bronchitis to overwhelming necrotizing pneumonia. Burkholderia pseudomallei is one of the lifethreatening causes of pneumonia in Southeast Asia and northern Australia. The initial step in the pathogenesis of respiratory infection is the attachment of the bacteria to the pharyngeal epithelial cells. Until now no studies have been done on the adherence of this bacteria to respiratory cells. A recent study has shown that B. pseudomallei was present in the pharynx of approximately half of the patients with pulmonary melioidosis, but absent in the controls. 4 This indicates that colonization of the pharynx by B. pseudomallei might be associated with the pathogenesis of this infection. Therefore, this study was conducted to describe the basic aspects of attachment of B. pseudomallei to pharyngeal epithelial cells. This will lay the groundwork for exploring the pathogenic mechanisms of melioidosis. MATERIALS AND METHODSBacteria. All eight strains of B. pseudomallei used were obtained from Chiang Mai University (Chiang Mai, Thailand) and their identification was confirm...
BackgroundMultidrug/extensively drug-resistant tuberculosis (M/XDR-TB) is a major public health problem, and early detection is important for preventing its spread. This study aimed to demonstrate the distribution of genetic site mutation associated with drug resistance in M/XDR-TB in the northern Thai population.MethodsThirty-four clinical MTB isolates from M/XDR-TB patients in the upper northern region of Thailand, who had been identified for drug susceptibility using the indirect agar proportion method from 2005 to 2012, were examined for genetic site mutations of katG, inhA, and ahpC for isoniazid (INH) drug resistance and rpoB for rifampicin (RIF) drug resistance. The variables included the baseline characteristics of the resistant gene, genetic site mutations, and drug susceptibility test results.ResultsAll 34 isolates resisted both INH and RIF. Thirty-two isolates (94.1%) showed a mutation of at least 1 codon for katG, inhA, and ahpC genes. Twenty-eight isolates (82.4%) had a mutation of at least 1 codon of rpoB gene. The katG, inhA, ahpC, and rpoB mutations were detected in 20 (58.7%), 27 (79.4%), 13 (38.2%), and 28 (82.3%) of 34 isolates. The 3 most common mutation codons were katG 315 (11/34, 35.3%), inhA 14 (11/34, 32.4%), and inhA 114 (11/34, 32.4%). For this population, the best genetic mutation test panels for INH resistance included 8 codons (katG 310, katG 340, katG 343, inhA 14, inhA 84, inhA 86, inhA 114, and ahpC 75), and for RIF resistance included 6 codons (rpoB 445, rpoB 450, rpoB 464, rpoB 490, rpoB 507, and rpoB 508) with a sensitivity of 94.1% and 82.4%, respectively.ConclusionThe genetic mutation sites for drug resistance in M/XDR-TB are quite variable. The distribution of these mutations in a certain population must be studied before developing the specific mutation test panels for each area. The results of this study can be applied for further molecular M/XDR-TB diagnosis in the upper northern region of Thailand.
Background: Extensively drug resistant tuberculosis (XDR-TB) is a serious problem in public health and XDR-TB patients usually develop from multi-drug resistance tuberculosis (MDR-TB) and pre-XDR-TB. The rapid molecular test for drug susceptibility testing (DST) can be used for early detection to prevent XDR-TB. Methods: We examined 34 clinical Mycobacterium tuberculosis (M. tuberculosis) isolates from MDR/XDR-TB patients in the upper north of Thailand that were identified with drug susceptibility profiles by indirect agar proportion method from 2005-2012. Our study investigated the genetic mutations in gyrA for ofloxacin resistance and rrs for kanamycin resistance. The genetic mutations and drug susceptibility test results were analyzed using the exact test. Results: The majority of the ofloxacin resistance was detected in gyrA 21, gyrA 70, gyrA 87, gyrA 102, gyrA 162, and gyrA 187 were at 0%, 12.5%, 37.5%, 0%, 50.0% and 25.0% sensitivity, respectively, and at 96.2, 96.2%, 20.1%, 96.2%, 57.7% and 61.5% specificity, respectively. Kanamycin resistance was found in rrs 512, rrs 241, rrs 223, rrs 414 and rrs 408 at 16.7%, 0%, 0%, 16.7% and 16.7% sensitivity, respectively, and at 96.4%, 92.9%, 82.1%, 82.1% and 71.4% specificity, respectively. This study found no significant correlation between gyrA mutations and ofloxacin resistance and also no correlation between the rrs gene and kanamycin resistance. Conclusion: These primer sequences and PCR products in our study such as gyrA and rrs might be unsuitable to detect ofloxacin and kanamycin resistance in the upper How to cite this paper:
Background: Molecular diagnosis based on the detection of mutations conferring genetic drug resistance is useful for early diagnosis and treatment of Pre-XDR and XDR-TB patients. However, the study of mutation as a marker to predict Pre-XDR and XDR-TB is rare. Methods: Thirty-four Mycobacterium tuberculosis (MTB) isolates from MDR, Pre-XDR and XDR-TB patients in the upper north of Thailand, who had been identified for drug susceptibility using the indirect agar proportion method from 2005-2012, were examined for genetic site mutations of katG, inhA, and ahpC for isoniazid (INH) drug resistance, rpoB for rifampicin (RIF) drug resistance, gyrA for ofloxacin (OFX), and rrs for kanamycin (KAN). Associations between resistant genes and Pre-XDR and XDR-TB in the MDR patients were performed using exact probability tests. Univariable logistic regression was used to quantify the strength of association between the gene mutation with Mycobacterium tuberculosis and the prevalence of Pre-XDR and XDR-TB in the MDR patients. Results: The mutations in the region of the rpoB gene at codon 445 (C445T) in the Pre-XDR or XDR-TB patients were significantly 20.6 times more prevalent among the MDR-TB patients. The inhA gene mutation at codon 114 (T114G) was also significantly 8.1 times more prevalent. Conclusion: The findings can be used to predict the odds of Pre-XDR and XDR-TB in MDR-TB patients, as a guide for prevention and treatments.
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