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Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor.

ABSTRACT: Burkholderia pseudomallei is the causative agent of melioidosis, a serious infection associated with high mortality and relapse. Current antimicrobial therapy using ceftazidime (CAZ) is often ineffective. Inhibitors of LpxC, the enzyme responsible for lipid A biosynthesis, have potential antimicrobial activity against several Gram-negative bacteria in vivo, but their activity against B. pseudomallei is unclear. Herein, we investigated the susceptibility of B. pseudomallei clinical isolates to LpxC-4, an LpxC inhibitor, and LpxC-4 in combination with CAZ. Time-kill assays for bactericidal activity were conducted for B. pseudomallei K96243, revealing growth inhibition and bactericidal effect at LpxC-4 concentrations of 2 ?g/mL and 4 ?g/mL, respectively. No significant synergistic effect was observed with the combination of LpxC-4 and CAZ. LpxC-4 susceptibility was tested on three groups of clinical isolates:1) CAZ- and trimethoprim-sulfamethoxazole (SXT)-susceptible (N = 71), 2) CAZ-resistant (N = 14), and 3) SXT-resistant (N = 23) isolates, by broth microdilution. The minimum concentration of LpxC-4 required to inhibit the growth of 90% of organisms was 2 ?g/mL for all isolates. The median minimum inhibitory concentration of both the CAZ/SXT-susceptible and CAZ-resistant groups was 1 ?g/mL (interquartile range [IQR] = 1-2 ?g/mL), compared with 2 ?g/mL (IQR = 2-4 ?g/mL) for the SXT-resistant group. Cell morphology was observed after drug exposure by immunofluorescent staining, and a change from rod-shaped to cell wall-defective spherical cells was observed in surviving bacteria. LpxC-4 is a potent bactericidal agent against B. pseudomallei and warrants further testing as a new antibiotic to treat melioidosis.

SUBMITTER: Sengyee S

PROVIDER: S-EPMC5508901 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor.

Sengyee Sineenart S Saiprom Natnaree N Paksanont Suporn S Limmathurotsakul Direk D Wuthiekanun Vanaporn V Chantratita Narisara N

The American journal of tropical medicine and hygiene 20170701 1

Burkholderia pseudomallei is the causative agent of melioidosis, a serious infection associated with high mortality and relapse. Current antimicrobial therapy using ceftazidime (CAZ) is often ineffective. Inhibitors of LpxC, the enzyme responsible for lipid A biosynthesis, have potential antimicrobial activity against several Gram-negative bacteria in vivo, but their activity against B. pseudomallei is unclear. Herein, we investigated the susceptibility of B. pseudomallei cl ...[more]

PMID: 28719324

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Project description:BackgroundMelioidosis is a neglected tropical disease with rising global public health and clinical importance. Melioidosis is endemic in Southeast Asia and Northern Australia and is of increasing concern in Malaysia. Despite a number of reported studies from Malaysia, these reports are limited to certain parts of the country and do not provide a cohesive link between epidemiology of melioidosis cases and the nation-wide distribution of the causative agent Burkholderia pseudomallei.Methodology/principle findingsHere we report on the distribution of B. pseudomallei sequence types (STs) in Malaysia and how the STs are related to STs globally. We obtained 84 culture-confirmed B. pseudomallei from confirmed septicaemic melioidosis patients from all over Malaysia. Prior to performing Multi Locus Sequence Typing, the isolates were subjected to antimicrobial susceptibility testing and detection of the YLF/BTFC genes and BimA allele. Up to 90.5% of the isolates were sensitive to all antimicrobials tested while resistance was observed for antimicrobials typically administered during the eradication stage of treatment. YLF gene cluster and bimABp allele variant were detected in all the isolates. The epidemiological distribution patterns of the Malaysian B. pseudomallei isolates were analysed in silico using phylogenetic tools and compared to Southeast Asian and world-wide isolates. Genotyping of the 84 Malaysian B. pseudomallei isolates revealed 29 different STs of which 6 (7.1%) were novel. ST50 was identified as the group founder followed by subgroup founders ST376, ST211 and ST84. A low-level diversity is noted for the B. pseudomallei isolates described in this study while phylogenetic analysis associated the Malaysian STs to Southeast Asian isolates especially isolates from Thailand. Further analysis also showed a strong association that implicates agriculture and domestication activities as high-risk routes of infection.Conclusions/significanceIn conclusion, MLST analysis of B. pseudomallei clinical isolates from all states in Malaysia revealed low diversity and a close association to Southeast Asian isolates.

Project description:Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a major community-acquired infection in tropical regions. Melioidosis presents with a range of clinical symptoms, is often characterized by a robust inflammatory response, may relapse after treatment, and results in high mortality rates. Lipopolysaccharide (LPS) of B. pseudomallei is a potent immunostimulatory molecule comprised of lipid A, core, and O-polysaccharide (OPS) components. Four B. pseudomallei LPS types have been described based on SDS-PAGE patterns that represent the difference of OPS-type A, type B, type B2 and rough LPS. The majority of B. pseudomallei isolates are type A. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) and gas chromatography to characterize the lipid A of B. pseudomallei within LPS type A isolates. We determined that B. pseudomallei lipid A is represented by penta- and tetra-acylated species modified with 4-amino-4-deoxy-arabinose (Ara4N). The MALDI-TOF profiles from 171 clinical B. pseudomallei isolates, including 68 paired primary and relapse isolates and 35 within-host isolates were similar. We did not observe lipid A structural changes when the bacteria were cultured in different growth conditions. Dose-dependent NF-?B activation in HEK cells expressing TLR4 was observed using multiple heat-killed B. pseudomallei isolates and corresponding purified LPS. We demonstrated that TLR4-dependent NF-?B activation induced by heat-killed bacteria or LPS prepared from OPS deficient mutant was significantly greater than those induced by wild type B. pseudomallei. These findings suggest that the structure of B. pseudomallei lipid A is highly conserved in a wide variety of clinical and environmental circumstances but that the presence of OPS may modulate LPS-driven innate immune responses in melioidosis.

Dataset Information

Susceptibility of Clinical Isolates of Burkholderia pseudomallei to a Lipid A Biosynthesis Inhibitor.

Publications

Susceptibility of Clinical Isolates of <i>Burkholderia pseudomallei</i> to a Lipid A Biosynthesis Inhibitor.

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