Involvement of the Efflux Pumps in Chloramphenicol Selected Strains of Burkholderia thailandensis: Proteomic and Mechanistic Evidence

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections

Analyses of the detergent-insoluble membrane proteins of chloramphenicol-resistant variants.

<p>SDS-PAGE analysis was performed on ATCC700388 and PHLSE082, two wild-type strains of <i>B. thailandensis</i>, and on 64CM16 and 132CM128, their respectively chloramphenicol resistant derivative strains. Proteins were stained with Coomassie blue. The variants presented different additional bands at around 51 kDa (band A), 48 kDa (band B), 43 kDa (bands C and band E) and at 95 kDa (band D). Molecular weight standards are indicated in kilodaltons.</p

Identification of two drug efflux pumps overproduced in the resistant variants.

<p>Identification of two drug efflux pumps overproduced in the resistant variants.</p

Effects of PAβN on antibiotic susceptibility of the <i>B. thailandensis</i> strains.

<p>Abbreviations: PAβN, phenylalanine-arginine β-naphthylamide; CM, chloramphenicol; NAL, nalidixic acid; CLO, cloxacillin.</p

Antibiotic susceptibility of the <i>B. thailandensis</i> strains.

<p>Antimicrobial agent abbreviations: CM, chloramphenicol; NAL, nalidixic acid; NFX, norfloxacin; CIP, ciprofloxacin; FOX, cefoxitin; CAZ, ceftazidime; OXA, oxacillin; CLO, cloxacillin; PIP, piperacillin; IMI, imipenem; ERY, erythromycin; TC, tetracycline; DC, doxycycline; TP, trimethoprim; TS, trimethoprim/sulfamethoxazole; POL B, polymyxin B. Values are means of three independent determinations.</p

A spontaneous mutation in <i>kdsD</i>, a biosynthesis gene for 3 Deoxy-_D-<i>manno</i>-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of <i>Francisella tularensis</i> and caused a high level of attenuation in murine models of tularemia

<div><p><i>Francisella tularensis</i>, a gram–negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, <i>F</i>. <i>tularensis</i> subspecies <i>tularensis</i> is considered a potential biowarfare agent. Due to its <i>in vitro</i> efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of <i>F</i>. <i>tularensis</i> by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within <i>kdsD</i> which encodes for an enzyme necessary for the production of 3-Deoxy-_D-<i>manno</i>-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A <i>kdsD</i> mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the <i>kdsD</i> mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for <i>Francisella</i> virulence and may be an attractive therapeutic target for developing novel medical countermeasures.</p></div

Pathology of mice challenged intranasally with <i>F</i>. <i>tularensis</i> 5 days post-challenge.

<p>Mice were challenged intranasally with Schu S4 WT (131 CFU), CipR mutant (1,750 CFU), or <i>kdsD</i>::<i>ltrB</i>_<i>L1</i> (6,000 CFU). Also included as a control for these studies were mice receiving PBS alone by intranasal administration. The strains used for challenge and HE stained organ (lung, spleen, and liver) are as indicated. All samples shown are at 5 days post-challenge when the Schu S4 challenged mice were moribund; in contrast, the mutant challenged mice displayed no clinical signs of infection at this time point. Schu S4 WT, Lung (4x)–multifocal areas of inflammation and necrosis (*); arrow indicates the inset area. Inset (40x)–necrosis admixed with inflammatory cells. Spleen (4x)–coalescing areas of necrosis (*) affecting red pulp and white pulp; arrow indicates the inset area. Inset (40x)–necrosis admixed with inflammatory cells. Liver (4x)–Single focus of necrosis (*); arrow indicates the inset area. Insert (40x)–necrosis with few inflammatory cells. CipR mutant, Lung (4x)–diffuse coalescing necrotic areas. Inset (40x)–necrosis admixed with inflammatory cells. Spleen (4x)–normal. Liver (4x)–normal. <i>kdsD</i>::<i>ltrB</i>_<i>L1</i>, Lung (4x)–few foci of necrosis (*); arrow indicates the inset area. Inset (40x)–necrosis admixed with inflammatory cells extends to the surface of the lung. Spleen (4x)–normal. Liver (4x)–normal.</p

Growth assays.

<p><i>F</i>. <i>tularensis</i> (A) or <i>F</i>. <i>novicida</i> (B) strains were grown in CDM at 37^°C with (empty) or without (filled) the presence of A5P at a concentration of 400 μM. Growth was monitored by optical density. OD measurements were based upon quadruplicate samples and bars represent standard error of the mean. These data represent at least two separate experiments. The <i>F</i>. <i>tularenesis kdsD</i>::<i>ltrB</i>_<i>L1</i> and <i>F</i>. <i>novicida kpsF</i>::T20 mutants were severely altered for growth in Chamberlain’s medium. However, the addition of A5P to the medium significantly increased the growth of these strains. In contrast, the presence of A5P did not affect the growth of the <i>F</i>. <i>tularensis</i> CipR mutant as the two lines completely overlapped.</p