Characterization of genes encoding for acquired bacitracin resistance in Clostridium perfringens.

Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC(90) (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC(50) was higher in turkey isolates (6 µg/ml) than in chicken isolates (3 µg/ml). Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml) and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens

Electrografted P4VP for High Aspect Ratio Copper TSV Insulation in Via-Last Process Flow

International audienceVia-Last metallization of High Aspect Ratio Through Silicon Via (HAR TSV) for 3D integration is challenging. Indeed, the formation of a uniform and conformal dielectric to insulate HAR TSVs in Via-Last process flow is difficult to achieve for any physical deposition process. In this study, we present the first reported HAR copper TSVs, insulated by highly conformal electrografted poly-4-vinylpyridine (P4VP) in Via-Last process-flow. As a demonstration, this allowed the metallization of HAR copper TSVs for die-to-die 3D integration of a 22 × 22 photodiode array tier onto a CMOS control electronics ASIC

Bacitracin MIC values (µg/mL) of <i>C. perfringens</i> strain c1261_A grown with different concentrations of efflux pump inhibitors.

<p>Blank area, concentrations not tested; –, no bacterial growth due to efflux pump inhibitors.</p

Comparison between the <i>C. perfringens bcrABDR</i> genes, the <i>E. faecalis bcrABD</i> operon and the <i>B. licheniformis bcrABC</i> operon.

<p>Organization of <i>C. perfringens</i> c1261_A resistance genes <i>bcrA, bcrB, bcrD,</i> and <i>bcrR</i> and comparison with the <i>bcrABD</i> operon of <i>E. faecalis</i> ARO1/DCVS (AY496968) and the <i>bcrABC</i> operon of <i>B. licheniformis</i> ATCC 9945A (L20573). Open arrows indicate ORFs. The amino acid percentages indicated relate to the identity between the amino acid sequences of the proteins encoded by the ORFs. The <i>bcrABC</i> operon of <i>B. licheniformis</i> is inverted to facilitate the comparison.</p

PFGE and hybridization analysis of I-CeuI and MluI double-digested DNA of the bacitracin resistant <i>C. perfringens</i> strain c1261_A.

<p>PFGE analysis of <i>C. perfringens</i> strain c1261_A total DNA (A). Southern blot of <i>C. perfringens</i> isolate c1261_A total DNA probed with <i>rrn</i> (B) and with <i>bcrB</i> (C). Sizes (in kilobases) are indicated on the left.</p

Expression and cotranscription of <i>bcrABDR</i> genes in presence of bacitracin.

<p>A) Semi-quantitative RT-PCR analysis of strain c1261_A grown in various concentrations of bacitracin. Expression of 16S rRNA gene (<i>rrn</i>) was used as a control. B) Amplification of intergenic regions by RT-PCR of strain c1261_A grown in presence of 256 µg/mL of bacitracin. Lane L, DNA ladder; Lane 1, <i>bcrA</i>; Lane 2, <i>bcrB</i>; Lane 3, <i>bcrD</i>; Lane 4, <i>bcrR</i>; Lane 5, <i>bcrD</i>/<i>bcrB</i> intergenic region; Lane 6, <i>bcrB</i>/<i>bcrA</i> intergenic region; Lane 7, <i>bcrA</i>/<i>bcrR</i> intergenic region.</p