Highly sensitive real-time PCR for specific detection and quantification of -0

<p><b>Copyright information:</b></p><p>Taken from "Highly sensitive real-time PCR for specific detection and quantification of "</p><p>BMC Microbiology 2006;6():2-2.</p><p>Published online 19 Jan 2006</p><p>PMCID:PMC1360083.</p><p>Copyright © 2006 Klee et al; licensee BioMed Central Ltd.</p> targeting the and sequences of . With the respective targets, 10 and 6.5 genome equivalents per reaction can be detected with a probability of 95 %

Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.

<p>Guinea pigs were inoculated with graded spore inocula of each strain by subcutaneous route in the flank (four animals per dose). The presence of pBCXO1 and PBCXO2 and the gene inactivated on pBCXO1 is specified where applicable. Results are expressed as mean lethal dose (LD50) and mean time to death in days (MTD, mean ± SD). Each experiment was performed at least twice.</p><p>Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and CA derivative strains by the subcutaneous route in guinea pigs.</p

<i>B. cereus</i> bv <i>anthracis</i> and <i>B. anthracis</i> strains used in this study.

<p><i>atxA</i>*: Inactive <i>atxA</i> through spontaneous mutations; <i>Ba</i>: <i>B</i>. <i>anthracis</i></p><p>**Kindly provided by Wolfgang Beyer, Hohenheim University, Stuttgart, Germany.</p><p><i>B. cereus</i> bv <i>anthracis</i> and <i>B. anthracis</i> strains used in this study.</p

Oligonucleotides for expression analysis used in this study.

<p>Oligonucleotides for expression analysis used in this study.</p

The <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain expresses a PDGA and a HA capsule, and toxins.

<p><b>(A)</b> Capsule expression in the CAP(Δ<i>pagA</i>), the CAR and the CAR-H(Δ<i>hasA</i>) strains in inducing conditions; the polyglutamate (PDGA) and hyaluronic acid (HA) capsule was visualised by immunofluorescence with a polyclonal anti-PDGA immune serum or by India ink staining; degradation of the HA capsule was achieved by incubation with hyaluronidase as described in the Materials and Methods section. (<b>B)</b> The production of toxin components PA and LF in overnight bacterial culture supernatants was determined by western blot with or without CO₂/bicarbonate as described in the Materials and Methods section.</p

Local role of the HA capsule and <i>in vivo</i> dissemination of the <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain during intranasal infection in mice.

<p>Mice were inoculated intranasally with spores of <b>(A)</b> CARP-<i>lux</i> (13 mice, inoculum 1 x 10⁸), <b>(B)</b> CAR-<i>lux</i> (16 mice, inoculum 1 x 10⁶; all mice died), or <b>(C)</b> CAP-<i>lux</i> (14 mice, inoculum 1 x 10⁶; all mice died) strains and bioluminescence was analysed at the indicated times after infection as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003455#pntd.0003455.g005" target="_blank">Fig. 5</a> (D: days). (<b>Ab-e)</b> Histological characterisation of the infected brain tissue shown in <b>Aa</b>, bottom panel; <b>(Ab)</b> Diffuse inflammatory lesion centred on leptomeninges (LM), multifocally extending to the brain parenchyma (star); <b>(Ac)</b> high density of bacteria in the leptomeninges and Virchow-Robin spaces; <b>(Ad)</b> inflammatory infiltrates consisting of neutrophils, haemorrhages and oedema provoking a marked distension of leptomeninges and <b>(Ae),</b> at higher magnification, extending to the cerebral parenchyma with the presence of bacteria in the neuropil (arrowhead) highly suggestive of a rupture of the blood-brain barrier. <b>(Ab & d)</b>: HE staining; <b>(Ac & e)</b>: Gram staining.</p

Ultrastructural analysis of the capsules of the <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain and its derivatives.

<p>Bacterial cells from the PDGA and HA capsule-expressing CA strain and its derivatives expressing a HA capsule (CAR), a PDGA capsule (CA-H(Δ<i>hasA</i>)) or no capsule (CAR-H(Δ<i>hasA</i>)) were prepared for Transmission Electron Microscopy as described in the Materials and Methods section. Scale bar: 500nm</p

Characterisation of the spontaneous mutations in AtxA.

<p>*The AtxA domains were defined according to (Hammerstrom <i>et al</i>., 2011) WH, Winged Helix-turn-helix; HTH, Mga-like Helix-Turn-helix; PRD, PhosphoTransferase Regulation Domain.</p><p>**SNP, Single Nucleotide Polymorphism; INS, Insertion; DEL, deletion; VNTR, Variable Number Tandem Repeat</p><p>^§AA, amino Acid</p><p>Characterisation of the spontaneous mutations in AtxA.</p

<i>In vivo</i> dissemination of the <i>B</i>. <i>cereus</i> bv <i>anthracis</i> CA strain during cutaneous infection in mice.

<p>Mice were inoculated into the ear pinna with spores of <b>(A)</b> CARP-<i>lux</i> (9 mice, inoculum 1 x 10⁷; all mice survived), <b>(B)</b> CAR-<i>lux</i> (8 mice, inoculum 1 x 10⁵; all mice died), or <b>(C)</b> CAP-<i>lux</i> (11 mice, inoculum 1 x 10⁵; all mice died) strains and bioluminescence was analysed at the indicated times after infection (D: days). These image series show a representative dorsal and ventral view of the same mouse for the various strains. Black and white photographs are overlaid with false-colour representation of luminescence intensity expressed in photons s -1 cm² sr -1.</p

Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and the CA derivative strains in mice.

<p>Mice were inoculated with graded spore inocula of each strain in the flank or by the intranasal route (six animals per dose). The following information is specified where applicable: (i) the presence of pBCXO1, PBCXO2 for the CI and CA-derived strains, and pXO1, pXO2 for the <i>B</i>. <i>anthracis (Ba)</i> Sterne 7702 and wild-type 9602 strains; (ii) the gene inactivated on pBCXO1; and iv) the virulence factors expressed—lethal and edema toxins (Tox), hyaluronic acid capsule (HA) and polyglutamic acid capsule (PDGA)—is indicated for each mutant. Results are expressed as mean lethal dose (LD50) and mean time to death in days (MTD, mean ± SD). Each experiment was performed at least twice. The asterisk denotes absence of mortality at the highest inoculum tested. NA, not applicable.</p><p>Virulence of the <i>Bacillus cereus</i> bv <i>anthracis</i> and the CA derivative strains in mice.</p