IgA and IgG serum antibody responses to CS5 and CS6 in individual patients.

<p>The responses to CS5 (broken lines) and CS6 (solid lines) were measured by ELISA in sera from patients infected with strains E1777, E1785 and E1779, on different days after hospitalization. Serum from the patient infected with strain E2265 was not available.</p

Levels of <i>csfD</i> and <i>cssB</i> transcription compared to in LB alone after one hour of culture.

<p>Level of <i>csfD</i> (A) and <i>cssB</i> (B) transcription in LB supplemented with crude bile or individual bile salts standardized to the level of transcription in LB medium alone, after one hour of culture. Transcription was measured by reverse transcriptase real time PCR. Bars show means and standard errors of the means of three separate experiments (strains E1785, E2265, and E3003, respectively). *, P = <0.05</p

Primers used for PCR and real-time Reverse Transcriptase PCR (rt RT-PCR).

a<p>CS5 minor subunit<b>;</b> GenBank accession number <u>AJ224079</u>.</p>b<p>CS6 structural subunit CssB; GenBank accession number <u>UO4846</u>.</p>c<p>Primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035827#pone.0035827-Nicklasson1" target="_blank">[44]</a>.</p>d<p><i>E. coli</i> D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH); GenBank accession number <u>U014639.1.</u></p

Transcriptional <i>csfD</i>:<i>ccsB</i> ratio <i>in vivo</i> and <i>in vitro</i>.

<p>Transcription of <i>csfD</i> and <i>cssB</i> was quantified by real time reverse transcriptase PCR (rt RT-PCR) in <i>in vivo</i> bacterial samples (strains E1777, E1785, E2265; closed bar) and after one (strains E1785, E2265, E3003; open bars) and two (E1777, E1779; hatched bars) hours of <i>in vitro</i> culture in LB medium alone and in LB supplemented with 0.15% crude bile or 0.2% individual bile salts. Bars show the means and standard errors of the means of two (after two hours of culture) or three (<i>in vivo</i> and after one hour of culture) separate experiments.</p

Strains used in the study and results from culturing^a of clinical stool specimens.

a<p>Culture was performed on MacConkey agar plates to detect <i>E. coli</i> and on Taurocholate-tellurite-gelatin agar (TTGA) for detection of vibrios; all cultures were performed overnight at 37°C.</p>b<p>Department of Microbiology and Immunology, University of Gothenburg, enterotoxigenic <i>Escherichia coli</i> (ETEC) strain collection number.</p

Phenotypic CS5 levels in LB alone and LB supplemented with bile or individual bile salts.

<p>CS5 expression was quantified by inhibition ELISA after overnight culture to stationary phase of strains E1777, E1779, E1785, E2265, and E3003. CS5 surface expression tifwas induced by NaGCH, but not by the corresponding unconjugated bile salt NaCH or its tauro-conjugated counterpart TCA (representative data).</p

Dose-dependent induction of phenotypic CS5 expression by NaGCH.

<p>Expression of CS5 was determined by inhibition ELISA in strain E1777 after overnight culture to stationary phase in LB medium supplemented with NaGCH or crude bile. Bars indicate means and standard errors of the means of two measurements in one experiment.</p

Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray

<div><p>Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarray's effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of <i>Acinetobacter baumannii</i>, <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of β-lactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ∼25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and –negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens.</p></div

ARDM content allocations for genes conferring resistance to different classes of antimicrobial compounds.

<p>Two hundred thirty-nine different resistance determinants are represented by probes on each sub-array.</p

ARDM v.1, PCR and phenotypic β-lactamase/ESBL profiles of the clinical and reference strains used in this study.

<p>N/A – not available. R – resistant, I – intermediate, S – sensitive.</p>a<p>The identified family to which the CTX-M allele belongs is indicated in parentheses.</p>b<p>While no official ATM resistance criteria for <i>A. baumannii</i> are available, all isolates of this species designated as resistant (R) in the above table showed no zone of inhibition around ATM disks.</p>c<p>Weakly positive detection by PCR.</p