Fitness of CFT073 capsule (<i>kpsD</i>) and O antigen (<i>waaL</i>) mutants during <i>in vivo</i> competition with CFT073 WT and corresponding complemented (<i>kpsD</i>^C, <i>waaL</i>^C) strains.

<p>C57B/L6 mice were infected transurethally with a 1∶1 mixture of CFT073<i>kpsD</i> and CFT073 WT or CFT073<i>kpsD</i>^C; CFT073<i>waaL</i> and CFT073 WT or CFT073<i>waaL</i>^C. (A) Each marker represents LOG₁₀ total CFU recovered from each mouse per 0.1 g of bladder tissue. Lines connect data points for the same mouse, and horizontal bars represent median values. (B) Each marker represents the LOG₁₀ competitive index calculated for each individual mouse; competitive indices are the ratio of the mutant 0.1 g of bladder tissue to that of WT or complemented strain. Dashed lines represent hypothetical competitive index of 1 (LOG₁₀1 = 0), which indicates no difference in fitness between the two strains. Horizontal bars represent group medians, and each competition group had 6 to 8 mice. All mutants were significantly outcompeted by CFT073 WT or their respective complemented strains for bladder colonization (<i>P</i><0.05). The O6 antigen was significantly more important than the K2 capsular antigen for bladder colonization (<i>P<</i>0.05).</p

Effect of <i>fis</i> and <i>hns</i> double deletion on <i>sslE</i> expression in UTI89 determined via western blot and qRT-PCR analysis.

<p>(A) Western blot analysis of SslE using (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>hns</i>, UTI89<i>fis</i> and UTI89<i>fis hns</i>. (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. The overall level of SslE was reduced in UTI89<i>fis hns</i> compared to wild-type UTI89. (B) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>hns</i>, UTI89<i>fis</i> and UTI89<i>fis hns</i>. All mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation.</p

Effect of <i>fis</i> and <i>typA</i> deletion in UTI89 on <i>sslE</i> expression determined via western blot and qRT-PCR analysis

<p>(A) Western blot analysis of SslE using (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>fis</i> and UTI89<i>fis</i>(pFis). (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. (B) Western blot analysis of SslE using whole-cell lysates and supernatant fractions prepared from UTI89, UTI89<i>typA</i> and UTI89<i>typA</i>(pTypA). (ii) Western blot loading control for whole cell lysate samples using an OmpA antibody. (C) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>fis</i> and UTI89<i>fis</i>(pFis); mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. (D) Relative fold-difference of <i>sslE</i> transcript levels of UTI89, UTI89<i>typA</i> and UTI89<i>typA</i>(pTypA); mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p

Table_3_The Performance of an Oral Microbiome Biomarker Panel in Predicting Oral Cavity and Oropharyngeal Cancers.XLSX

<p>The oral microbiome can play a role in the instigation and progression of oral diseases that can manifest into other systemic conditions. These associations encourage the exploration of oral dysbiosis leading to the pathogenesis of cancers. In this study, oral rinse was used to characterize the oral microbiome fluctuation associated with oral cavity cancer (OCC) and oropharyngeal cancers (OPC). The study cohort consists of normal healthy controls (n = 10, between 20 and 30 years of age; n = 10, above 50 years of age), high-risk individuals (n = 11, above 50 years of age with bad oral hygiene and/or oral diseases) and OCC and OPC patients (n = 31, HPV-positive; n = 21, HPV-negative). Oral rinse samples were analyzed using 16S rRNA gene amplicon sequencing on the MiSeq platform. Kruskal–Wallis rank test was used to identify genera associated with OCC and OPC. A logistic regression analysis was carried out to determine the performance of these genera as a biomarker panel to predict OCC and OPC. In addition, a two-fold cross-validation with a bootstrap procedure was carried out in R to investigate how well the panel would perform in an emulated clinical scenario. Our data indicate that the oral microbiome is able to predict the presence of OCC and OPC with sensitivity and specificity of 100 and 90%, respectively. With further validation, the panel could potentially be implemented into clinical diagnostic and prognostic workflows for OCC and OPC.</p

Schematic representation of the mutagenesis strategy utilizing <i>rpsL</i> counter selection.

<p>A 100(shown as patterned region in WT sequence) was selected for the <i>kpsD</i> and <i>waaL</i>; Strep^R strains harboring the pRedET plasmid were transformed with PCR product consisting of the <i>rpsL-neo</i> cassette flanked by 50 bp regions homologous to sequences on either side of the chosen point of insertion; double cross-over events during homologous recombination make the mutant strains Strep^S Kan^R due to integration of the <i>rpsL-neo</i> cassette; for complementation, mutant strains were transformed with 100 bp oligonucleotides bearing the same sequence as the WT across the region of insertion; complemented strains were again Strep^R due to loss of the <i>rpsL-neo</i> cassette and restoration of the parental genotype; streptomycin resistant (Strep^R), streptomycin sensitive (Strep^S), kanamycin resistant (Kan^R). The methodology depicted in this figure is a modification of the <i>rpsL</i> counter-selection technique previously used for site-directed mutagenesis in bacterial artificial chromosomes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094786#pone.0094786-Bird1" target="_blank">[57]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094786#pone.0094786-White1" target="_blank">[59]</a>.</p

Effect of <i>fis</i> deletion on <i>sslE</i> expression in strains IHE3034, 536 and EC958 determined via western blot and qRT-PCR analysis.

<p>Western blot analysis of SslE using preparations from (A) IHE3034, (B) 536 and (C) EC958; each with their respective <i>fis</i> mutant and complemented strains. In each analysis, (i) whole-cell lysates and (iii) supernatant fractions were examined. In addition, (ii) a control for whole cell lysate samples was performed using an OmpA antibody. (D) Relative fold-difference of <i>sslE</i> transcript levels of IHE3034, 536 and EC958, and their respective <i>fis</i> mutant and complemented strains. All mRNA levels were calculated relative to the level of IHE3034 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p

UPEC survival in human serum.

<p>Bacteria (approximately 4×10⁸ CFU/ml) were incubated in fresh human serum for 90 min and colony counts were used to determine serum sensitivity index (SSI) for each strain, shown at the top of the graph. SSI of 0 represents a resistant strain; 1,2,3,4,5 represent equivalent reduction in log values of colony forming units (CFU/ml) post incubation with serum. The WT strains CFT073, 1177 and RS218 were resistant; CFT073<i>waaL</i>, 1177<i>waaL</i> and RS218<i>waaL</i> mutants were all highly sensitive to human serum (SSI; <i>P</i><0.05, paired t test) and the complemented strains (<i>waaL</i>^C) were resistant. CFT073<i>kpsD</i> and RS218<i>kpsD</i> mutants displayed reduced sensitivity (SSI = 2 and 1, respectively); complemented strains (<i>kpsD</i>^C) were resistant. The 1177<i>kpsD</i> mutant was completely resistant to serum killing.</p

Locus positions of chromosome-borne CU fimbrial operons relative to the genome of <i>E.coli</i> MG1655.

<p>The <i>E. coli</i> K-12 MG1655 chromosome (outer black ring) was used as a reference map to visualise the locus position of 30 chromosome-borne CU fimbrial types. Types highlighted in blue are present in <i>E. coli</i> K-12 MG1655, types in red are absent in this strain. Fimbrial types associated with PAIs are indicated by an asterisk. A number of PAI associated fimbrial gene clusters occupy different locus positions relative to the MG1655 genome. tRNA sites that flank CU-containing PAIs are indicated on the inner blue ring.</p

Western blot and qRT-PCR analysis to examine <i>sslE</i> expression in ExPEC strains.

<p>(A) Western blot analysis of SslE from (i) whole-cell lysates and (iii) supernatant fractions prepared from UTI89, UTI89<i>sslE</i>, IHE3034, IHE3034<i>sslE</i>, 536, EC958 and MG1655. SslE has a predicted size of approximately 167 kDa. Two major cross-reacting bands of this size were detected (indicated by arrows), possibly representing full-length and processed SslE. No cross-reacting bands were detected in samples prepared from UTI89<i>sslE</i> and IHE3034<i>sslE</i>, demonstrating the specificity of antibody. (ii) Loading control for whole cell lysate samples. The same samples used above were examined by western blot using an OmpA antibody. Similar levels of OmpA were detected in all samples, indicating equivalent loading of total protein. (B) Relative fold-difference of <i>sslE</i> transcript levels of ExPEC strains UTI89, IHE3034, 536, EC958 as determined by qRT-PCR. All mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA. The relative <i>sslE</i> mRNA levels were consistent with the data observed from the western blot analysis. The data was obtained from three independent experiments; error bars indicate standard deviation.</p

Effect of <i>hns</i> deletion on <i>sslE</i> transcription in UTI89 and EC958 at 22°C and 37°C via qRT-PCR analysis.

<p>Relative fold-difference of <i>sslE</i> transcript levels of UTI89 and EC958 with their respective <i>hns</i> mutant and complemented strains grown at (A) 22°C (black bars) and (B) 37°C (grey bars). All mRNA levels were calculated relative to the level of UTI89 <i>sslE</i> mRNA at the respective temperature. The data was obtained from three independent experiments; error bars indicate standard deviation. Statistical analysis was performed using an unpaired, two-tailed t-test.</p