Summary of BAC clones made from human Saliva DNA recovered from functional-based screens.

1<p>Accession numbers for the clone sequences are: AMP4 (KF982313), AMP5 (KF982314), AMP7 (KF982315), SUL3 (KF982316), SUL5 (KF982317), SUL6 (KF982318), SUL8 (KF982319), SUL9 (KF982320), SUL10 (KF982321), SUL11 (KF982322), SUL15 (KF982323), and SUL20 (KF982324).</p>2<p>ORF prediction by RAST server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Aziz1" target="_blank">[27]</a>.</p>3<p>Amp^I = intermediate ampicillin resistance; Sul^R = resistant to sulphonamide compounds; Sul^RS = reduced susceptibility to sulphonamide compounds compared to EPI300; Sxt^R = resistant to trimethoprim/sulphamethoxazole 1∶19; Sxt^RS = reduced susceptibility to trimethoprim/sulphamethoxazole 1∶19 compounds compared to <i>E. coli</i> EPI300.</p

Alignment of the DHPS amino acid sequences from sulphonamide resistant BAC clones and representative DHPS sequences.

<p>The numbering above the alignment is based on the DHPS sequence of the <i>N. meningitidis</i> strain BT054 and amino acids identical to this sequence are indicated by a dot. Gaps are indicated by a hyphen. Amino acids discussed in the text are indicated by an asterisk above the numbering. SUL-R = sulphonamide resistant; SUL-S = sulphonamide susceptible; SUL-RS = reduced susceptibility to sulphonamide. The nucleotide accession number and reference for the representative DHPS sequences used in the alignments are: <i>N. meningitidis</i> BT054 (X68067; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Radstrom1" target="_blank">[54]</a>), <i>N. meningitidis</i> MO035 (X68062; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Radstrom1" target="_blank">[54]</a>), <i>N. meningitidis</i> NM419 (AY722006; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Fiebelkorn1" target="_blank">[39]</a>), <i>N. subflava</i> NJ9703 (ACEO02000001; direct submission), <i>V. parvula</i> Te3^T (CP001820; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Gronow1" target="_blank">[49]</a>), <i>S. pneumoniae</i> 708 (U16156; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Lopez1" target="_blank">[55]</a>), <i>S. pneumoniae</i> WA-152 (AJ311336; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Haasum1" target="_blank">[41]</a>), <i>S. pyogenes</i> G1 (AJ000686; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Swedberg1" target="_blank">[40]</a>), <i>S. pyogenes</i> G56 (AJ000685; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086428#pone.0086428-Swedberg1" target="_blank">[40]</a>), and <i>S. infantis</i> SK1302 (NZ_AEDY01000064; direct submission).</p

Difference in melanisation of <i>Galleria mellonella</i> following infection with <i>E. coli</i> isolates.

<p>Images of <i>Galleria mellonella</i> infected with ARD1257 and ARD1258, demonstrating the melanisation that occurred following infection with ARD1257.</p

Gel picture showing the electrophoretic separation of plasmids.

<p>The plasmids carried by ARD1257 (B), ARD1258 (C), and ARD1258C (D) migrating in 0.8% agarose, for 270 min at 150 v, 4°C, are shown. Lane A shows the reference plasmid bands from strain 39R861. The reference plasmid bands are from top to bottom; 148 kb, 63 kb, 36 kb, genomic DNA band, 6.8 kb. Lane A–C were run on a separate occasion to lane D but under identical run conditions with a reference plasmid.</p

Compounds identified using the Omnilog Phenotype Microarray (Biolog), where a significant difference in respiration, denoted by AUC values, were detected between ARD1257 and ARD1258.

<p>T-Test performed to compare AUC values for ARD1257 and wild-type ARD1258.</p

Growth curves in LB media.

<p>The growth curves for ARD1258, ARD1257, ARD1258C and MG1655 determined over 24°C in LB media are shown. The absorbance in corrected for the blank and represents the mean of triplicate wells from three individual experiments; error bars indicate a 95% confidence interval.</p

Survival of <i>Galleria mellonella</i> with <i>E. coli</i> isolates.

<p>The mean percentage survival rate of <i>Galleria mellonella</i> infected with different <i>E. coli</i> strains after 24 h. Error bars indicate the Standard deviation from replicate experiments.</p

Virulence determinants microarrays data for 106 <i>Salmonella</i> strains analysed.

<p>At the top, the analysed genes are grouped according to their particular genomic location or function (fimbrial). The order of strains represents their relatedness according to the UPGMA dendrogram type performed in BioNumerics 5.1. The hybridization result of a distinct strain is shown by row. A white box indicates the absence and a black box indicates the presence of the target sequence in the strain.</p

Adhesion and invasion of HT-29 cells by control strain <i>S</i>. Typhimurium LT2, <i>S</i>. Infantis Sal147, and <i>S</i>. Infantis Sal280.

<p>Data shown are means ± SEM from four independent experiments.</p

Table_5_Identification of a New Antimicrobial Resistance Gene Provides Fresh Insights Into Pleuromutilin Resistance in Brachyspira hyodysenteriae, Aetiological Agent of Swine Dysentery.XLSX

<p>Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.</p