Detection of <i>vapN</i> in <i>Rhodococcus equi</i> isolates cultured from humans

<div><p><i>Rhodococcus equi</i> can cause severe infections in people, particularly in immunocompromised individuals. The <i>R</i>. <i>equi</i> virulence plasmids (<i>vap</i>) encoding <i>vapA</i> and <i>vapB</i> are linked to development of infections in domestic animals. Recently, a novel virulence plasmid, <i>vapN</i>, was identified in isolates cultured from cattle, but its prevalence or significance in human <i>R</i>. <i>equi</i> infections has not been extensively studied. To determine the prevalence of <i>vapN</i> in a diverse collection of human-derived isolates from different countries, 65 <i>R</i>. <i>equi</i> isolates collected by various institutions from 1984 to 2002 were screened for the presence of <i>vapN</i> and other virulence plasmids through polymerase chain reaction (PCR) using redesigned primer sets. Of the isolates that carried plasmids, 43% (16/37) were <i>vapN</i>-positive and fewer were <i>vapB</i> or <i>vapA</i>-positive (30 and 16%, respectively). This is the first report of <i>vapN</i> carriage in <i>R</i>. <i>equi</i> isolated from human infections. One isolate (H-30) carried <i>vapN</i> but did not amplify the conjugal plasmid transfer gene <i>traA</i> associated with carriage of <i>vap</i>, which could be explained by sequence variation within the <i>traA</i> gene. Another isolate (H-55) amplified <i>traA</i>, but did not amplify <i>vapA</i>, <i>B</i>, or <i>N</i> (<i>traA</i>⁺ <i>vapABN</i>^-) with previously described primer sets or those developed for this study. The H-55 <i>traA</i> sequence had 98% identity to <i>traA</i> sequences in <i>vapA</i> plasmids, which suggests that it may carry a variant of previously characterized virulence plasmids or a novel virulence plasmid. Carriage of <i>vapN</i> in <i>R</i>. <i>equi</i> isolates derived from people is not uncommon and more research is needed to determine its significance in the epidemiology and pathogenesis of human <i>R</i>. <i>equi</i> infections.</p></div

Detection of <i>vapN</i> in <i>Rhodococcus equi</i> isolates cultured from humans - Fig 1

<p><b>Agarose gel electrophoresis comparison of previously published primer sets (A) and those designed for this study (B) for the <i>vapA</i>, <i>vapB</i>, <i>and vapN</i> genes.</b> The expected amplicon sizes for the A sets are <i>vapA</i> (286 bp), <i>vapB</i> (477 bp), and <i>vapN</i> (625 bp). Reactions for <i>vapA</i> and <i>vapB</i> were performed individually and not in multiplex as originally described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190829#pone.0190829.ref012" target="_blank">12</a>]. The human isolates depicted are H-5 (<i>traA+ vapN+ vapAB-</i>), H-30 (<i>traA- vapN+ vapAB-</i>), H-32 (<i>traA+ vapB+ vapAN-</i>), H-55 (<i>traA+ vapABN-</i>), and H-56 (<i>traA+ vapA+ vapBN-</i>). The control strains used were ATCC^® 33701^™ (<i>vapA</i>-positive), clinical porcine isolate EIDL 99–213 (<i>vapB</i>-positive), clinical canine isolate TAMU 49–33 (<i>vapN</i>-positive), and ATCC^® 6939^™ (avirulent genotype). The corresponding <i>choE</i> DNA amplification control band for each reaction is below the panels. The DNA ladder used was Ready-to-use 100 bp ladder (Biotium; Fremont, CA) with fragments from 100 to 1500 bp. Both <i>vapA</i> primer sets were specific for <i>vapA</i>. There is non-specific amplification in the non-<i>vapB-</i>positive and non-<i>vapN</i>-positive isolates with the previous primer sets. The primer sets developed for this study (B) amplified a single product.</p

Oligonucleotide primer sequences.

<p>Oligonucleotide primer sequences.</p

Expression of AJC proteins in the intestinal mucosa of control dogs (ED group) (A, D, G) and dogs treated with VSL#3 (B, E, H) or CT (C, F, I).

<p>No discernible differences in the distribution or staining intensity of E-cadherin are observed between normal mucosa (A) and IBD samples (B and C); the overall intensity of E-cadherin staining decreased from the luminal epithelium to the distal crypts. Occludin-specific labelling is most intense at the epithelial cell AJC (arrows) of the luminal epithelium covering the apical portion of villi in ED (D) and VSL#3 (E); a weak to absent expression is observed in the luminal epithelium and in some intestinal glands of the small intestine of the CT sample (F). In colonic samples belonging to ED (G) and VSL#3 (H) groups, claudin-2 is readily detectable only in the colonic crypt epithelium, decreasing in intensity from the distal to the proximal crypt and becoming barely detectable at the luminal surface of the colon. In contrast, claudin-2 expression is increased in the proximal crypt and luminal epithelium of all samples from CT dogs (I).</p

Results for histology scores, CIBDAI, CD3+ cells, FoxP3+ cells, TGF-β cells, and plasma citrulline concentrations.

<p>Significant differences between baseline (T0) and 30 days after the end of therapy (T1) were observed for all parameters in both treatment groups except the expression of FoxP3+ T-cells in the CT group (P = 0.3296). While TGF-β increased significantly in both treatment groups, the magnitude of the increase was significantly higher in dogs treated with VSL#3 (P = 0.0008). Data for CD3+ cells, FoxP3+ cells, TGF-β cells expressed as cells per 62,500 μm².</p

Summary characteristics of enrolled dogs.

<p>m = male, mn = neutered male, f = female, fs = spayed female; CT = combination therapy;</p><p>n/a-not applicable.</p

Histology of intestinal mucosa of dogs with IBD after treatment with VSL#3 (A, C, E) and CT (B, D, F).

<p>A residual inflammatory infiltrate with lymphocytic-plasmacytic cells (arrows) is evident after the therapy in both samples (H&E, 40X). In both treatment groups ssimilar patterns of mucosal infiltrations with CD3+ T-lymphocytes are evident (C and D). Infiltration with Fox-P3+ cells are proportionally increased in a sample belonging to a VSL#3 treated dog (E) compared to the sample from CT treated dog (F). Note the particular Fox-P3+ T-cells concentrations at the apical portion of villi in the VSL#3 treated dog (E) (arrow-heads) (IHC, ABC method, Harris haematoxylin nuclear counterstain, 40X).</p

Expression of AJC proteins.

<p>Mucosal biopsies were evaluated after the end of treatment (T1) either with the probiotic (VSL) or combination drug therapy (CT), and compared to archived mucosal samples from dogs euthanized for non-gastrointestinal disorders (ED). (*significantly different to the other 2 groups; line denotes median).</p

Oligonucleotides primers/probes used in this study.

a<p>Originally described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094699#pone.0094699-Walter1" target="_blank">[52]</a>.</p>b<p>Originally described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094699#pone.0094699-Heilig1" target="_blank">[53]</a>.</p

Median and range proportion of foals with sequences detected in the fecal DNA of rectal swab samples (Phylum, class, order, and family).

<p>Fecal swab samples collected from 37 Quarter Horse foals on days 2 and 30 of life. *P values represent the results of McNemar’s test for paired dichotomous data, adjusted by the method of Hochberg. NP  =  Not Performed.</p