Alignment of <i>phsA</i> sequences in 21 <i>S</i>. Choleraesuis.

<p>The deletion of G at position 760 resulted in a frame-shift mutation. The first sequence is H₂S-positive <i>S</i>. Choleraesuis strain SC-B67 (NC_006905.1).</p

The methodology of <i>Salmonella</i> detection procedure.

<p>The methodology of <i>Salmonella</i> detection procedure.</p

Dendrogram displaying the PFGE profiles of the 43 isolates.

<p>The strain number, origin, source, sequence type (ST), and H₂S phenotype are shown for each strain. +, H₂S-producing isolate; −, non-H₂S-producing isolate.</p

Sequence alignment of the <i>phs</i> gene and the protein.

<p>A nonsense mutation at position 208 of the <i>phsA</i> gene results in the replacement of a sense codon (CAG) with a termination codon (UAG) leading to the premature termination of <i>phsA</i>. The first sequence, <i>phsA</i>, is based on <i>S</i>. enterica serotype Typhimurium strain LT2 (GenBank AE006468). *, termination codon; +, H₂S-producing isolate; −, non-H₂S-producing isolate.</p

Phylogenetic relationships of all <i>S</i>. Choleraesuis STs from MLST database by eBURST analysis.

<p>All <i>S</i>. Choleraesuis STs were divide into three clonal complexes. The blue solid circle represents the founder clonal complex. The red lines indicate SLVs between STs.</p

Antimicrobial Resistance and Molecular Investigation of H₂S-Negative <i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Choleraesuis Isolates in China

<div><p><i>Salmonella enterica</i> subsp. <i>enterica</i> serovar Choleraesuis is a highly invasive pathogen of swine that frequently causes serious outbreaks, in particular in Asia, and can also cause severe invasive disease in humans. In this study, 21 <i>S</i>. Choleraesuis isolates, detected from 21 patients with diarrhea in China between 2010 and 2011, were found to include 19 H₂S-negative <i>S</i>. Choleraesuis isolates and two H₂S-positive isolates. This is the first report of H₂S-negative <i>S</i>. Choleraesuis isolated from humans. The majority of H₂S-negative isolates exhibited high resistance to ampicillin, chloramphenicol, gentamicin, tetracycline, ticarcillin, and trimethoprim-sulfamethoxazole, but only six isolates were resistant to norfloxacin. In contrast, all of the isolates were sensitive to cephalosporins. Fifteen isolates were found to be multidrug resistant. In norfloxacin-resistant isolates, we detected mutations in the <i>gyrA</i> and <i>parC</i> genes and identified two new mutations in the <i>parC</i> gene. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and clustered regularly interspaced short palindromic repeat (CRISPR) analysis were employed to investigate the genetic relatedness of H₂S-negative and H₂S-positive <i>S</i>. Choleraesuis isolates. PFGE revealed two groups, with all 19 H₂S-negative <i>S</i>. Choleraesuis isolates belonging to Group I and H₂S-positive isolates belonging to Group II. By MLST analysis, the H₂S-negative isolates were all found to belong to ST68 and H₂S-positive isolates belong to ST145. By CRISPR analysis, no significant differences in CRISPR 1 were detected; however, one H₂S-negative isolate was found to contain three new spacers in CRISPR 2. All 19 H₂S-negative isolates also possessed a frame-shift mutation at position 760 of <i>phsA</i> gene compared with H₂S-positive isolates, which may be responsible for the H₂S-negative phenotype. Moreover, the 19 H₂S-negative isolates have similar PFGE patterns and same mutation site in the <i>phs</i>A gene, these results indicated that these H₂S-negative isolates may have been prevalent in China. These findings suggested that surveillance should be increased of H₂S-negative <i>S</i>. Choleraesuis in China.</p></div

Primers for PCR amplification of the <i>phsABC</i> genes.

<p>Primers for PCR amplification of the <i>phsABC</i> genes.</p

CRISPR spacer content of the 21 <i>S</i>. Choleraesuis isolates.

<p>^#Novel spacer identified in this study.</p><p>CRISPR spacer content of the 21 <i>S</i>. Choleraesuis isolates.</p

Mutations detected in the <i>gyrA</i> and <i>parC</i> gene of H₂S-negative <i>S</i>. Choleraesuis isolates.

<p>Ser, serine. Gly, glycine. Ala, alanine. Tyr, tyrosine. Cys, cysteine. Arg, arginine. Pro, proline.</p><p>Mutations detected in the <i>gyrA</i> and <i>parC</i> gene of H₂S-negative <i>S</i>. Choleraesuis isolates.</p

Supplementary Material for: <i>Mycobacterium avium</i> Complex Infections: Detailed Phenotypic and Functional Immunological Work-Up Is Required despite Genetic Analyses

Introduction: Cervical scrofulous lymphadenitis due to Mycobacterium avium complex (MAC) in immunocompetent adults is a rare disease. The presence of MAC infections demands meticulous clinical evaluation of patients along with detailed phenotypic and functional evaluation of their immune system including next-generation sequencing (NGS) analyses of target genes. Methods: Exact clinical histories of the index patients both suffering from retromandibular/cervical scrofulous lymphadenitis were obtained along with phenotypic and functional immunological evaluations of leukocyte populations followed by targeted NGS-based sequencing of candidate genes. Results: Immunological investigations showed normal serum immunoglobulin and complement levels, but lymphopenia, which was caused by significantly reduced CD3+CD4+CD45RO+ memory T-cell and CD19+ B-cell numbers. Despite normal T-cell proliferation to a number of accessory cell-dependent and -independent stimuli, the PBMC of both patients elaborated clearly reduced levels of a number of cytokines, including IFN-γ, IL-10, IL-12p70, IL-1α, IL-1β, and TNF-α upon TCR-dependent T-cell stimulation with CD3-coated beads but also superantigens. The IFN-γ production deficiency was confirmed for CD3+CD4+ helper and CD4+CD8+ cytotoxic T cells on the single-cell level by multiparametric flow cytometry irrespective of whether PMA/ionomycin-stimulated whole blood cells or gradient-purified PBMC was analyzed. In the female patient L1, targeted NGS-based sequencing revealed a homozygous c.110T>C mutation in the interferon-γ receptor type 1 (IFNGR1) leading to significantly reduced receptor expression on both CD14+ monocytes and CD3+ T cells. Patient S2 presented with normal IFNGR1 expression on CD14+ monocytes but significantly reduced IFNGR1 expression on CD3+ T cells, despite the absence of detectable homozygous mutations in the IFNGR1 itself or disease-related target genes. Exogenous addition of increasing doses of IFN-γ resulted in proper upregulation of high-affinity FcγRI (CD64) on monocytes from patient S2, whereas monocytes from patient L1 showed only partial induction of CD64 expression after incubation with high doses of IFN-γ. Conclusion: A detailed phenotypic and functional immunological examination is urgently required to determine the cause of a clinically relevant immunodeficiency, despite detailed genetic analyses