Additional file 1 of A systematic review of the clinical impact of small colony variants in patients with cystic fibrosis

Additional file 1: Table S1. PubMed search strategy. Table S2. Web of Science search strategy. Table S3. Embase search strategy. Table S4. Scopus search strategy. Table S5. Bias assessment of cohort studies. Table S6. Bias assessment of cross-sectional studies. Table S7. Bias assessment of case series studies. Table S8. Bias assessment for prevalence studies. Figure S1. Flow diagram of search procedure. Figure S2. Funnel plot for prevalence of SCVs. Figure S3. Sensitivity analysis forest plot for prevalence of SCVs. Figure S4. Sensitivity analysis funnel plot of prevalence of SCVs. Figure S5. Funnel plot for mean difference of FEV1% between SCV and NCV participants. Figure S6. Sensitivity analysis forest plot for mean difference of FEV1% between SCV and NCV participants. Figure S7. Sensitivity analysis funnel plot for the mean difference of FEV1% between SCV and NCV participants. Table S9. Specimens used for SCV collection. Table S10. Growth characteristics of SCVs. Table S11. Agar mediums for SCV cultivation. Table S12. Incubation conditions for SCVs. Table S13. Tests used for SCV confirmation

Genetic variants following ceftazidime experimental evolution in <i>Pseudomonas aeruginosa</i> strain PAO1 and PAO1::<i>lacZ</i> (n = 25).

Genetic variants following ceftazidime experimental evolution in Pseudomonas aeruginosa strain PAO1 and PAO1::lacZ (n = 25).</p

Ceftazidime and meropenem MIC for engineered mutants.

Ceftazidime and meropenem MIC for engineered mutants.</p

Minimum inhibitory concentration for <i>P</i>. <i>aeruginosa</i> PAO1 transposon mutants.

Minimum inhibitory concentration for P. aeruginosa PAO1 transposon mutants.</p

S1 File -

Pseudomonas aeruginosa causes a wide range of severe infections. Ceftazidime, a cephalosporin, is a key antibiotic for treating infections but a significant proportion of isolates are ceftazidime-resistant. The aim of this research was to identify mutations that contribute to resistance, and to quantify the impacts of individual mutations and mutation combinations. Thirty-five mutants with reduced susceptibility to ceftazidime were evolved from two antibiotic-sensitive P. aeruginosa reference strains PAO1 and PA14. Mutations were identified by whole genome sequencing. The evolved mutants tolerated ceftazidime at concentrations between 4 and 1000 times that of the parental bacteria, with most mutants being ceftazidime resistant (minimum inhibitory concentration [MIC] ≥ 32 mg/L). Many mutants were also resistant to meropenem, a carbapenem antibiotic. Twenty-eight genes were mutated in multiple mutants, with dacB and mpl being the most frequently mutated. Mutations in six key genes were engineered into the genome of strain PAO1 individually and in combinations. A dacB mutation by itself increased the ceftazidime MIC by 16-fold although the mutant bacteria remained ceftazidime sensitive (MIC ampC, mexR, nalC or nalD increased the MIC by 2- to 4-fold. The MIC of a dacB mutant was increased when combined with a mutation in ampC, rendering the bacteria resistant, whereas other mutation combinations did not increase the MIC above those of single mutants. To determine the clinical relevance of mutations identified through experimental evolution, 173 ceftazidime-resistant and 166 sensitive clinical isolates were analysed for the presence of sequence variants that likely alter function of resistance-associated genes. dacB and ampC sequence variants occur most frequently in both resistant and sensitive clinical isolates. Our findings quantify the individual and combinatorial effects of mutations in different genes on ceftazidime susceptibility and demonstrate that the genetic basis of ceftazidime resistance is complex and multifactorial.</div

Frequencies of predicted change-of-function variants in ceftazidime-sensitive and -resistant clinical isolates^b'*'.

Frequencies of predicted change-of-function variants in ceftazidime-sensitive and -resistant clinical isolatesb'*'.</p

Genetic variants identified following ceftazidime experimental evolution studies in <i>Pseudomonas aeruginosa</i> strain PA14 (n = 10).

Genetic variants identified following ceftazidime experimental evolution studies in Pseudomonas aeruginosa strain PA14 (n = 10).</p

Additional file 2 of CD161 expression defines new human γδ T cell subsets

Additional file 2: Supp. Table 1. Flow cytometry staining panel

Additional file 1 of CD161 expression defines new human γδ T cell subsets

Additional file 1: Sup Fig. 1. Gating strategy followed for biaxial gating. Sup Fig. 2 CD161 expressing cell percentages in (A) total γδ T cell, (B) Vδ1+γδ T cell and (C) Vδ1−γδ T cell populations. CD161 expression shows no correlation with age in any of the analyzed subsets (R squared/ p value for each subset R2 = 0.133/p = 0.125, R2 = 0.027/ p = 0.465, R2 = 0.015/ p = 0.579 respectively). Sup Fig. 3 Fluorescence minus one (FMO) staining controls for CD161, HLA-DR, CD45RA and CD27 are shown together with fully stained sample showing staining pattern on γδ T cells

Table_1_Characterizing and correcting immune dysfunction in non-tuberculous mycobacterial disease.xlsx

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) is a chronic, progressive, and growing worldwide health burden associated with mounting morbidity, mortality, and economic costs. Improvements in NTM-PD management are urgently needed, which requires a better understanding of fundamental immunopathology. Here, we examine temporal dynamics of the immune compartment during NTM-PD caused by Mycobacterium avium complex (MAC) and Mycobactereoides abscessus complex (MABS). We show that active MAC infection is characterized by elevated T cell immunoglobulin and mucin-domain containing-3 expression across multiple T cell subsets. In contrast, active MABS infection was characterized by increased expression of cytotoxic T-lymphocyte-associated protein 4. Patients who failed therapy closely mirrored the healthy individual immune phenotype, with circulating immune network appearing to ‘ignore’ infection in the lung. Interestingly, immune biosignatures were identified that could inform disease stage and infecting species with high accuracy. Additionally, programmed cell death protein 1 blockade rescued antigen-specific IFN-γ secretion in all disease stages except persistent infection, suggesting the potential to redeploy checkpoint blockade inhibitors for NTM-PD. Collectively, our results provide new insight into species-specific ‘immune chatter’ occurring during NTM-PD and provide new targets, processes and pathways for diagnostics, prognostics, and treatments needed for this emerging and difficult to treat disease.</p