Journal: PLoS Pathogens

Article Title: PknG senses amino acid availability to control metabolism and virulence of Mycobacterium tuberculosis

doi: 10.1371/journal.ppat.1006399

Figure Lengend Snippet: garA is required for growth of M . tuberculosis in vitro , survival in macrophages, and virulence in mice. ( A ) M . tuberculosis lacking garA was unable to grow on standard 7H10 medium unless supplemented with asparagine. Plasmid-borne garA restored the defect, but variants of garA with mutations at threonine 21 in the phosphorylation motif (ETTS) gave only partial complementation. Strains were grown in Middlebrook 7H9 plus 30 mM asparagine then washed and diluted in standard 7H9 and spotted onto standard 7H10 with or without 10 mM asparagine. Photographs are representative of at least 3 independent experiments. ( B ) M . tuberculosis lacking garA (red squares) had a defect in growth and survival in differentiated THP-1 cells compared to parental M . tuberculosis H37Rv (black circles). Re-introduction of GarA (black triangles) or variants of GarA lacking a single phosphorylation site (grey crosses and squares) restored growth but variant GarA lacking both phosphorylation sites (green triangles) did not. Data points show the mean and standard deviation from four replicates and are representative of two independent experiments. ( C ) M . tuberculosis lacking garA was avirulent in mice as it was eliminated from the lungs. BALB/C mice were infected intranasally with 10 5 bacilli and bacterial burden was measured on days 1, 7, 21 and 28. Data points show the bacterial burden in individual animals. The bacterial burden of mice infected with Δ garA Mt (red squares) was significantly lower than those infected with M . tuberculosis H37Rv (black circles), or complemented Δ garA Mt (black triangles) at all time points from day 7 (p

Article Snippet: Bacterial strains, media, and culture M . tuberculosis H37Rv and M . smegmatis mc2 155 were routinely cultured on Middlebrook 7H10 agar (Oxoid) with 10% ADN (0.5% bovine serum albumin, 0.2% dextrose, 0.085% NaCl) and Middlebrook 7H9 medium (Oxoid) with 10% ADN and 0.05% Tween 80.

Techniques: In Vitro, Mouse Assay, Plasmid Preparation, Variant Assay, Standard Deviation, Infection

Journal: PLoS ONE

Article Title: The Effect of Hyperglycaemia on In Vitro Cytokine Production and Macrophage Infection with Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0117941

Figure Lengend Snippet: Effects of euglycaemic and hyperglycaemic culture conditions on H37RV infection and cytokine production in vitro. (A) CD14 + selected monocytes were differentiated into M2 macrophages in the presence of 5 mmol/L glucose, 5 mmol/L glucose and 20 mmol/L mannitol or 25 mmol/L glucose. The macrophages were infected for 1 hour with H37Rv at an MOI of 10. After infection the macrophages were washed and fresh media containing the different glucose media was added. After 24 hours supernatants were collected and the cells were lysed by osmotic pressure. (B) Cell lysates were serially diluted and plated on Middlebrook 7H10 agar. CFUs were counted after 2–3 weeks of growth at 37°C. Data are shown as mean ± SEM of two independent experiments (n = 4). (C) Pro-inflammatory cytokines TNF-α and IL-6 were measured along with the anti-inflammatory cytokines IL-10 and IL-1RA. Data are shown as mean ± SEM.

Article Snippet: M2 macrophages were lysed in water for 5 minutes and plated on Middlebrook 7H10 agar (Difco, Becton-Dickinson) supplemented with OADC.

Techniques: Infection, In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Meropenem-Clavulanic Acid Has High In Vitro Activity against Multidrug-Resistant Mycobacterium tuberculosis

doi: 10.1128/AAC.00171-15

Figure Lengend Snippet: The MIC distribution of meropenem-clavulanic acid for the M. tuberculosis isolates ( n = 68) tested using Middlebrook 7H10 medium, shown by bars shaded as follows: non-MDR/XDR-TB (gray); XDR-TB (black), and MDR-TB (hatched). The MIC distribution shows

Article Snippet: Middlebrook 7H10 agar (Becton Dickinson AB, Stockholm, Sweden) enriched with 10% oleic acid–albumin–dextrose–catalase (OADC) and 5% glycerol was prepared in 14-cm petri dishes, each dish containing 60 ml of agar.

Techniques:

Journal: PLoS ONE

Article Title: Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0092799

Figure Lengend Snippet: Effect of inhibition of ROS and NO production on the survival of Mtb strains inside MØs. MØs were incubated with apocynin or L-NIL for 30 minutes, then infected with Mtb wild-type or Δ( ku,ligD,recA ) for 2 hours (MOI = 10) and washed with HBSS. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; #p≤0.04, Δ( ku,ligD,recA ) vs. Δ( ku,ligD,recA ) + apocynin or L-NIL; Mann-Whitney U test).

Article Snippet: A sample of each strain was taken during exponential and stationary phases of Mtb growth, and serial dilutions were prepared and plated on Middlebrook 7H10 agar supplemented with 10% OADC.

Techniques: Inhibition, Incubation, Infection, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Either Non-Homologous Ends Joining or Homologous Recombination Is Required to Repair Double-Strand Breaks in the Genome of Macrophage-Internalized Mycobacterium tuberculosis

doi: 10.1371/journal.pone.0092799

Figure Lengend Snippet: Survival of Mtb strains inside MØs. (A) MØs were infected with Mtb wild-type or its mutants, Δ( ku,ligD,recA ), Δ( ku,ligD ) or Δ recA , for 2 hours (MOI = 10), washed with HBSS, and cultured for 2, 4, and 6 days. (B and C) MØs were infected with Mtb wild-type or its mutants (B) or complemented strains Δ( ku,ligD,recA )- P recA recA – complemented strain I or Δ( ku,ligD,recA )- P kuligD ku-ligD – complemented strain II (C) for 6 days. On the day of infection and after 6 days of culture, MØs were lysed with Triton X-100 and cell lysates were plated onto Middlebrook 7H10 agar supplemented with 10% OADC. After 21 days of culture, CFUs were counted. Data are presented as fold increase in CFUs, expressed as means ± SEM (n = 5; *p≤0.05, Δ( ku,ligD,recA ) vs. wild-type; #p≤0.05, complemented strain I and II vs. Δ( ku,ligD,recA ) Mann-Whitney U test).

Article Snippet: A sample of each strain was taken during exponential and stationary phases of Mtb growth, and serial dilutions were prepared and plated on Middlebrook 7H10 agar supplemented with 10% OADC.

Techniques: Infection, Cell Culture, MANN-WHITNEY

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Multidrug Resistance of a Porin Deletion Mutant of Mycobacterium smegmatis

doi: 10.1128/AAC.48.11.4163-4170.2004

Figure Lengend Snippet: MspA-dependent sensitivities and permeabilities of M. smegmatis to hydrophobic compounds. (A) The sensitivities of wild-type M. smegmatis (black bars), the Δ mspA mutant (white bars), and the Δ mspA mutant transformed with the mspA expression vector pMN014 (gray bars) to erythromycin were determined by serial dilution in Middlebrook 7H10 agar (c, concentration). The number of surviving cells was normalized to the number of cells counted on plates without antibiotic for each strain, and the results are expressed as relative CFU (percent CFU). (B) The accumulation of [ 14 C]chenodeoxycholate by M. smegmatis SMR5 (wild type; closed circles) and M. smegmatis MN01 (Δ mspA ; open circles) was measured. Regression analysis yielded uptake rates of 0.09 and 0.03 nmol mg −1 min −1 for 20 μM chenodeoxycholate for the wild-type and mutant strains of M. smegmatis , respectively. The assay was performed at 37°C. The structures of the compounds are shown in the corresponding graphs. The sensitivity and uptake experiments were done in triplicate, and the results are shown with their standard deviations. The asterisks denote the datum points that differed significantly ( P

Article Snippet: MICs were determined by agar dilution with Middlebrook 7H10 plates.

Techniques: Mutagenesis, Transformation Assay, Expressing, Plasmid Preparation, Serial Dilution, Concentration Assay

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Multidrug Resistance of a Porin Deletion Mutant of Mycobacterium smegmatis

doi: 10.1128/AAC.48.11.4163-4170.2004

Figure Lengend Snippet: MspA-dependent sensitivity and permeability of M. smegmatis to isoniazid. (A) The sensitivities of wild-type M. smegmatis (black bars), the Δ mspA mutant (white bars), and the Δ mspA mutant transformed with the mspA expression vector pMN014 (gray bars) to isoniazid were determined by serial dilution in Middlebrook 7H10 agar (c, concentration). The number of surviving cells was normalized to the number of cells counted on plates without antibiotic for each strain, and the results are expressed as relative CFU (percent CFU). The structure of isoniazid is shown. (B) The accumulation of 20 μM [ 14 C]isoniazid by M. smegmatis SMR5 (wild type; closed circles) and M. smegmatis MN01 (Δ mspA ; open circles) was measured. The assay was performed at 37°C. The sensitivity and uptake experiments were done in triplicate, and the results are shown with their standard deviations. The asterisks denote the datum points that differed significantly ( P

Article Snippet: MICs were determined by agar dilution with Middlebrook 7H10 plates.

Techniques: Permeability, Mutagenesis, Transformation Assay, Expressing, Plasmid Preparation, Serial Dilution, Concentration Assay

Journal: PLoS ONE

Article Title: Novel Monoclonal Antibody and Peptide Binders for Mycobacterium avium subsp. paratuberculosis and Their Application for Magnetic Separation

doi: 10.1371/journal.pone.0147870

Figure Lengend Snippet: Outgrowth of milk bacteria non-specifically adhering to four types of coated magnetic beads in Middlebrook 7H9 OADC broth without (■) and with antibiotic supplements (NOA ◆ and PANTA ▼) after MS was applied to raw milk.

Article Snippet: Numbers of each Mycobacterium sp. before and after MS were determined by plating 100 μl of the initial cell suspensions and the bead suspensions after MS on Middlebrook 7H10/OADC agar (both Difco).

Techniques: Magnetic Beads, Mass Spectrometry

Journal: AMB Express

Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

doi: 10.1186/s13568-017-0373-6

Figure Lengend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

Article Snippet: By contrast, 65% (13/20) of soils inoculated onto Modified Middlebrook 7H10 agar with 250 mL/L MG produced mycobacterial growth.

Techniques: Modification, Isolation

Journal: Frontiers in Microbiology

Article Title: Functional Studies of Five Toxin-Antitoxin Modules in Mycobacterium tuberculosis H37Rv

doi: 10.3389/fmicb.2016.02071

Figure Lengend Snippet: Toxic effect of the five toxins on the growth of M. smegmatis . (A) Transformation of pMV306AC-toxin plasmid into M. smegmatis mc 2 155. One microgram of DNA was electrophorated and cells were plated on Middlebrook 7H10 solid media. The plates were incubated for 3 days at 37°C. (B) Effect of toxin on the induction on colony formation of M. smegmatis mc 2 155. Each transformant from (A) was grown for 3 days at 37°C. The culture was diluted 100-fold into Middlebrook 7H9 liquid media and streaked on Middlebrook 7H10 solid media in the absence and presence of 0.2% acetamide. The numbers in the circle correspond to those in (A) .

Article Snippet: M. smegmatis mc2 155 was grown in Middlebrook 7H9 (a liquid medium, Difco) containing 0.2% glycerol and 0.02% Tween 80 or Middlebrook 7H10 (a solid agar medium, Difco) containing 0.2% glycerol.

Techniques: Transformation Assay, Plasmid Preparation, Incubation

Journal: AMB Express

Article Title: Isolation of nontuberculous mycobacteria from soil using Middlebrook 7H10 agar with increased malachite green concentration

doi: 10.1186/s13568-017-0373-6

Figure Lengend Snippet: Modified Middlebrook 7H10 medium containing 250 mg/L MG with mycobacteria primarily isolated from soil. a Sample collected at campus, b sample collected at community, and c collected at natural park. Black arrows indicate mycobacterial colonies, red arrows indicate dried soils

Article Snippet: PANTA-containing Middlebrook 7H10 agar was prepared similarly, except that MG was replaced with polymyxin B (40,000 U/L)–amphotericin B (4 mg/L)–nalidixic acid (16 mg/L)–trimethoprim (4 mg/L)–azlocillin (4 mg/L) (PANTA; Becton, Dickinson and Company, Spark, USA) reconstituted with oleic acid–albumin–dextrose–catalase (OADC; Becton, Dickinson and Company, Spark, USA) enrichment as recommended by the manufacturer.

Techniques: Modification, Isolation