Gc agar base

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GC agar base is a culture media formulation used for the isolation and cultivation of Neisseria species and other fastidious microorganisms. It provides nutrients and growth factors required for the growth of these microorganisms.

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Lab products found in correlation

Vitox, by Thermo Fisher Scientific (2 mentions) Polyvitox, by Thermo Fisher Scientific (1 mentions) Sheep blood, by BD (1 mentions) Colistin sulfate, by Thermo Fisher Scientific (1 mentions) Vancomycin, by Thermo Fisher Scientific (1 mentions) Isovitalex, by Thermo Fisher Scientific (1 mentions)

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GC agar (23 citations) GC base (2 citations) Agar base (2 citations)

7 protocols using gc agar base

Cultivation of Pathogenic Bacterial and Yeast Strains

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N. meningitidis B1940 [B:NT:P1.3,6,15; LOS immunotype L3,7,9] is piliated and expresses Opa and Opc adhesins [13 (link)]. MC58 is an international reference strain [14 (link)]. The meningococcal strains were cultured on GC agar base (OXOID) supplemented with 1% (v/v) Polyvitox (OXOID) at 37 °C in a 5% CO₂ incubator as described [15 (link), 16 (link)].
Escherichia coli strain DH5α [F⁻ Φ80d lacZΔM15 endA1 recA1 hsdR17 supE44 thi-1 l⁻gyrA96 Δ(lacZYA-argF) U169] was used in cloning procedures. This strain was grown in Luria Bertani (LB) medium. To allow plasmid selection, LB medium was supplemented with ampicillin (75 µg ml⁻¹) or kanamycin (50 µg ml⁻¹). E. coli strain BL21 (DE3) [B F^–ompT gal dcm lon hsdS_B(r_B^–m_B^–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB⁺]_K-12(λ^S)] was used for expression of His-tagged proteins.
Saccharomyces cerevisiae AH109 strain (MATa, trp1-901, leu2-3, 112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1) was used for two-hybrid assay [17 (link), 18 (link)]. This strain was cultivated with synthetic dropout medium (SD).

Talà A., Guerra F., Calcagnile M., Romano R., Resta S.C., Paiano A., Chiariello M., Pizzolante G., Bucci C, & Alifano P. (2022). HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis. Journal of Biomedical Science, 29, 45.

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Antibiotic Susceptibility Testing Protocol

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AST was performed by the KB disk-diffusion technique using a GC agar base (Oxoid, Basingstoke, UK) with 1% BBL IsoVitalex Enrichment (BD Diagnostics, Franklin Lakes, NJ, USA) with following concentration disks (Oxoid) of ceftriaxone 30 μg and cefixime 5 μg. The results were interpreted by measuring inhibition-zone diameters and categorizing as susceptible and insusceptible in accordance with the Clinical and Laboratory Standards Institute. For ceftriaxone, we categorized inhibition-zone diameters as susceptible at ≥35 mm and insusceptible at <35 mm. For cefixime, we categorized inhibition-zone diameters as susceptible at ≥31 mm) and insusceptible at <31 mm. This work was carried out after timely isolation and purification of the collected strains.

Han Y., Yin Y.P., Xu W.Q., Zhu X.Y., Chen S.C., Dai X.Q., Yang L.G., Zhu B.Y., Zhong N., Cao W.L., Zhang X.H., Wu Z.Z., Yuan L.F., Zheng Z.J., Liu J, & Chen X.S. (2020). Disk-Diffusion Testing Is an Inappropriate Screening Tool for Cephalosporin-Resistant Gonorrhoea Strains in Clinical Practice in China. Infection and Drug Resistance, 13, 2417-2423.

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Antibiotic Susceptibility Profiling of N. gonorrhoeae

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Beta-lactamase production of N. gonorrhoeae isolates was tested by a nitrocefin disk (BD Diagnostics, Franklin Lakes, NJ, USA) as per the manufacturer’s instructions. All beta-lactamase-positive isolates were known penicillinase-producing N. gonorrhoeae (PPNG) isolates.
The MICs (μg/ml) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method on GC agar base (Oxoid Ltd., Hampshire, UK) supplemented with 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, MD, USA). The antibiotic powder was purchased from Sigma–Aldrich (Milwaukee, WI, USA). The practice of conducting the agar dilution method was performed following the Clinical and Laboratory Standards Institute guidelines [20 ]. The agar dilution was performed in duplicate, and the obtained MIC values were interpreted based on the interpretative criteria of the CLSI guidelines [21 ]. N. gonorrhoeae ATCC 49226 was used as the quality control strain for the agar dilution test.

Nokchan N., Wongsurawat T., Jenjaroenpun P., Nitayanon P, & Tribuddharat C. (2022). Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand. PLoS ONE, 17(7), e0271657.

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Genotypic Analysis of Multidrug-Resistant Neisseria gonorrhoeae

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Bacterial experiments were performed with the international N. gonorrhoeae reference strain ATCC49226 and its isogenic ΔmtrE deletion mutant, for which construction has been published previously [18 (link)]. Shortly, mtrE was replaced with the kanamycin resistance gene kanR through homologous recombination using a linearized vector expressing kanR flanked by mtrE up- and downstream regions. In addition, the contemporary multidrug-resistant clinical isolates SZ20 (CRO^HLR, CFM^HLR, PEN^R, CIP^R, TET^R), SRRSH240 (CRO^HLR, CFM^HLR, PEN^R, CIP^R, TET^R), ZJXSH89 (AZM^HLR, PEN^R, CIP^R, TET^R), ZJXSH86 (PEN^R, CIP^R, TET^R), ZXH83 (CRO^R, CFM^R, PEN^R, CIP^R, TET^R), SRRSH64 (CRO^RS, CFM^R, AZM^HLR, PEN^R, CIP^R, TET^R) and HTPH74 (CRO^R, CFM^R, PEN^R, CIP^R, TET^R) were used [12 (link),18 (link),30–32 (link)]. Experiments were initiated by reviving −80 °C glycerol stocks onto GC agar base (Oxoid) containing 1% (v/v) Vitox (Oxoid) and bacteria were grown overnight at 37 °C in the presence of 5% CO₂.

Song S., Wang S., Jiang X., Yang F., Gao S., Lin X., Cheng H, & van der Veen S. (2023). Th1-polarized MtrE-based gonococcal vaccines display prophylactic and therapeutic efficacy. Emerging Microbes & Infections, 12(2), 2249124.

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Antibiotic Susceptibility in N. gonorrhoeae

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The effect of different growth supplements on the MIC of eight antibiotics (zoliflodacin, penicillin, gentamicin, tetracycline, spectinomycin, azithromycin, ciprofloxacin, and ceftriaxone) on N. gonorrhoeae isolates was determined. For this GC agar base (OXOID, USA) supplemented with 10% sterile defibrinated sheep blood (Lezhen, China) or 2% bovine hemoglobin (BD, USA) or 1% Vitox (OXOID, USA) were added separately to the media containing different concentrations of the antibiotics. Thereafter, 1 µL of bacterial suspension was transferred onto the agar surface using a replicator. Eventually, each medium was cultured for 18–24 hours at 36°C in a 5% CO₂-enriched atmosphere. The growth of N. gonorrhoeae in different groups was observed and recorded. All steps strictly followed the WHO standard operation of the agar dilution method except for the other two additional growth supplements (sheep blood and hemoglobin).9 (link) Each test was repeated for each bacteria strain to confirm the results.

Zhou Q., Xu W., Xia D., Zhu X., Han Y., Chen K, & Yin Y. (2022). Impact of Alternative Growth Supplements on Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae. Infection and Drug Resistance, 15, 5475-5481.

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Isolation and Genomic Sequencing of N. meningitidis

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Neisseria meningitidis reference strain Z2491 (DSM No. 15465) was obtained from DSMZ (Braunschweig, Germany). N. meningitidis isolates were previously collected over a time period of ~10 years during meningococcal meningitis epidemics in Sub-Saharan Africa (two sequence types ST2859 and ST7). Isolates underwent typically 2 rounds of single colony sub-culturing and over-night expansion in vitro. For genomic DNA preparation, strains were grown on supplemented GC agar base (Oxoid) plates for 20–24 hours in 5% CO₂ at 37°C. Single colonies were transferred into liquid Brain Heart Infusion (BactoTM) medium and again incubated overnight in 5% CO₂ at 37°C. Genomic DNA was extracted as described previously [28 (link)]. Genomic DNA samples of two strains (isolate NM1264 and reference Z2491) were subjected to SMRT sequencing. The material of strain NM1264 represented aliquots of a genomic DNA sample previously subjected to the Illumina sequencing method [29 (link)].

Sater M.R., Lamelas A., Wang G., Clark T.A., Röltgen K., Mane S., Korlach J., Pluschke G, & Schmid C.D. (2015). DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates. PLoS ONE, 10(12), e0144612.

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Gonococcal Isolates from Thai Patients

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134 isolates collected from urethral swabs of patients with positive N. gonorrhoeae infections at Thai Red Cross Anonymous Clinic, Thai Red Cross AIDS Research Centre, and King Chulalongkorn Memorial Hospital, Bangkok, Thailand, from patients with gonococcal infections during 2016–2019. For culture preservation, all isolates were grown on TM medium (GC agar base supplemented with 1% haemoglobin, 1% IsoVitaleX, and vancomycin, colistin sulfate, and nystatin selective supplement (Oxoid, United Kingdom). All N. gonorrhoeae isolates were preserved at − 80 °C. N. gonorrhoeae ATCC 49226 reference strain was used for quality control of all phenotypic and molecular characterizations.

Kueakulpattana N., Wannigama D.L., Luk-in S., Hongsing P., Hurst C., Badavath V.N., Jenjaroenpun P., Wongsurawat T., Teeratakulpisan N., Kerr S.J., Abe S., Phattharapornjaroen P., Shein A.M., Saethang T., Chantaravisoot N., Amarasiri M., Higgins P.G, & Chatsuwan T. (2021). Multidrug-resistant Neisseria gonorrhoeae infection in heterosexual men with reduced susceptibility to ceftriaxone, first report in Thailand. Scientific Reports, 11, 21659.

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