Six Pseudomonas aeruginosa strains, PAS71, PAS72, PAS81, and PAS82, were isolated and identified by the Department of Clinical Laboratory, the First Affiliated Hospital of Sun Yat-sen University, and conserved in our laboratory. P. aeruginosa ATCC27853 was purchased from ATCC (Manassas, VA, USA) and conserved in our laboratory. P. aeruginosa PAO1 was provided by the CAS Key Laboratory of Pathogenic Microbiology and Immunology, Beijing, China and conserved in our laboratory. Mueller–Hinton (MH) medium and Luria-Bertani (LB) media were purchased from Guangdong Huankai Microbial Sci. and Tech. Co., Ltd. (Guangzhou, China), following our previous study (Li et al., 2021 (link)). MH agar (MHA) medium was prepared by the addition of 1.5% agar to the MH medium. Cation-adjusted Mueller–Hinton broth (CAMHB) was purchased from Beijing Solarbio Sci. Tech. Co., Ltd. (Beijing, China), which contained the following (g/L): beef dip powder, 3.0; acid hydrolyzed casein, 17.5; soluble starch, 1.5; calcium ion, 20–25 mg; magnesium ion, 10–12.5 mg; pH, 7.3 ± 0.1. MH, LB, and CAMHB media were used for aerobic cultivation at 37°C and shaken at 180 rpm. LVX susceptibility discs (Product no. 21055, 5 μg/tablet) were purchased from Beijing Bolyou Biotech. Co. Ltd. (Beijing, China). LVX (CAS: 100986–85-4, S17134-25 g, purity ≥98%) was purchased from Shanghai Yuanye Biotech. Co. Ltd. (Shanghai, China).
Atcc 27853
Manufactured by American Type Culture Collection
Sourced in United States
ATCC 27853 is a bacterial strain that belongs to the species Staphylococcus aureus. It is a gram-positive, catalase-positive, and coagulase-positive coccus. This strain is commonly used as a reference organism in microbiological and antimicrobial testing procedures.
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Lab products found in correlation
P aeruginosa, by American Type Culture Collection (2 mentions)
Cetrimide agar, by Merck Group (1 mentions)
E coli, by American Type Culture Collection (1 mentions)
S aureus atcc 25923, by American Type Culture Collection (1 mentions)
Atcc 25922, by American Type Culture Collection (1 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (1 mentions)
Luria bertani medium, by Merck Group (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
C57bl 6 mice, by Beijing Huafukang Biotechnology (1 mentions)
Imipenem, by BD (1 mentions)
Meropenem, by BD (1 mentions)
Ertapenem, by BD (1 mentions)
Mueller hinton agar (mha), by BD (1 mentions)
K pneumoniae, by BD (1 mentions)
Sheep blood agar, by bioMérieux (1 mentions)
Atcc 15442, by American Type Culture Collection (1 mentions)
11 protocols using atcc 27853
1
Levofloxacin Susceptibility of Pseudomonas aeruginosa
2
Pseudomonas aeruginosa Nosocomial Infections
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Over a 6-month period (October 2020–March 2021), 115 NIs-causing P. aeruginosa isolates were collected from hospitalized patients referred to teaching hospitals of three cities of Khuzestan province (Ahvaz, Abadan and Khorramshahr), southwest Iran. NIs were defined according to the diagnostic criteria of the Centers for Disease Control and Prevention (CDC) [15 ]. The isolates were related to four types of NIs including UTI, VAP, SSI and BSI, which were collected from different wards including intensive care unit (ICU), burn, infectious diseases, nephrology, emergency, pediatrics, gastroenterology, skin diseases, dialysis, internal medicine, and critical care unit (CCU). Initial identification of the isolates was based on typical growth on a selective medium Cetrimide agar (Merck, Darmstadt, Germany) and different biochemical tests including colony characteristics, sulphur indole and motility (SIM), triple sugar iron (TSI), urease, citrate, oxidase, and growth at 42 °C [16 (link)]. PCR of the gyrB gene was used for final identification [17 ]. P. aeruginosa ATCC® 27,853™ was used as a positive control. A stock of all bacterial isolates was prepared in a microtube containing tryptic soy broth (TSB) with 20% glycerol and stored at −80 °C until further investigation.
3
Isolation and Identification of Pathogenic Bacteria
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All collected biopsies were intensively washed with 5 ml of normal saline. Twenty µl suspension of each saline-washed specimen was suspended on to each three plates of meat peptone agar.
MacConkey agar, a selective media, was also employed to isolate common pathogenic bacteria like Salmonella and Shigella species. Colony forming unit (CFU) count, morphological characteristics of bacterial isolates at average logarithmic growth phase and identification of bacterial species using a series of biochemical tests were aseptically performed. Sterility and performance of the prepared media were checked by parallel inoculation of localy available control strains of American Type Culture Collection: S. aureus (ATCC ® -25923), P. aeruginosa (ATCC ® -27853) and E. coli (ATCC ® -25922).
MacConkey agar, a selective media, was also employed to isolate common pathogenic bacteria like Salmonella and Shigella species. Colony forming unit (CFU) count, morphological characteristics of bacterial isolates at average logarithmic growth phase and identification of bacterial species using a series of biochemical tests were aseptically performed. Sterility and performance of the prepared media were checked by parallel inoculation of localy available control strains of American Type Culture Collection: S. aureus (ATCC ® -25923), P. aeruginosa (ATCC ® -27853) and E. coli (ATCC ® -25922).
4
Isolation and Identification of XDR-Acinetobacter baumannii
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XDR-A. baumannii strains were isolated from clinical specimens collected in three tertiary hospitals affiliated to Shandong University (Qilu Hospital, Jinan Central Hospital and Shandong Provincial Qianfoshan Hospital; all in Jinan, China) from November 2014 to December 2015. For patients from whom A. baumannii strains were isolated more than once, only one strain from each patient was included. VITEK® 2 microbial analysis instruments were used to identify the XDR-A. baumannii isolates (bioMérieux, Inc., Marcy l'Etoile, France). The Kirby-Bauer method (8 ) was applied to re-evaluate the strains to meet the criteria for XDR-A. baumannii (4 (link)). As a result, a total of 50 strains were included, of which 36 strains were from sputum, 5 from lavages, 3 from blood, 3 from skin wounds or surgical incisions of skin, 2 from cerebrospinal fluid and 1 from urine. ATCC 25922 and ATCC 27853 were used as quality controls (American Type Culture Collection, Manassas, VA, USA). The Ethics Committee of Qilu Hospital of Shandong University approved the present study (approval no. KYLL-2017-612). All patients provided written informed consent.
5
Antibiotic Susceptibility of Pseudomonas
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The experiments were performed using P. aeruginosa reference strains: PAO1 and ATCC 27853 (American Type Culture Collection) and four clinical isolates: 15/3, 82/3, 9/5, and 14/3 showing different susceptibility patterns to antibiotics (Table S4 ) [18 (link)].
6
Pseudomonas aeruginosa Strain Isolation and Inoculum Preparation
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P. aeruginosa ATCC 27853 was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and a clinical P. aeruginosa strain was isolated from an eye-infected and hospitalized patient from a previous study [31 (link)]. The bacterial isolates were steaked on the Mueller Hinton Agar (MHA, Oxoid, Basingstoke, UK) and then incubated overnight at 35 ± 2 °C. The turbidity of inoculum was adjusted to 0.5 McFarland standard around 1–2 × 108 CFU/mL [29 (link),31 (link),32 (link)].
7
Molecular Identification of P. aeruginosa from Burn Wounds
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In this cross-sectional study, 100 isolates of P. aeruginosa have been gathered from wound infections of burn patients admitted to Imam Mosa Kazem Burn Hospital in Isfahan, Iran, between March and July 2015. Each isolate has been determined due to the standard bacteriological methods including Gram-staining, growth at 42°C in cetrimide agar, oxidation-fermentation (OF), TSI, and oxidase tests. Furthermore, polymerase chain reaction (PCR) of exoA gene has been carried out to confirm the bacteriologic identification. A 397-bp fragment of the exoA gene has been selected with specified primers (forward: 5’-GACAACGCCCTCAGCATCACCAGC-3,' reverse: 5’-CGCTGGCCCATTCGCTCCAGCGCT-3’).[23 (link)] Each PCR reaction was prepared in 20 μL volume include 10 μL the commercial Master Mix (containing Taq DNA polymerase, dNTPs, and MgCl2) (Ampliqon Denmark), 1 μL DNA sample, 0.5 μL of each primer (Metabion, Germany), and 8 μL distilled. Samples were then subjected to one cycle of 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 68°C for 30 s, and 72°C for 45 s and one final cycle of 72°C for 5 min. P. aeruginosa strain ATCC27853 (American Type Culture Collection) was included as the control.
8
Murine Model of Pseudomonas Infection
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All the protocols were performed in accordance with the approved guidelines. Female C57/BL6 mice (all 6–8 weeks of age) were obtained from the Beijing HFK Bioscience Co. Ltd. The mice were maintained at a twelve hours light and night cycle under specific pathogen free (SPF), temperature controlled conditions, and were fed with a standard laboratory diet during the entire experiment. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Sichuan University.
Anti-CD4 mAb hybridoma cell (clone GK1.5, rat IgG), anti-CD8 mAb hybridoma cell (clone 2.43, rat IgG), and bacteria strains (ATCC 27853, PAO-1 and PAO-6) were all purchased from the American Type Culture Collection (Rockville, MD, USA). IL-17 antibody and the control IgG were purchased from Sigma (St. Louis, MO, USA). Hybridoma cell clone 2.43 was cultured in DMEM medium with 10% fetal bovine serum (FBS), and GK1.5 cultured in Iscove’s Modified Dulbecco’s (20% FBS) medium (Invitrogen). P. aeruginosa bacteria were all cultured with Luria–Bertani (LB) medium (Sigma-Aldrich, Shanghai, Trading Co. Ltd).
Anti-CD4 mAb hybridoma cell (clone GK1.5, rat IgG), anti-CD8 mAb hybridoma cell (clone 2.43, rat IgG), and bacteria strains (ATCC 27853, PAO-1 and PAO-6) were all purchased from the American Type Culture Collection (Rockville, MD, USA). IL-17 antibody and the control IgG were purchased from Sigma (St. Louis, MO, USA). Hybridoma cell clone 2.43 was cultured in DMEM medium with 10% fetal bovine serum (FBS), and GK1.5 cultured in Iscove’s Modified Dulbecco’s (20% FBS) medium (Invitrogen). P. aeruginosa bacteria were all cultured with Luria–Bertani (LB) medium (Sigma-Aldrich, Shanghai, Trading Co. Ltd).
9
Antimicrobial Susceptibility Testing Protocol
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During the antimicrobial susceptibility testing step, the following were used: the diffusion method on Mueller– Hinton Agar (Becton Dickinson, Frankfurt, Germany) and imipenem (10 µg) and meropenem (10 µg) discs plus ertapenem (10 µg) for K. pneumoniae only (Becton Dickinson, Germany). The results of the antimicrobial susceptibility tests were interpreted according to the European Committee on Antimicrobial Susceptibility Testing recommendations (EUCAST, Breakpoint tables for bacteria, Clinical breakpoints—bacteria v. 12.0) [22 ]. P. aeruginosa was purchased from American Type Culture Collection (ATCC 27853) and E. coli ATCC 25922 strains served as a susceptibility testing quality control.
10
Isolation and Characterization of Multidrug-Resistant P. aeruginosa Strains
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The six P. aeruginosa clinical strains (PAS1-6) were kindly provided by Kamphaeng Phet Hospital. These strains were isolated from specimens, such as blood, pus, and sputum, obtained from hospitalized patients at Kamphaeng Phet Hospital, Kamphaeng Phet, Thailand in 2022. A reference strain of drug-sensitive (DS) P. aeruginosa ATCC 27853 (PA-27853) was purchased from the American Type Culture Collection (ATCC).
A colony of each bacterium tested in this study was cultured on 5% sheep blood agar (BioMérieux, Inc. Hazelwood, MO, USA) at 35 ± 2 °C for 18–24 h. For subculturing, isolated bacterial colonies were re-streaked on the Mueller–Hinton Agar (MHA, Oxoid, Basingstoke, UK) and then incubated at 35 ± 2 °C for 24 h. The turbidity of the inoculum was adjusted to 0.5 McFarland standard, approximately 1–2 × 108 CFU/mL [15 (link),17 (link)].
A colony of each bacterium tested in this study was cultured on 5% sheep blood agar (BioMérieux, Inc. Hazelwood, MO, USA) at 35 ± 2 °C for 18–24 h. For subculturing, isolated bacterial colonies were re-streaked on the Mueller–Hinton Agar (MHA, Oxoid, Basingstoke, UK) and then incubated at 35 ± 2 °C for 24 h. The turbidity of the inoculum was adjusted to 0.5 McFarland standard, approximately 1–2 × 108 CFU/mL [15 (link),17 (link)].
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