Midstream clean catch urine samples were inoculated on CLED agar (Oxoid, UK) plates using a calibrated loop delivering 0.001ml of urine. Inoculated plates were incubated at 37°C for 24 to 48 hrs [27 ]. The samples were considered positive for UTI if pure culture of 105CFU/ml were obtained from uncentrifuge urine sample and ≥5 pus cells observed in urine sample per field under microscope [8 (link),28 (link),29 ]. The presumptive identification of the isolates was based on the cultural characteristics on CLED agar (Oxoid, UK) plates and identification confirmed by standard identification protocol namely; Gram staining, motility test and conventional biochemical tests (i.e. oxidase, catalase, coagulase, IMViC, TSI agar, urease, gelatinase and the ability to grow in KCN [30 –33 ]. The isolates were preserved using 15%v/v glycerol at −70°C.
Cled agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
CLED agar is a microbiological culture medium used for the isolation and differentiation of various bacterial species, particularly those associated with urinary tract infections. The medium supports the growth of a wide range of bacterial organisms and allows for the identification of certain species based on their colony morphology and metabolic characteristics.
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Lab products found in correlation
Macconkey agar, by Thermo Fisher Scientific (3 mentions)
Blood agar, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Cystine lactose electrolyte deficient, by Thermo Fisher Scientific (1 mentions)
Countbright absolute counting beads, by Thermo Fisher Scientific (1 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Recombinant human c5a, by R&D Systems (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Microflex, by Bruker (1 mentions)
Bact alert 3d microbial detection system, by bioMérieux (1 mentions)
Chocolate agar, by Thermo Fisher Scientific (1 mentions)
Sabouraud agar, by Thermo Fisher Scientific (1 mentions)
10 protocols using cled agar
1
Urine Culture and Identification Protocol
2
Urine Culture and Identification Protocol
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Midstream clean catch urine samples were inoculated on CLED agar (Oxoid, UK) plates using a calibrated loop delivering 0.001ml of urine. Inoculated plates were incubated at 37°C for 24 to 48 hrs [27 ]. The samples were considered positive for UTI if pure culture of 105CFU/ml were obtained from uncentrifuge urine sample and ≥5 pus cells observed in urine sample per field under microscope [8 (link),28 (link),29 ]. The presumptive identification of the isolates was based on the cultural characteristics on CLED agar (Oxoid, UK) plates and identification confirmed by standard identification protocol namely; Gram staining, motility test and conventional biochemical tests (i.e. oxidase, catalase, coagulase, IMViC, TSI agar, urease, gelatinase and the ability to grow in KCN [30 –33 ]. The isolates were preserved using 15%v/v glycerol at −70°C.
3
Urine Culture Procedure for UTI Diagnosis
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Well-mixed urine samples were cultured on a plate containing approximately 25 ml of CLED agar (Oxoid, England), blood agar (Oxoid, England), and MacConkey agar (Oxoid, England) within 2 hours after collection using a 0.002-ml sterile loop. The plates were then incubated overnight at 37°C under aerobic conditions. Given the significant risks associated with the use of strict cut-offs, the standard agar-based clinical culture value of 105 colony-forming unit (CFU)/mL was used to represent an arbitrary cut-off [10 –12 (link)]. Thus, upon inspection, bacteria growth ≥105 cfu/ml was taken a “positive” for UTI infection, whereas bacteria growth <105 cfu/ml and mixed bacteria growth were taken as a contaminant. Escherichia coli ATCC 25299 was used as a quality control strain. Isolates were identified using Gram staining, morphological characteristics, and standard biochemical methods [13 ].
4
Urine Culture Protocol for Detecting Significant Bacteriuria
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Participants were asked to provide a midstream urine sample according to the clean-catch procedure. Samples were collected using a sterile container that was refrigerated (4°C), transported in an ice-pack to the medical laboratory, and processed within 1 hour of collection. Using a standard quantitative loop, urine samples (1 μL and 10 μL) were used to inoculate Cysteine lactose electrolyte deficient (CLED) agar (Oxoid, Basingstoke, UK), MacConkey, 5% Sheep Blood agar, and chromogenic UTI (Oxoid) agar plates. Plates were incubated for 24 h at 37°C and the outcome was judged as significant/non-significant growth, or contaminated (discarded). Significant bacteriuria was defined as urine culture plates showing ≥105 colony-forming units (CFU)/mL of single bacterial species. Symptomatic bacteriuria was defined as significant bacteriuria in addition to symptoms related to UTI, while asymptomatic bacteriuria was defined as significant bacteriuria in the absence of UTI symptoms. MDR bacteria were defined as isolates resistant to ≥2 antimicrobial agents.
5
Midstream Urine Sample Collection and Inoculation
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Clean voided midstream urine samples were collected in sterile special urine collection cups with the assistance of trained laboratory staff at DDI. Before sample collection, each patient was provided with a brochure and instructions explaining how to collect a correct midstream urine sample to avoid contamination. All urine samples were inoculated using a calibrated inoculation needle with 10 μL of urine and each sample was inoculated on three types of media: blood agar, MacConkey agar plates, and CLED agar (Oxoid, Basingstoke, UK). All plates were incubated at 37°C for 24–48 hours for visible growth.
6
Comprehensive Bacterial Identification Protocol
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Urine, sputum, and pus samples were directly plated onto MacConkey agar (Oxoid, UK). Furthermore, urine samples were cultured using CLED agar (Oxoid, UK). The cultured plates were then incubated aerobically at 37 °C to be inspected for growth after 18–24 h [11 ].
Blood and ascitic fluid samples were inserted in Bactec blood culture bottles (Becton Dickinson International, Belgium) and put in a BACTEC 9010 device, followed up for a maximum of five days to detect a positive alarm signal in Bactec. Subcultures were subsequently done on MacConkey agar [11 ].
All GNB growing on MacConkey agar and CLED agar were identified systematically using conventional biochemical reactions [11 ]. Bacterial isolates that were not conclusively identified to species level by the conventional biochemical reactions were tested using the analytical profile index API-20E for Enterobacterales and API-20NE for non- Enterobacterales (Bio-Mérieux, France).
7
Immunohistochemical analysis of C5aR1 expression
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The following reagents were used in this study: tryptone, yeast extract, cystine lactose electrolyte deficient (CLED) agar (Oxoid, Basingstoke, UK); monoclonal rat anti-mouse Ly6G, polyclonal goat anti-mouse C5aR1 (M-19) (Santa Cruz Biotechnology, Heidelberg, Germany); polyclonal rabbit anti-mouse cytokeratin (Abcam); Alexa Fluor 488 donkey anti-goat IgG (Jackson Immunology Research Lab., West Grove, USA); fluorescein-labeled Galanthus nivalis lectin (GNL, which binds to mannosyl residues) (Vector Laboratories, Peterborough, UK); polyclonal rabbit anti-human ZO-1 and 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies Ltd., Beijing, China); protease inhibitor cocktail, tetramethylrhodamine (TRITC) (Sigma-Aldrich, St. Louis, USA); anti-phospho-ERK1/2 (Thr202/Tyr204), -IκB (Ser 32), and anti-ERK1/2, -IκB antibodies used for Western blot and signaling pathway studies (Cell Signaling Technology, Danvers, USA); cell culture medium, fetal calf serum, gentamicin, TRIzol, CountBright™ absolute counting beads, Fast SYBR® Green Master Mix, M-PER mammalian protein extraction reagent, RIPA lysis buffer and BCA protein assay kit (Thermo Fisher, Waltham, USA); human recombinant C5a (R&D Systems); C5aR1 peptide antagonist (PMX53, Ac-Phe-cyclo [Orn-Pro-dCha-Trp-Arg]); and control peptide (random sequence) (synthesized by GenScript, Shanghai, China).
8
Characterization of Multidrug-Resistant E. coli
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BCs were incubated (BacTAlert® 3D microbial detection system, Biomerieux) and E. coli colonies identified by matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometry (Microflex, Bruker) following growth on cysteine lactose electrolyte deficient (CLED) agar (Oxoid). Antimicrobial susceptibilities were determined using Metascan Elite (MAST) with British Society for Antimicrobial Chemotherapy (BSAC) breakpoints.14 Isolates resistant to amoxicillin/piperacillin plus cefotaxime were screened for extended-spectrum beta-lactamase (ESBL) production utilising antimicrobial/inhibitor discs (Rosco).
Antimicrobial resistance scores comprised the number of antimicrobial agents to which the isolate was resistant. MDR was defined in line with international guidelines (non-susceptible to ≧1 agent in ≧3 antimicrobial categories).15 (link)
Urine microscopy (Sedimax platform, Menarini Diagnostics), culture and sensitivity testing (Metascan Elite) was performed. A urinary WCC >10/µl was considered elevated. Urinary isolates were confirmed as E. coli using MALDI-TOF mass spectrometry.
Bacteraemia and, where available, linked urinary isolates were sequenced.
Antimicrobial resistance scores comprised the number of antimicrobial agents to which the isolate was resistant. MDR was defined in line with international guidelines (non-susceptible to ≧1 agent in ≧3 antimicrobial categories).15 (link)
Urine microscopy (Sedimax platform, Menarini Diagnostics), culture and sensitivity testing (Metascan Elite) was performed. A urinary WCC >10/µl was considered elevated. Urinary isolates were confirmed as E. coli using MALDI-TOF mass spectrometry.
Bacteraemia and, where available, linked urinary isolates were sequenced.
9
Isolation and Identification of Enterobacteriaceae from Mulago Hospital
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Samples from 25 wards of Mulago Hospital were aseptically collected by purposive consecutive sampling from patients who gave informed written consent; properly labelled and taken to the Microbiology Laboratory for Enterobacteriaceae culture and isolation. The originating ward, patient’s gender and age were recorded. The samples were inoculated by streaking on Blood agar (Oxoid, UK), MacConkey agar (Oxoid, UK) and CLED agar (Oxoid, UK) plates. The plates were incubated aerobically at 37 °C for 18–24 h to allow development of bacterial colonies. Preliminary identification of the isolates was done using phenotypic colonial characteristics. Confirmatory identification of the suspect colonies was carried out by conventional biochemical tests as described by Cheesbrough [10 ]. These were: indole, Methyl red, Voges-Proskauer, citrate utilization and urease production tests as well as triple sugar iron and oxidase tests.
10
Pathogen Identification from Wound Secretions
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Once the surgeon confirmed the failure of the per secundam suture, wound secretions were aseptically obtained. Specimens were collected on sterile cotton swabs without contaminating them with skin commensals. The samples were inoculated on Columbia 5% blood agar, Chocolate agar, CLED agar, Sabouraud agar (all media produced by OXOID Ltd., UK) and incubated for 18 -24 hours at 37°C. After the incubation period, colonies suggestive of pathogenically significant germs were subjected to species identification in the automatic Vitek 2C/Phoenix BD. system or biochemical tests. For identifying both Gram-negative and Gram-positive microorganisms, fluorogenic and chromogenic conventional tests were automatically performed. All the probes were treated uniformly in the hospital's laboratory.
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