This experimental study was conducted at the Microbiology Laboratory of the University Hospital of Martinique. During eighteen months, all B. lanceolatus snakes captured by personnel of the National Office of Forests in Martinique were studied. Snakes were grouped according to their geographical origin, i.e., wet (forest and near water areas) versus dry regions (peri-urban areas). The snake’s oral cavity was opened and sampled from the vicinity of the fangs using sterile cotton swabs. Samples were subjected to Gram staining and examined for bacterial growth. They were plated on non-selective blood agar and chocolate agar and cultured at 37 °C for 2–7 days and the color and shape of the colonies were observed. Species identification was performed with API-20E and API-20NE systems (BioMérieux, Marcy L’Etoile, France). Antimicrobial susceptibilities of all isolates to beta-lactams were determined by the disk diffusion methods based on the definitions of the Antibiogram Committee of the French Microbiology Society [10 (link)]. The inhibition zone diameters of each drug for each isolate were determined after overnight incubation at 35.8 °C in ambient air. The interpretive criteria of the inhibition zone and minimum inhibitory concentrations were in accordance with those of the Antibiogram Committee of the French Society of Microbiology.
Api 20ne system
Manufactured by bioMérieux
Sourced in France
The API 20NE system is a standardized identification system for the identification of non-fastidious Gram-negative rods. It consists of a plastic strip containing 20 microtubes, each filled with a dehydrated substrate, that allows the identification of more than 120 different bacterial species. The results are interpreted using an identification table or database.
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Lab products found in correlation
Api zym, by bioMérieux (2 mentions)
Mueller hinton broth (mhb), by Merck Group (2 mentions)
Mueller hinton agar (mha), by Thermo Fisher Scientific (2 mentions)
Jem 1400flash, by JEOL (1 mentions)
Gram staining kit, by Solarbio (1 mentions)
Jem 1010, by JEOL (1 mentions)
Bordet gengou agar, by BD (1 mentions)
Mueller hinton agar plate, by Merck Group (1 mentions)
Api 20e system, by bioMérieux (1 mentions)
Tryptic soy agar, by Bio-Rad (1 mentions)
Vitek 2 automated system, by bioMérieux (1 mentions)
Phoenix system, by BD (1 mentions)
Etest, by bioMérieux (1 mentions)
Sheep blood agar, by Thermo Fisher Scientific (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Oxidase strips, by Thermo Fisher Scientific (1 mentions)
Trypticase soy agar tsa, by Thermo Fisher Scientific (1 mentions)
Trypticase soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Vitek 2 automated microbial identification system, by bioMérieux (1 mentions)
Sabouraud dextrose agar, by Thermo Fisher Scientific (1 mentions)
48 protocols using api 20ne system
1
Antimicrobial Susceptibility of B. lanceolatus Snake Oral Microbiome
2
Comprehensive Bacterial Characterization Protocol
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Gram staining was performed using a Gram staining kit (Solarbio, Hangzhou, China) by following the manufacturer’s instructions. The shape of cells was observed by transmission electron microscopy (JEM-1400FLASH; JEOL; Tokyo, Japan). The motility of cells was identified by the development of turbidity in a tube containing a semi-solid APA medium. Endospores were examined according to the Schaeffer–Fulton staining method [24 ]. Growth at different temperatures (4, 15, 20, 29, 37, 45, 55 and 65 °C) was determined in liquid APA medium. The tolerance to salinity and alkalinity was determined in a liquid APA medium with various NaCl concentrations (0, 2, 5, 8, 10, 15 and 20%, w/v) and pH range (pH 6.0–12.0, at intervals of 1.0 pH unit). The pH of the basal medium was adjusted using the buffer system, as described by Narsing Rao et al. [25 (link)]. Catalase and oxidase activities were tested using 3% (v/v) H2O2 and the oxidase reagent, respectively [26 (link)]. Other enzyme activities, biochemical characteristics and utilization of carbon source were detected using API ZYM, API 20NE systems (bioMérieux, Grenoble, France) and GEN Ⅲ Microplate (Biolog, Newark, NJ, USA), by following the manufacturer’s instructions.
3
Characterization of a Novel Bacterial Strain
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Cell morphology, Gram reaction, anaerobic growth, pH range for growth, growth at various concentrations of NaCl, hydrolysis of gelatin and urea and susceptibility to antibiotics were investigated as described by Park et al. (2014) . For transmission electron microscopy (JEM1010; JEOL), cells were negatively stained with 1% (w/v) phosphotungstic acid and air-dried. Grids were then examined. Growths at 4, 10, 20, 25, 28, 30, 35, 37, and 40 °C on MA were measured to estimate the optimal temperature and temperature range for its growth. Nitrate reduction and hydrolysis of aesculin or Tween 80 were investigated as described previously (Lányí 1987 ) using arti cial seawater (Bruns et al. 2001) for the preparation of the media. Hydrolysis of other substrates was tested as described by Barrow and Feltham (1993) with the modi cation that MA was used. Activities of catalase and oxidase were determined as described by Lányí (1987) . Utilization of various substrates (each 0.2%) for growth was investigated as described by Kämpfer et al. (1991) . Other biochemical and physiological properties were determined using API ZYM and API 20NE systems (bioMérieux; France). Enzyme activities by the API ZYM system were determined after incubation at 30 ºC for 8 h. Other physiological and biochemical properties by the API 20NE system were determined after incubation at 30 ºC for 2 days.
4
Profiling A. baumannii Isolates in Iran
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A total of 95 non-repetitive A. baumannii isolates were collected from inpatients mostly with respiratory tract infections, bacteraemia and wound infections between August 2016 and February 2017 from an educational hospital in Tehran, Iran. Species identi cation was performed by API20NE system (Biomérieux, Marcy-l'Étoile, France) and PCR ampli cation of the bla OXA-51-like gene [23] and rpoB sequencing [24] . This study was approved by the local ethics committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1396.22).
5
Bordetella spp. Isolation and Identification
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The samples were cultured into a fresh Bordet Gengou agar or Regan Low medium (Difco Laboratories, Detroit, USA) prepared in our laboratory, which included 15% of defibrinated horse’s blood and 40 mg/L of cephalexin (Selective supplement Bordetella, Oxoïd-Unipath, Dardilly, France). The growth media used for bacterial culture were validated using B. pertussis (CIP 8132), B. parapertussis (CIP 12822) and B. holmesii (CIP104394) reference strains.
They were incubated at 35 ± 2 °C in a moist atmosphere and maintained for three to ten days under aerobic conditions. Colonies were identified based on characteristic morphology, Gram stain, catalase and oxidase testing and by using the API 20NE system (bioMérieux, Marcy L’Etoile, France). The colonies suspected to be Bordetella spp. were tested by molecular tools as described below.
They were incubated at 35 ± 2 °C in a moist atmosphere and maintained for three to ten days under aerobic conditions. Colonies were identified based on characteristic morphology, Gram stain, catalase and oxidase testing and by using the API 20NE system (bioMérieux, Marcy L’Etoile, France). The colonies suspected to be Bordetella spp. were tested by molecular tools as described below.
6
Identification and Antibiotic Profiling of Acinetobacter baumannii
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A total of 48 clinical isolates (see Additional files 1 and 2 ) of A. baumannii were collected from the Southwest Hospital of China from January 2012 to January 2014 (all the isolates were collected as part of standard patient care and informed content of the patients or their families was obtained). All 48 isolates were phenotypically identified to genus level by the API 20-NE system (Biomerieux, France). Genotypic identification was performed by PCR detection of the intrinsic blaOXA-51-like gene [13 (link)] and sequence analysis of the 16S rRNA gene [14 (link)]. The antimicrobial susceptibility of these isolates was determined by the K-B method and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) criteria (http://clsi.org/ ). Multilocus sequence typing (MLST) was used to investigate the molecular epidemiology of the isolates with seven housekeeping genes comprising gltA, gyrB, ghdB, recA, cpn60, gpi, and rpoD. The primer sequences, PCR reaction conditions and MLST analysis that were used have all been previously described [15 (link)].
7
Phenotypic and Genotypic Characterization of XDR Acinetobacter baumannii
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A. baumannii were identified by API 20 NE system (bio-Merieux, Marcy l'Etoile, France) and PCR amplification of bla OXA-51 [9] .
Antimicrobial susceptibility testing was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2019) [10] and analyzed by the SIRscan system. Bacterial suspension at 0.5-McFarland standard turbidity was inoculated on a Mueller-Hinton agar plate (Merck, Frankfurt, Germany). For the quality control in susceptibility testing, Escherichia coli CIP 7624 (ATCC 25922) was used as reference strain for internal quality control [10] , and external quality controls were conducted regularly by the Tunisian Health Ministry.
XDR-Ab was defined as resistant, in addition to carbapenems, to at least three classes of antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and fluroquinolones [11] .
The minimum inhibitory concentrations (MIC) of imipenem was determined by E-test method (Biom erieux ® ) and the MIC of colistin was determined by broth dilution method (UMIC Biocenric ® ).
Antimicrobial susceptibility testing was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2019) [10] and analyzed by the SIRscan system. Bacterial suspension at 0.5-McFarland standard turbidity was inoculated on a Mueller-Hinton agar plate (Merck, Frankfurt, Germany). For the quality control in susceptibility testing, Escherichia coli CIP 7624 (ATCC 25922) was used as reference strain for internal quality control [10] , and external quality controls were conducted regularly by the Tunisian Health Ministry.
XDR-Ab was defined as resistant, in addition to carbapenems, to at least three classes of antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and fluroquinolones [11] .
The minimum inhibitory concentrations (MIC) of imipenem was determined by E-test method (Biom erieux ® ) and the MIC of colistin was determined by broth dilution method (UMIC Biocenric ® ).
8
Antimicrobial Resistance Profiling of Gram-Negative Isolates
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Gram-negative isolates were tested for oxidase activity and screened for resistance to MEM using the Kirby–Bauer disk diffusion susceptibility test following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations [78 ]. Zone inhibition diameters of oxidase-negative strains were evaluated on the bases of the clinical and ECOFF breakpoints proposed for the Enterobacterales [79 ]. For oxidase-positive isolates, the clinical breakpoints for Pseudomonas were followed, as they were the only ones available. Resistant isolates or isolates with zone diameters smaller than the ECOFF value (available for Enterobacterales only) were identified at the species level by using API® 20 E system (bioMérieux, Marcy l’Etoile, France) or API® 20 NE system (bioMérieux). After identification, the isolates were frozen at −80 °C for further testing.
9
Revitalization of 14-year-old P. aeruginosa
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P. aeruginosa cells, maintained during 14 years in sterilised seawater, were revitalised by the addition of 100 ml of sterilised nutrient broth to the salt crystal in the Erlenmeyer flask and incubated at 37 °C with 100 rpm of shaking. Subsequent plating of an aliquot from this culture on nutrient agar yielded observable colonies. A few isolated colonies from the different replicas were recovered and saved for further analysis. Biochemical profiles of P. aeruginosa ATCC 27853 and the resuscitated cells (T48 variant in this study) were characterised using API 20NE system (bio-Merieux, France).
10
Pertussis Diagnostic Protocol: Culture and PCR
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The Pertussis Reference Laboratory samples included nasopharyngeal specimens from 16,151 hospitalized patients from Buenos Aires with signs/symptoms of pertussis infection. These samples were routinely screened for B. pertussis by culture and PCR. B. pertussis culture was performed on Regan-Lowe agar (Difco, https://www.fishersci.com ) supplemented with 15% (vol/vol) defibrinated fresh sheep blood at 36.5°C and monitored for 10 days. Suspected colonies were replicated in Bordet-Gengou agar (Difco) supplemented with 15% (vol/vol) defibrinated fresh sheep blood. Colonies exhibiting hemolysis were gram-stained and tested by using agglutination with B. pertussis–specific antiserum (Murex Diagnostic Products, https:///www.murex-ph.com ) and PCR (22 (link),23 (link)). The isolates were also biochemically typed by using the API-20-NE system (bioMérieux, https://www.biomerieux.com ).
Isolates were stored at –80°C in 1% (wt/vol) casaminoacid solution containing 20% (vol/vol) glycerol. B. pertussis strain Tohama phase I (Collection de l’Institut Pasteur) was also grown on Bordet-Gengou agar at 36.5°C for 72 h.
Isolates were stored at –80°C in 1% (wt/vol) casaminoacid solution containing 20% (vol/vol) glycerol. B. pertussis strain Tohama phase I (Collection de l’Institut Pasteur) was also grown on Bordet-Gengou agar at 36.5°C for 72 h.
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