In brief, BD8 was identified by Gram-staining and characterized biochemically using API Coryne at the species level according to the manufacturer's instructions (API Coryne, BioMerieux, France). Partial 16S rRNA gene sequencing analysis was used to characterize BD8 at the molecular level. Briefly, genomic DNA was isolated from a 1-day liquid culture using a GeneMATRIX Bacterial and Yeast Genomic DNA Purification kit 50 (EURx, Gdansk, Poland) and then the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using primers 27F (5′-AGR GTT YGA TYM TGG CTC AG-3′) and 1492R (5′-GGT 96 TAC CTT GTT ACG ACT T-3′). The PCR products were sequenced and analyzed at the Institute of Biochemistry and Biophysics, Polish Academy of Sciences (Warsaw, Poland), using the standard shotgun sequencing reagents and a 454 GS FLX Titanium Sequencing System (Roche, Basel, Switzerland), according to the manufacturer's instructions. The gene sequences obtained from the BD2 isolate were compared in the National Center for Biotechnology Information (NCBI) for homology using BLAST and multiple-aligned with 16S rRNA gene sequences of different strains.
Api coryne
Manufactured by bioMérieux
Sourced in France
The API Coryne is a biochemical identification system designed for the identification of Corynebacterium species and related organisms. It provides a standardized procedure for the biochemical characterization of these bacteria based on a series of enzymatic and metabolic tests.
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Colistin sulfate, by Thermo Fisher Scientific (1 mentions)
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Streptococcal grouping kit, by Thermo Fisher Scientific (1 mentions)
Columbia blood agar base, by Thermo Fisher Scientific (1 mentions)
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Maldi tof ms, by Bruker (1 mentions)
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22 protocols using api coryne
1
Identification and Characterization of Bacterial Isolate BD8
2
Corynebacterial Identification and Toxin Analysis
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We cultured samples on 5% sheep blood and serum tellurite agar plates (both Becton Dickinson, Heidelberg, Germany). Colonies suggestive of coryneform bacteria were subjected to MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry (Microflex; Bruker Daltonics, Bremen, Germany) and API Coryne (bioMérieux, Marcy-L’Ètoile, France) according to published methods (16 (link)) for species and biovar determination. We analyzed presence of the toxin gene by using a quantitative PCR approach (17 (link)).
3
Characterizing Optimal Growth Conditions
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To determine the optimal temperature and pH for growth, the strain was incubated for 7 days in BactoTM Tryptic Soy Broth (TSB; Becton, Dickinson and Company, Sparks, MD, USA) at 5 °C, 10 °C, 20 °C, 25 °C, 28 °C, 37 °C, and 45 °C and at pH 3 to pH 13, respectively. Growth in various concentrations of NaCl was also examined after 7 days of incubation in TSB. Chemotaxonomic experiments were conducted on the basis of a previous report [7 (link)]. Physiological and biochemical characteristics were evaluated using API ZYM, API Coryne, and API 50CH Biochemical Test Kits (bioMérieux, Marcy I’Etoile, France) according to the manufacturer’s instructions. Assimilation of carbon sources at a final concentration of 1% (w/v) was tested using ISP 9 agar as the basal medium according to Pridham and Gottlieb [8 (link)].
4
Bacterial Isolation and Identification Protocol
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Samples were plated on Blood Agar Base and Columbia Blood Agar Base with nalidixic acid and colistin sulfate (Oxoid ltd., Hampshire, UK), supplemented with 5% sterile defibrinated sheep blood and incubated both in aerobic and microaerophilic (5% CO2) conditions at 37 °C for 48 h. Colonies were selected and identified as previously described [7 (link)]. Further biochemical identification was performed using commercial identification galleries (API®Coryne and API®20Strep, bioMérieux, Marcy-l’Etoile, France) according to manufacturer’s instructions. Isolates were identified as a species only if identification scores in the multi-substrate identification systems were excellent, very good or good (90.0–99.9% ID); otherwise, identification was made only at the genus level (spp.). Latex agglutination test (Streptococcal grouping kit, Oxoid ltd, Hampshire, UK) and Christie Atkins Munch-Petersen test (CAMP test) were used for identification according to previous reports [30 (link)]. Pure cultures of each isolate were stored at − 70 °C.
5
Comprehensive Parasitic and Bacterial Identification
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Parasitological examination of the intestine was carried out resourcing to direct wet mount, sedimentation and filtration techniques. Liver, spleen and lung samples were analysed using standard bacteriological methods. Enterobacteriaceae and non-Enterobacteriaceae were tested using the ID 32E (Biomerieux®, Lisbon, Portugal) test and the API 20NE kit (Biomerieux®) test respectively. The presence of Streptococcus and Staphylococcus was investigates using the ID 32 STREPT (Biomerieux®) and the ID 32 STAPH kits (Biomerieux®), respectively. The API CORYNE (Biomerieux®) kit was used for the identification of Corynebacteria and coryne-like organisms. For Salmonella, peptone water and Rappaport Vassiliadis semi solid culture media were used. Whenever there was a suspicion of Salmonella, the agarose SMID2 and XLD culture media were used. Other culture media for bacterial identification in the samples included the MacConkey agar and the Blood agar culture media.
6
Nitrate Reductase Gene Screening in Isolates
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All isolates were screened for the presence of the nitrate reductase gene as previously reported by Almeida et al., using the primers narG_F and narG_R [20 (link)]. The DSM 7177 reference strain biovar Equi was used as positive control. The PCR mixture contained 5 μL of QIAGEN HotStarTaq mix, 1.5 μL H2O, 1 μM of each primer and 2.5 μL of the genomic DNA. Amplification was performed in a final volume of 10 μL using a thermal cycler (Applied Biosystems SimpliAmpTM Thermal Cycler, Thermo Fisher Scientific) as follows: first denaturation step at 95 °C for 15 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 90 s and a final extension of 10 min at 72 °C. The amplified products were analyzed with a capillary electrophoresis system (QIAxcel Advanced, QIAGEN, Hombrechtikon, Switzerland). In addition, the nitrate reduction reaction generated by the API Coryne (bioMérieux, Marcy-l’Etoile, France) test was retained as biochemical control for each isolate.
7
Reviving Diphtheria Bacteria from Clinical Isolates
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We revived 54 C diphtheriae C. diphtheriae
8
Biotyping and Genetic Characterization of C. pseudotuberculosis
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Biotyping of isolates was performed using API Coryne (BioMerieux, France) according to the manufacturer’s instructions. All isolates were identified for starch hydrolysis activity (Moussa et al., 2014 ). Briefly, brain heart infusion agar mixed with 0.4% starch was spot-inoculated with tested bacteria. Plates were incubated at 37 °C for 48 h, and then were covered with gram’s iodine. Iodine reacts with starch to form a dark blue background and clear areas around the spots of inoculum will appear. The total DNA of all strains were isolated as described previously by Pallen et al. (1994) (link) and used for the identification of 16s rRNA and PLD genetic characteristics of C. pseudotuberculosis. The detection of 16s rRNA gene of different isolates was carried out using PCR with purified DNA and the primer sequence of Dorella et al. (2006) (link). Additionally, all isolates were examined for the PLD gene using PCR technology according to Moussa et al. (2014) .
9
Isolating and Identifying C. diphtheriae
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C. diphtheriae CHUV2995 was isolated from the bronchoalveolar lavage (BAL) of a patient hospitalized in Lausanne University Hospital and subsequently identified using a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker, US). For Ion Torrent as well as for API Coryne (bioMérieux) identification tests (see below), bacteria were grown on blood agar plates at 37°C in a 5% CO2 humidified atmosphere for 24–48 h. For PacBio sequencing (see below), bacteria were grown in Todd-Hewith Broth (THB) at 37°C in ambient atmosphere for 48–72 h.
10
Taxonomic Identification of Bacterial Isolates
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For the taxonomic identification, additional to the 16S rRNA gene sequences and morphological traits, physiological and biochemical features of the isolates DK15 and ST13 were determined. Physiological criteria such as a sodium chloride resistance was tested on ISP9 medium supplemented with 2.5, 5, 7.5 and 10% of NaCl, utilization of different carbon sources (glucose, mannitol, arabinose, inositol, mannose, fructose, galactose, rhamnose, sucrose, xylose) was tested on ISP9 medium with the final concentration of carbon sources adjusted to 1%, pH (levels of 2-9) and temperature (4, 25, 28, 30, 37 and 42 °C) tolerance was tested on the ISP2 medium. Commercially available test kits such as an ApiZym® and ApiCoryne® (bioMérieux, France) were used for the biochemical characterisation. The Api stripes were inoculated following by manufacturer's manual. The observed characteristics were compared with the phylogenetically related type cultures obtained from German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany.
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