Bacteriological examination of milk samples was performed according to Malinowski and Kołosowska [49 ]. Briefly, 10 μl of milk were cultivated on Blood Agar Base (bioMlrieux Poland), MacConkey Agar (BTL, Poland), Mueller-Hinton Agar (BTL, Poland), and Edwards Medium (Oxoid Ltd., England). Plates were incubated at 37°C and read at 24 and 48 h later. Colonies were identified by their colony morphology and Gram-staining. Detailed identification of isolated bacteria was performed using API tests (bioMerieux Poland).
Api test
Manufactured by bioMérieux
Sourced in France, Poland
API tests are a line of identification systems designed for the rapid and accurate identification of microorganisms. These tests provide a standardized, reproducible, and easy-to-use method for the biochemical identification of a wide range of bacterial and yeast species from clinical and industrial samples.
Automatically generated - may contain errors
Lab products found in correlation
Edwards medium, by Thermo Fisher Scientific (2 mentions)
Mannitol salt agar, by Thermo Fisher Scientific (1 mentions)
Blood agar base, by bioMérieux (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Api 50ch, by bioMérieux (1 mentions)
Agarose gel electrophoresis, by Merck Group (1 mentions)
Tbe buffer, by Merck Group (1 mentions)
Genomic mini kit, by A&A Biotechnology (1 mentions)
Mj mini gradient thermal cycler, by Bio-Rad (1 mentions)
Redtaq readymix dna polymerase, by Merck Group (1 mentions)
Clean up mini kit, by A&A Biotechnology (1 mentions)
Plate count agar pca, by Merck Group (1 mentions)
Bigdye terminator cycle sequencing ready reaction kit, by Thermo Fisher Scientific (1 mentions)
Dg18 medium, by Merck Group (1 mentions)
Tween 40, by Merck Group (1 mentions)
Iron 3 chloride hexahydrate, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
β carotene, by Merck Group (1 mentions)
Iron 2 sulfate heptahydrate, by Merck Group (1 mentions)
Etest, by bioMérieux (1 mentions)
13 protocols using api test
1
Milk Microbiological Examination Protocol
2
Milk Bacterial Profiling Protocol
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Milk samples were hand-mixed and opened in a biosafety level II cabinet. Bacteriological examination of milk samples was performed as recommended previously [59 ,60 (link)]. Briefly, 10 μL of milk were streaked by the quadrant streaking method over Blood Agar Base (bioMérieux, Warsaw, Poland), Mac Conkey Agar (BTL, Warsaw, Poland), Mannitol salt agar (Oxoid Ltd., Basingstoke, UK), and Edwards Medium (Oxoid Ltd., Basingstoke, UK) plates. Plates were incubated at 37 °C, and then read after 24 and 48 h. The bacteria were tentatively identified according to their cultural and morphological appearance, and Gram’s reaction [61 ]. Detailed identification of isolated bacteria was performed using standard biochemical tests and API tests (bioMérieux, Warsaw, Poland) [62 ,63 (link)].
3
Microbial Identification and Characterization
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All bacteria and yeasts isolated from samples were transferred onto individual culture plates. Following this, they were macroscopically and microscopically characterized using Gram-staining, catalase test and oxidase test (Microbiologie Bactident Oxydase, Merck, Germany). Next, isolates with the same morphology and biochemical features were grouped into strains and identified using API tests (BioMérieux, France): API 50 CH, API STAPH and API 20 NE (for bacteria) and API C AUX (for yeasts). Identified bacteria were genetically confirmed using the 16S rRNA gene nucleotide sequence (Jensen et al. 1993 (link)).
Isolated filamentous fungi were cultured on CYA (Czapek Yeast Extract Agar, Difco, USA) and YES medium (yeast extract with supplements) and visually identified, macroscopically and microscopically, using taxonomic keys (Bensch et al. 2012 (link); Frisvad and Samson 2004 ; Pitt and Hocking 2009 ; Klich 2002 ). Identity of moulds and yeasts was confirmed using ITS1/2 sequence of the rDNA region (White et al. 1990 ). Genomic DNAs were extracted using a method described previously (Stępień et al. 2011 (link)). The resulting nucleotide sequences of the studied micro-organisms were analysed and compared to the sequences published in the National Center for Biotechnology Information (NCBI) database, using the BLASTN 2.2.27+ program (Zhang et al. 2000 (link)).
Isolated filamentous fungi were cultured on CYA (Czapek Yeast Extract Agar, Difco, USA) and YES medium (yeast extract with supplements) and visually identified, macroscopically and microscopically, using taxonomic keys (Bensch et al. 2012 (link); Frisvad and Samson 2004 ; Pitt and Hocking 2009 ; Klich 2002 ). Identity of moulds and yeasts was confirmed using ITS1/2 sequence of the rDNA region (White et al. 1990 ). Genomic DNAs were extracted using a method described previously (Stępień et al. 2011 (link)). The resulting nucleotide sequences of the studied micro-organisms were analysed and compared to the sequences published in the National Center for Biotechnology Information (NCBI) database, using the BLASTN 2.2.27+ program (Zhang et al. 2000 (link)).
4
Evaluation of Bacterial Strains for Research
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The following standard strains were used in the study: E. coli ATCC 25922, NCTC 10538 and NCTC 8196; K. pneumoniae ATCC 13883 and ATCC 700603; Proteus vulgaris ATCC 13315; P. aeruginosa ATCC 27853, NCTC 6749 and PAO1; S. maltophilia ATCC 13637 and ATCC 12714; A. baumannii ATCC 19606; and Burkholderia cepacia ATCC 25416. This study also included 180 clinical strains, i.e., 36 strains of 5 Gram-negative rod species (E. coli, K. pneumoniae, P. aeruginosa, S. maltophilia, and A. baumannii). Clinical strains were isolated from samples of different materials derived from hospitalized patients in Warsaw in the period 2007–2010 and were identified in hospital microbiological laboratories by routine microbiological methods using API tests (bioMérieux, Marcy l’Etoile, France). All strains were stored at −80 °C until analysis. Prior to testing, each strain was subcultured twice on TSA (bioMérieux) medium for 24–48 h at 30 °C to ensure viability.
5
Antimicrobial Susceptibility of Clinical Isolates
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The following standard strains were used in the study: E. coli ATCC 25922, NCTC 10538 and NCTC 8196; Klebsiella pneumoniae ATCC 13883 and ATCC 700603; Proteus mirabilis ATCC 12453; Proteus vulgaris ATCC 13315 and NCTC 4635; Enterobacter cloacae DSM 6234; P. aeruginosa ATCC 27853, ATCC 25616, NCTC 6749 and PAO1; Stenotrophomonas maltophilia ATCC 13637 and ATCC 12714; Acinetobacter baumannii ATCC 19606; and Burkholderia cepacia ATCC 25416. These studies also included 72 clinical strains, i.e. a dozen strains of six Gram-negative rod species (E. coli, K. pneumoniae, P. mirabilis, P. aeruginosa, S. maltophilia, and A. baumannii). Clinical strains were isolated from different hospitalized patient samples obtained in Warsaw from 2007 to 2010 and were identified by routine methods using API tests (bioMérieux). All strains were stored at -80°C until analysis. Prior to testing, each strain was sub-cultured twice on TSA (bioMérieux) medium for 24 to 48 h at 30°C to ensure viability.
6
Identification of Bacterial Isolates
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Identification of colonies was initially performed by means of Gram staining and biochemical tests; specifically: catalase test, coagulase test and oxidase test. Then, API tests (bioMérieux S.A., France) were also performed. Two API tests were included in the present study. On the one hand, the API Staph was used to identify Staphylococcus spp., Micrococcus spp. and Kocuria spp. On the other hand, API 20E was used to identify microorganisms belonging to the genera Enterobacteriaceae and other non-fastidious Gram-negative rods.
7
Antioxidant and Microbial Analyses Protocol
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2-2-Diphenyl-1-picrylhydrazyl (DPPH); Folin-Ciocalteu’s reagent (2 N); (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox); (+)-catechin; and 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), linoleic acid, Tween 40, iron (II) sulfate heptahydrate, iron (III) chloride hexahydrate, β-carotene, RedTaq ReadyMix DNA polymerase, agarose gel electrophoresis and TBE buffer were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Plate count agar (PCA), DG18 medium, and bullion agar were purchased from Merck (Darmstadt, Germany). The oxidase test kit was obtained from Merck (Darmstadt, Germany). API Tests were purchased from BioMérieux (France). Genomic Mini kit and Clean Up Mini Kit were purchased from A&A Biotechnology (Gdynia, Poland). MJ Mini Gradient Thermal Cycler was obtained from Bio-Rad, (Hercules, CA, USA). BigDye Terminator Ready Reaction Cycle Sequencing kit was purchased from Applied Biosystems (Foster City, CA, USA). Unless indicated otherwise, all chemicals were purchased from Avantor Performance Materials Poland S.A. (Gliwice, Poland).
8
ESBL/AmpC Enterobacteria in Wild Animals
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A total of 17 positive ESBL/AmpC enterobacterial isolates recovered from 75 fecal samples of wild animals at pet market, Taif, Western Saudi Arabia (5 rock hyrax, 4 Yemen Linnet, 3 common kestrel, 3 red foxes, 3 long-tailed finches, 2 caracal, 2 peacock, 1 rock dove, 1 hamadryas baboon, 1 orange-winged parrot, 1 Burmese python, 1 Hill Mynah, 1 African gray parrot, 1 common myna) were included. Wild animals are caught or bought for pet, shops, local breeder or traded (sometimes illegally). The enterobacterial isolates were 9 E. coli, and single isolates of Klebsiella pneumonia, Klebsiella oxytoca, Proteusmirabilis, Proteus vulgaris, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Citrobacter youngae. Isolates were identified and confirmed by commercially available biochemical test (API tests; bioMérieux). The ESBLs and AmpC beta-lactamase production were achieved by commercially available Etest (bioMérieux).
9
Pseudomonas aeruginosa Isolates from Warsaw
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Nine hundred clinical isolates of P. aeruginosa obtained from 4 Warsaw hospital microbiological laboratories were examined in this study. These strains were isolated from samples of various clinical materials derived from different patients according to the principle of one isolate from one patient. The isolates were collected in the period 2000-2014 and were initially identified as ceftazidime or/and cefepime resistant P. aeruginosa strains in microbiological laboratories by routine microbial methods, including API tests (bioMérieux). All strains were stored at -80°C until analysis. Prior to testing, each strain was subcultured twice on TSA (bioMérieux) medium for 24 to 48 h at 30°C to ensure viability.
10
Microbial Characterization of Environmental Samples
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All bacteria and yeasts isolated from the samples (air, biomass, FFRs) were transferred onto individual culture plates. Following this, they were macroscopically and microscopically characterized using Gram-staining, catalase test and oxidase test (Microbiologie Bactident Oxidase, Merck, Darmstadt, Germany). Next, isolates with the same morphology and biochemical features were grouped into strains and identified using API tests (bioMérieux, Marcy-l’Étoile, France): API 50 CH, API STAPH and API 20 NE (for bacteria) and API C AUX (for yeasts). Isolated filamentous fungi were cultured on Czapek yeast extract agar (CYA, Difco, Franklin Lakes, NJ, USA) and yeast extract with supplements (YES) media and visually identified, macroscopically and microscopically, using taxonomic keys [21 ,22 ,23 ,24 (link)].
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