Elizabethkingia anophelis strain EM361-97 was isolated from the blood of a 46-year-old male patient with advanced nasopharyngeal carcinoma and lung cancer. During admission, the patient suffered from pneumonia, respiratory failure, and profound shock. He initially received empirical antibiotics with levofloxacin. Unfortunately, the patient died several days after this infection. One blood culture from the patient yielded a gram-negative bacillus that was initially identified as E. meningoseptica using API/ID32 GN (bioMérieux S.A., Marcy l’Etoile, France) by the clinical microbiology laboratory. This isolate was named strain EM361-97 and was stored at −80 °C as a glycerol stock for further experiments. We re-identified this isolate as E. anophelis using 16S ribosomal RNA (rRNA) gene sequencing as previously published15 (link). The minimum inhibitory concentration (MIC) of this isolate was examined using the broth microdilution method. The susceptibilities were determined according to the interpretive standards for “other non-Enterobacteriaceae” as suggested by the Clinical and Laboratory Standards Institute (CLSI) guidelines16 .
Api id32 gn
Manufactured by bioMérieux
Sourced in France
The API/ID32 GN is a manual microbiology identification system for the identification of Gram-negative bacilli. It provides a standardized, miniaturized, and easy-to-use format for the biochemical identification of a wide range of Gram-negative bacteria.
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3 protocols using api id32 gn
1
Isolation and Characterization of Elizabethkingia anophelis
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Elizabethkingia anophelis strain EM361-97 was isolated from the blood of a 46-year-old male patient with advanced nasopharyngeal carcinoma and lung cancer. During admission, the patient suffered from pneumonia, respiratory failure, and profound shock. He initially received empirical antibiotics with levofloxacin. Unfortunately, the patient died several days after this infection. One blood culture from the patient yielded a gram-negative bacillus that was initially identified as E. meningoseptica using API/ID32 GN (bioMérieux S.A., Marcy l’Etoile, France) by the clinical microbiology laboratory. This isolate was named strain EM361-97 and was stored at −80 °C as a glycerol stock for further experiments. We re-identified this isolate as E. anophelis using 16S ribosomal RNA (rRNA) gene sequencing as previously published15 (link). The minimum inhibitory concentration (MIC) of this isolate was examined using the broth microdilution method. The susceptibilities were determined according to the interpretive standards for “other non-Enterobacteriaceae” as suggested by the Clinical and Laboratory Standards Institute (CLSI) guidelines16 .
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Elizabethkingia anophelis strain EM361-97 was isolated from the blood of a 46-year-old male patient with advanced nasopharyngeal carcinoma and lung cancer. During admission, the patient suffered from pneumonia, respiratory failure, and profound shock. He initially received empirical antibiotics with levofloxacin. Unfortunately, the patient died several days after this infection. One blood culture from the patient yielded a gram-negative bacillus that was initially identified as E. meningoseptica using API/ID32 GN (bioMérieux S.A., Marcy l’Etoile, France) by the clinical microbiology laboratory. This isolate was named strain EM361-97 and was stored at −80 °C as a glycerol stock for further experiments. We re-identified this isolate as E. anophelis using 16S ribosomal RNA (rRNA) gene sequencing as previously published15 (link). The minimum inhibitory concentration (MIC) of this isolate was examined using the broth microdilution method. The susceptibilities were determined according to the interpretive standards for “other non-Enterobacteriaceae” as suggested by the Clinical and Laboratory Standards Institute (CLSI) guidelines16 .
2
Comprehensive Characterization of Novel Bacterium
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Gram-staining, oxidase and catalase reactions, and motility (the hanging drop method) were determined as described by Gerhardt et al. (1994) . The morphology of cells grown in MB and negatively stained with a 1% phosphotungstic acid on carbon-coated 200-mesh copper grids was examined by electronic transmission microscopy [Libra 120 FE (Carl Zeiss), provided by the Far Eastern Centre of electronic microscopy, Zhirmunsky Institute of Marine Biology, Far Eastern Branch of the Russian Academy of Sciences]. Hydrolysis of starch, casein, gelatin, Tween 80, DNA, L-tyrosine, chitin, nitrate reduction (sulfanilic acid/α-naphthylamine test), and growth at different salinities (0-12% NaCl), temperatures (5-45 °C), and pH values (4.5-10.0) were carried out using arti cial sea water (ASW) as described in a previous paper (Romanenko et al. 2013 ). The arti cial sea water (ASW) contained (per liter of distilled water): 30 g NaCl, 4.9 g MgCl 2 , 2.0 g MgSO 4 , 0.5 g CaCl 2 , 1.0 g KCl, 0.01 g FeSO 4 . Biochemical tests were performed using API 20E, API 20NE, API ID32 GN, and API ZYM test kits (bioMérieux, France) as described by the manufacturer except the cultures were suspended in ASW.
3
Comprehensive Bacterial Characterization Workflow
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Cell morphologies of new isolates were observed using transmission electron microscopy (JEOL, JEM1010) using the negative staining method. The Gram-staining reaction was performed using a kit, following the manufacturer's instructions (bioMérieux). Catalase activity was determined by adding 3% (w/v) H 2 O 2 solution and oxidase activity was examined using 1% (w/v) tetramethyl-p-phenylenediamine diamine. (Cappuccino and Sherman 2002) . The bacterial growth of strains BT186 T and BT505 was tested on Reasoner's 2A (R2A) agar, Luria-Bertani (LB) agar, Tryptic Soy Agar (TSA) Nutrient Agar (NA), and MacConkey (MAC) agar, respectively. Growth at 10, 15, 20, 25, 30 and 30 °C was assessed under various pH conditions (5 to 9, 1 pH intervals) and different effect of NaCl concentrations (1%to 5% [w/v %], 1% intervals). Enzyme activities, assimilation of carbon sources, acid production from substrates and other physiological characteristics were determined by inoculating API 20NE, API ZYM and API ID32GN strips performed according to the manufacturer's instructions (bioMérieux).
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