Gn card

Samples were collected from the tested surfaces (25 cm²), inside of a tap, drain traps and drain gratings using sterile swabs moistened in a sterile NaCl solution and/or using contact plates (Oxoid, Wesel, Germany). Identification to the level of genus was possible based on microscopic and macroscopic assessment of bacterial colonies as well as biochemical tests: ID Color Catalase (BioMérieux, Marcy-l’Étoile, France)—the reagent detects the presence of catalase, thus enabling the differentiation of bacteria that possess this characteristic, Bactident Oxidase test (MERCK, Darmstadt, Germany)—for the detection of cytochrome oxidase in microorganisms. Identification of the isolated strains to the level of species was carried out using an automatic identification system VITEK^® Compact 2 (BioMérieux, Durham, NC, U.S.). GN cards (BioMérieux, Durham, NC, U.S.) were used for identification of Enterobacterales and a select group of nonfermenting Gram-negative organisms, GP cards (BioMérieux, Durham, NC, U.S.) were applied for the identification of enterococci, streptococci, staphylococci and a selected group of gram-positive organisms. All identified strains were stored for further tests in temperature −80 ± 10 °C, in microbanks (Pro-Lab Diagnostics, Richmond Hill, ON, Canada).

Selective Brucella agar (SBA)-containing antibiotics were utilized as the selective media for Brucella species culture. Testing was performed using probable strains of short coccobacilli with very small colonies that were both catalase- and oxidase-positive to determine the formation of urease and H₂S. Agar plates (Sigma–Aldrich, Saint Louis, USA) were used to identify the isolates. A Vitek 2 instrument and GN cards (bioMe’rieux, France) were used as directed by the supplier. The rapid slide agglutination antigen and a complement-enzyme linked immunosorbent assay (cELISA) were used to identify the Brucella species obtained. As a confirmatory method of detection, a MALDI Biotyper (MBT) device was employed, as well as SYBR green real-time PCR analysis, which was verified by microfluidic electrophoresis investigations.

The suspected isolates that illustrated gram negative short coccobacilli from oxidase and catalase positive small colonies were examined for production of urease and H₂S. On Columbia agar (Sigma-Aldrich, Saint Louis, USA), the strains were recognized by the Vitek 2 device (bioMérieux SA F-69280 Marcy l’Etoile France), via GN cards (bioMérieux SA F-69280 Marcy l’Etoile France) as indicated in the company’s guidelines. Concisely, the bacterial suspension was prepared and balanced by McFarland standards (0.5 to 0.63). Vitek®2 cards were inoculated, and the cards were then submitted to the machine to for proper identification.

A total of 386 Enterobacteriaceae isolates (188 Klebsiella pneumoniae, 122 Escherichia coli, 76 Enterobacter cloacae) were collected according to random selection within group (ESBL negative isolates were random collected from ESBL negative group, ESBL positive isolates were random collected from ESBL positive group, CRE isolates were random collected from CRE group) from two different regions of China (Chengdu and Chongqing), all microorganisms were isolated from patient specimens (such as Urine, blood, sputum and secretion) during treatment, and then those isolates have to be preserved for scientific research. All strains were identified by GN card (bioMerieux, Durham, NC, USA) and ID32 GN (bioMerieux, Durham, NC, USA).