Api 20e strip

The ability of bacterial strains to use different organic compounds as sole energy and carbon source was evaluated using Biolog'S GN2 system, according to manufacturer's instructions. Biochemical characterization was performed using API 20E strips (Biomerieux), according to manufacturer'S instructions. Both systems were incubated at 37°C for 72 h.

Enterobacteriaceae strains were Gram strained and then identified with API 20E strips (bioMérieux, Marcy l’Etoile, France). Susceptibilities against antimicrobial agents and the detection of ESBL production were routinely performed in the two hospitals by the disc diffusion synergy method using discs containing cefepime, ceftazidime and cefotaxime each placed 30 mm apart around a disc containing clavulanic acid as recommended by the Antibiogram Committee of the French Microbiology Society [13 ].

Identification was achieved with API 20E strips (bioMerieux, France) in accordance to the Kit’s product insert.

We retrospectively collected 12,858 Gram-negative bacteria that were isolated from wildlife (6,226/12,858), patients (5,828/12,858), livestock and poultry (712/12,858), and environment (92/12,858) in 14 provinces (Anhui, Beijing, Gansu, Guangxi, Guizhou, Hainan, Hunan, Jiangxi, Ningxia, Qinghai, Sichuan, Tianjin, Yunnan, Zhejiang) of China from 2010 to 2019. The study was approved by the ethics review committee of the National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Informed consent was obtained from participants. All strains were identified using VITEK II Compact system (bioMérieux, France) or API 20E strips (bioMérieux, France). The mcr-1 or bla_NDM gene positive strains were identified again by VITEK II Compact system (bioMérieux, France), the results of which were consistent with the original ones.

As part of this study, reference strains originating from dogs were obtained from the University of Glasgow’s Veterinary Diagnostic Services laboratory for subsequent analysis. Identities of the strains were confirmed using API 20E strips (API system by bioMérieux, available at https://www.biomerieux.co.uk/product/apir-id-strip-range). A positive E. coli control and a negative Klebsiella species control were used for both genotypic and phenotypic confirmation of E. coli isolates. These two reference isolates were resistant to all antimicrobial agents used in this study.

The spleens of the rodents were tested for the presence of the Y. pestis pla gene by specific molecular-based detection (Charrel et al., 2004 (link)). The pla-positive spleen samples were inoculated onto two different culture media. Each spleen was crushed in 500 μl of brain-heart infusion broth, and 100 μl of this broth was inoculated onto both Columbia sheep blood agar (COS, BioMerieux, Marcy l'Etoile, France) and selective Yersinia BIN agar plates (Ber et al., 2003 (link)). The colonies obtained on both culture media were still tested for pla gene by PCR. The pla-positive colonies were checked by 16S rRNA and RpoB gene sequencing. One colony was picked for genome sequencing. The isolate pathogenicity was experimented using a mouse model, preparing a pure culture of the inoculum on COS plates. Colonies were harvested and suspended in sterile distilled water at a concentration of 10⁶/ml, and 100 μl of this suspension was inoculated subcutaneously into four Balb/c mice. The phenotypic profile of the isolate, subcultured on Mac Conkey agar and COP agar plates, was compared with that of the Y. pestis EV76 vaccinal strain using API20E strips form BioMerieux (Marcy l'etoile, France), according to the manufacturer's recommendations.

Specimens from cloacal/rectal swabs were directly streaked onto the MacConkey agar (Oxoid, Basingstoke, UK) without antibiotics and incubated at 37 °C aerobically for 24 h. A single colony from predominant morphologically similar colonies was picked from each plain MacConkey agar plate and subcultured in a nutrient agar (Hi media, Mumbai, India). Colonies on nutrient agar were identified by colonial morphology, Gram stain, catalase and oxidase production [85 (link)], and various biochemical tests (indole, methyl red, Voges–Proskauer, and citrate utilization test) and were later confirmed by API 20E following the manufacturer’s recommendations (BioMérieux, Marcyl’Etoile, France) [86 (link)]. Briefly, a single colony was emulsified into sterile saline and filled in the compartments and then incubated at 37 °C for 18 to 24 h aerobically in a wet chamber of analytical profile index (API), API 20E strips (BioMérieux, Marcy-Etoile, France). E. coli and other Enterobacteriaceae were identified at the species level.