Microflex lt

An antecedent frozen sample as close as possible to the day of diagnosis of NEC (D₀) from every case and their corresponding contemporaneous control were selected for culture of both Clostridium species and Enterobacteriaceae. For isolation of Clostridium species, an alcohol shock method [29 ] was used to eliminate non-spore-forming organisms, and the suspension plated on Fastidious Anaerobic Agar (Oxoid). Additionally, samples were inoculated on to CHROMagar (BD) to aid identification of Enterobacteriaceae. All bacterial isolates were identified by matrix-assisted laser desorption/ionization–time of flight using a Bruker Microflex LT (Bruker Daltonics).

Following 48 and 72 h of incubation, the Petri dishes were examined and a single colony structure was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Microflex LT, Bruker Daltonik GmbH, Bremen, Germany). Briefly, a single colony from each Petri dish was transferred onto a MALDI-TOF MS target plate, covered with 1 μL of α-cyano-4 hydroxycinnamic acid (i.e. matrix solution) dissolved with 50% acetonitrile-2.5% trifluoroacetic acid (HCCA matrix), and left to dry at room temperature before protein analysis by MS. Subsequently, it was covered with the target, and analyzed by the mass spectra represented in term of the mass change ratios (m/z) of each microorganism. According to the library matching, the MS spectrum of unknown microbial isolates was compared with the MS spectra of known microbial isolates contained in the MALDI-TOF MS database. In order to detect the species level identification of microbes, a typical mass range m/z of 2–20 kDa was used by Flex Control software, in which mainly ribosomal proteins were represented together with housekeeping proteins.

Bacterial culture on GAM-agar plates was performed for 24 meconium samples (5 corresponding negative field controls) and 24 amnion samples (4 negative field controls). The frozen samples were first transferred to an anaerobic workstation (Ruskinn Concept Plus), mixed thoroughly and plated, using an aseptic technique, at +37°C. After this, the samples were transferred to a laminar flow cabinet and plated in aerobic conditions at +37°C. The culture plates were checked for growth daily for 14 days. All visible bacterial colonies were subcultured until pure isolates were obtained. Fresh cultures were then identified using MALDI-TOF (Bruker Microflex LT) at the Central laboratory of the Faculty of Veterinary Medicine (Helsinki, Finland).
Samples were prepared using MALDI Biotyper MSP Identification Standard Method v 1.1. Mass spectra were analyzed in a mass/charge range from 2,000 to 20,000 Da with MBT Compass v4.1 on flexControl v3.4 (Bruker Daltonik GmbH) using BDAL-7311 as the reference library. The Bruker Bacterial Test Standard (RUO) (Bruker Daltonik GmbH) was used for instrument calibration. If the identification confidence score was < 2.00, further identification was done with 16S rRNA gene amplicon Sanger sequencing at the Institute of Biotechnology (University of Helsinki, Finland).

Non-duplicated Enterobacteriaceae strains that exhibited carbapenem resistance phenotype (meropenem MIC ≥ 4 μg/ml) were collected from hospitals located in 25 Provinces and Municipalities in China, namely Anhui, Beijing, Fujian, Gansu, Guangdong, Guangxi, Guizhou, Hainan, Hebei, Henan, Hubei, Hunan, Jilin, Jiangxi, Liaoning, Nanjing, Shandong, Shanxi, Shaanxi, Shanghai, Sichuan, Tianjing, Xinjiang, Zhejiang and Chengdu, during the period, June 2014 through June 2015. One representative hospital (normally the largest general hospital in the location) from each location was chosen for sample collection. All strains were subjected to species confirmation using the Vitek 2 system (bioMérieux, Marcy-l'E' toile, France), and the MALDI-TOF MS apparatus (Bruker Microflex LT, Bruker Daltonik GmbH, Bremen, Germany).

Blood cultures in automatic systems (BD BACTEC™ FX Top-Unit; Becton Dickinson, Eysins, Switzerland), Gram staining and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Bruker Microflex LT; Bruker, Hamburg, Germany) were used in the diagnosis of bacterial infections. These diagnostic tests were performed at the Institute of Clinical Microbiology and Hygiene, University Hospital Regensburg, and have been described in more detail [35 (link)]. Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis are common pathogenic microorganisms in sepsis [36 (link)], and were detected in the blood of our patients.

From September 2015 to July 2016, five hospitals (two in Xuzhou, two in Suqian, and one in Lianyungang) in Northern Jiangsu Province of China collected 1,185 E. coli isolates to examine the prevalence and molecular epidemiology of carbapenem-resistance isolates. Initial species identification and antimicrobial susceptibility testing was performed by the Vitek 2 system (bioMe'rieux, France) and MALDI-TOF MS (Bruker Microflex LT, Bruker Daltonik GmbH, Bremen, Germany) according to the manufacturer's instructions.