DNA, Bacterial

The cecal contents bacterial DNA of each sample was extracted using HiPure Stool DNA Kit B (Magen, Shanghai, China) following the manufacturer’s instructions. The DNA extractions were quantified by ultraviolet spectroscopy and amplified using universal primers of 341F (CCTACGGGNGGCWGCAG) and 806R (GGACTACHVGGGTATCTAAT) to target the V3–V4 domain of bacterial 16S rRNA. The amplicons were normalized, pooled, and sequenced on the Illumina GAIIx platform.
The raw Illumina fastq files were quality-filtered, de-multiplexed, and analyzed using quantitative insights into microbial ecology. Sequences with more than one ambiguous nucleotide or within correct barcodes or primers were removed. The Ribosomal Database Project classifier was used to classify tags into different taxonomies against Greengenes Database (version 20101006) with confidence threshold of 0.5. The software Mothur was used to cluster tags of more than 97% identity into operational taxonomic units (OTUs), and then the abundances of OTUs were calculated.

Bacterial genomic DNA was extracted using a rapid procedure described by Pitcher et al[18 (link)]. Plasmid DNA was extracted using GeneAid Hi-Speed Plasmid Mini kit (GeneAid, Taiwan). Standard PCR amplifications were performed with Biotools DNA polymerase (Biotools, Spain). All primers used for PCR were synthesized by 1^st Base Singapore and are listed in Additional file
1: Table S1. Electrocompetent cells were prepared from 6 ml overnight bacterial culture according to the procedure described by Choi et al (2005)
[19 (link)]. Electroporation was carried out by placing 100 μl electrocompetent cells and 3 μl plasmid DNA in a sterile cuvette (0.1 cm electrode gap, Bio-Rad) and pulsed at 1.8 V using settings for bacteria in a Bio-Rad MicroPulser.
The plasmid, pwFRT-Tel^R, was digested with XmaI and the 3.265 kb fragment carrying the tellurite-resistance cassette was isolated and ligated with XmaI-linearized pMo130 to produce the suicide plasmid, pMo130-Tel^R. The orientation of the tellurite-resistance cassette insert shown in Figure 
1A was ascertained by digesting the plasmid with Xho1 and BamHI which gave a 4.161 kb and a 5.231 kb band. An insertion of the tellurite-resistance cassette into pMo130-Tel^R in the opposite orientation would have produced two bands of 1.150 kb and 8.242 kb.

Individuals were grouped as follows. Group 1 (five D. reticulatum fed a lab diet for 7 days); Group 2 (five D. reticulatum fed a lab diet for 14 days); Group 3 (five D. reticulatum fed a lab diet and infected on Day 7 with P. hermaphrodita with feces collected 7 days postinfection—14 days in total); Group 4 (three A. valentianus fed a lab diet for 7 days); Group 5 (three A. valentianus fed a lab diet for 14 days); Group 6 (three A. valentianus infected with P. hermaphrodita with feces collected 7 days postinfection—14 days in total). Feces were collected from each slug for DNA extraction.
DNA was extracted from feces using DNeasy PowerSoil Pro Kit (Qiagen) following the manufacturer's instructions. The presence of bacterial DNA was checked after extractions using PCR amplification of the hypervariable regions of the 16S rRNA gene. This was carried out using the primers 27f (5′‐AGAGTTTGATCMTGGCTCAG‐3′) and 1492r (5′‐TACGGYTACCTTGTTACGACTT‐3′) (Lane, 1991 ) with the following thermocycler conditions: 3 min at 95°C followed by 35 cycles of 15 s at 95°C, 30 s at 55°C, 1.5 min at 72°C, and a final step of 8 min at 72°C. Amplicons were visualized using agarose gel electrophoresis to confirm that PCRs had worked; in all cases, bands of the correct size were present, and no amplification of bacterial DNA could be seen in the extraction negative control or the PCR negative control.
DNA samples were sent for 16S rRNA metagenomic sequencing (Novogene). The V4 hypervariable region of the 16S rRNA gene was amplified using the primers 515F (5′‐GTGCCAGCMGCCGCGGTAA‐3′) and 806R (5′‐GGACTACHVGGGTWTCTAAT‐3′). All PCR reactions were carried out with Phusion® High‐Fidelity PCR Master Mix (New England Biolabs). Sequencing libraries were generated with NEBNext® Ultra^TM DNA Library Prep Kit for Illumina and quantified via Qubit and Q‐PCR. Libraries were sequenced on an Illumina NovaSeq. 6000 platform to generate 2 × 250 bp paired‐end reads.
Analysis of the raw reads occurred at Novogene using the following method. Paired‐end reads were merged using FLASH (V1.2.7) (Magoč and Salzberg, 2011 (link)). Quality filtering on the raw tags was performed under specific filtering conditions to obtain high‐quality clean tags according to the QIIME (V1.7.0) (Caporaso et al., 2010 (link)). The tags were compared with the reference database (SILVA database) using the UCHIME algorithm (Edgar et al., 2011 (link)) to detect chimera sequences. Detected chimera sequences were then removed to obtain Effective Tags. All Effective Tags were processed by UPARSE software (v7.0.1090) (Edgar, 2013 (link)). Sequences with ≥97% similarity were assigned to the same Operational Taxonomic Units (OTUs).
For each OTU, QIIME (Version 1.7.0) in the Mothur method was performed against the SSU rRNA database of SILVA Database for species annotation at each taxonomic rank (Threshold:0.8~1) (Quast et al., 2012 (link)). MUSCLE (Version 3.8.31) (Edgar, 2004 (link)) was used to obtain the phylogenetic relationship of all OTUs.
OTUs abundance information was normalized using a standard of sequence number corresponding to the sample with the least sequences. OTUs were analyzed for Alpha diversity (Wilcoxon test function) and Beta diversity (AMOVA—Analysis of Molecular Variance) to obtain richness and evenness information in samples. AMOVA was also used to compare the taxonomic compositions of infected and noninfected slugs in weighted PCoA. Analysis of Alpha and Beta diversity were all performed on the normalized data and calculated with QIIME (Version 1.7.0). Significant intragroup variation is detected via MetaStats based on their abundance.

Between 2013 and 2019, the NRC identified 329 non-duplicate NDM-5-producing E. coli isolates, including 224 isolates of known geographical location, which were obtained from 224 single patients. For these 224 NDM-5-producing E. coli isolates, bacterial cultures were grown overnight in Luria-Bertani (LB) medium (PanReac AppliChem ITW Reagents, Darmstadt, Germany) supplemented with meropenem (2 mg/L). Total genomic DNA from 1 mL bacterial cultures was extracted (Easy-DNA gDNA Purification Kit, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) and prepared (Nextera XT DNA Library Preparation Kit, Illumina, Eindhoven, the Netherlands) for WGS. Libraries were paired-end sequenced on a HiSeq 1500 (2 × 251 bp) or NextSeq (2 × 151 bp) instrument (Illumina, San Diego, United States) and quality of raw sequence data was checked using FastQC v0.11.9 [25 ]. Mash Distance v2.1 and Mash Screen v2.1 were performed on raw reads for species identification and contamination check, respectively [26 (link),27 (link)]. Reads were de novo assembled using SPAdes v3.10.1 with default parameters in the careful mode [28 (link)]. Quality metrics of assemblies were assessed using QUAST v5.0.2 without a reference sequence [29 (link)]. This dataset also included raw data of two isolates co-harbouring bla_NDM-5, which were sequenced as part of a previous study [3 (link)].

Approximately 5 g of the middle section of the feces was collected and immediately frozen and stored at ‒80°C. The samples were transported on dry ice to Shenzhen Micro Health Gene Technology Co., Ltd. for high-throughput sequencing. MoBio's PowerSoil® DNA Isolation Kit was used to extract bacterial DNA from fecal samples. Amplification of the V3 – V4 region of the 16S rRNA gene in DNA was performed by polymerase chain reaction (PCR). Amplified samples were sequenced using the Illumina MiSeq high-throughput sequencing platform.