Bacterial dna extraction kit

Genomic DNA of 102 NDM-positive strains was extracted using the Tiangen Bacterial DNA Extraction Kit. The entire genomic DNA was fully sequenced using the Illumina HiSeq system (Novogene, Tianjin, China), and raw data were evaluated for quality and filtered by fastaQC v0.12.1 and Cutadapt v4.2 [17 (link),18 (link)]. Clean reads were assembled into contigs using SAPdes v.3.15.2 [19 (link)]. Whole genome sequencing of strains was performed using the Oxford Nanopore GridION (Novogene, Tianjin, China) and Illumina platforms. Using unicycler v0.5.0 [20 (link)], Illumina platform sequencing data and Nanopore sequencing data were subjected to mixed stitching.

The instrumentation used for the study is described in the Supplementary Materials S1. The bacterial DNA extraction kit, goat anti-mouse IgG antibody, rabbit anti-6-FAM polyclonal antibody (6-FAM), and anti-digoxin antibody used in this paper were purchased from Tiangen Biotech Co., Ltd. (Shanghai, China), BBI Sciences Corporation (Shanghai, China), Otwo Biotech (Shenzhen, China), and Abbkine (Wuhan, China), respectively. DNA marker 2000, isothermal amplification PCR mix, and bovine serum albumin (BSA) were provided by Sangong Biotech Co., Ltd. (Shanghai, China). SYTO9, streptavidin (SA) and polyethylenimine (PEI), MES monohydrate (MES), N(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), tetraethoxysilane (TEOS) and Tween-20 used in the synthesis of materials were obtained from Sigma-Aldrich (St. Louis, MO, USA). CdSe/ZnS-MPA QDs were ordered from Mesolight Inc. (Suzhou, China). The nitrocellulose membrane utilised in the immunochromatographic strips was procured from Sartorius (Madrid, Spain), while the remaining materials were sourced from Jieyi Biotechnology Co. (Shenzhen, China).

A Stool DNA Extraction Kit (Tiangen, Beijing, China) was used to extract DNA from 200 mg faeces and amniotic fluid following the kit instructions. A 10 ml sample of amniotic fluid was centrifuged at 10,000×g for 10 min, the supernatant was removed, and the pellet was used for extraction of bacterial DNA. Because the amount of bacterial in air, drinking water and colostrum samples may be very low, there may be fewer drug-resistant genes involved and can not be detected. Considering that the intestinal tract of newborns may also be a bacteria amplification environment, even a small amount of bacteria or drug-resistant genes may reproduce in large quantities. Therefore, air, drinking water and colostrum samples were cultivated and parallel blank samples run during the whole process. Further, in order to avoid contamination, the experiments were performed in the second-level biosafety laboratory and strictly followed the procedure of aseptic operation.
Colostrum, air and drinking water samples were cultured for 48 h and a Bacterial DNA Extraction Kit (Tiangen, Beijing, China) was used to extract DNA.

Sphingobium sp. ATCC 27551 was cultured in SP medium, as described by Ohmori et al. [31 ], and the genomic DNA was extracted using a bacterial DNA extraction kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. The full-length opd gene was amplified from the genomic DNA by polymerase chain reaction (PCR) to construct plasmid pUC-opd as described by Ohmori et al [31 ]. Using the plasmid pUC-opd as templates, the gene encoding OPH without N-terminus 29 amino acids was PCR-amplified with the designated primers (S1 Table), and overlap-extension PCR was performed to generate the mutants F132Y, L140Y, and F132Y/L140Y with primers listed in S1 Table. The PCR products were purified, digested with EcoRI and HindIII, and then ligated with vector pET-28. The constructed plasmids were transformed into E.coli DH5α for positive clone screening and DNA sequencing.

The in vitro experiments were divided into 4 groups based on the OD₆₀₀ value. Different groups were co-cultured in BHI in the presence of 10% glucose or 1% or 0.1% stevioside. The control group was cultured in BHI, and all bacterial solutions were filled to 2,000 µL with BHI and collected after 36 h. The bacteria were collected in centrifuged tubes and centrifuged at 10,000 rpm for 1 min, following which a bacterial DNA extraction kit (Tiangen, China) and SYBR Green kit (Cwbio, China) were used to evaluate the relative expressions of Fim-A and Hag-A in different groups via RT-PCR. The primers of different genes are all listed in Table 1.
Table 1

Primers sequences of different genes in the RT-PCR

Primers	Sequence (5’ to 3’)
Gapdh F	TGCACCACCAACTGTTA
Gapdh R	GATGCAGGGATGATGTT
TNF-α F	CCACGTCGTAGCAAACCAC
TNF-α R	TTGTCCCTTGAAGAGAACCTG
IL-1β F	CAGGCAGGCAGTATCACTCA
IL-1β R	TGTCCTCATCCTGGAAGGTC
IL-6 F	CCGGAGAGGAGACTTCACAG
IL-6 R	TCCACGATTTCCCAGAGAAC
U-16 S rRNA F	ACTCCTACGGGAGGCAGCAGT
U-16 S rRNA R	ATTACCGCGGCTGCTGGC
Pg 16 S rRNA F	TGGGTTTAAAGGGTGCGTAG
Pg 16 S rRNA R	CAATCGGAGTTCCTCGTGAT
HagA-F	ACAGCATCAGCCGATATTCC
HagA-R	CGAATTCATTGCCACCTTCT
FimA-F	TACTTCCACGCCTTCTCCTGTT
FimA-R	CATCTTTACTGTTGCCACTTCG

Genomic DNA of the bacterial community from each site was extracted using a bacterial DNA Extraction Kit (Tiangen Biotech, Inc., Beijing, China) according to the manufacturer’s protocols (Zhang et al., 2021a (link)). The DNA served as a template for PCR amplification of the V4 region of 16S rRNA using the primer set 515F/806R (Caporaso et al., 2011 (link); Walters et al., 2016 (link)). The sequencing library was set up when the amplicons of 16S rRNA were purified, and Ion S5™XL of Thermofisher was used in the sequencing. The raw fastq data were quality-filtered by low-quality parts and chimeric sequences to get clean reads (Martin, 2011 (link); Rognes et al., 2016 (link)). The clean reads were clustered into operational taxonomic units (OTUs) at the 97% similarity level using Uparse (Edgar, 2013 (link)). Since this study only focused on bacteria, we deleted all OTUs that did not belong to bacteria. The MUSCLE method and the SSU rRNA database of silva132 were used for the annotation species analysis (Wang et al., 2007 (link); Quast et al., 2013 (link)). We followed a previously reported method (Wemheuer et al., 2020 (link)) and applied Tax4Fun to reveal the functional and redundancy index (FRI) of the sequenced bacterial genome.