Reverse primer

The presence of bacterial macrolide resistance gene ermA was detected by PCR and conducted at Loyola University Chicago. Forward primer: 5′-ACA TGA TAT TCC CTG TTT ACC CA-3′. reverse primer: 5′-TGG AAA TGA GTC AAC GGG TG-3′. Each PCR reaction was carried out in a final volume of 50 μL consisting of molecular grade nuclease-free water (35 μL), 10× Taq buffer (5 μL) (Thermo, Waltham, MA, USA), 25 mM MgCl₂ (3 μL), DNA template (3 μL), 10 mM dNTPs (1 μL), forward primer (1 μL), reverse primer (1 μL), and Taq polymerase (1 μL) (Thermo). The PCR reactions were performed in a SimpliAmp thermocycler (Applied Biosystems, Waltham, MA, USA). Thermocycling conditions were as follows: initial denaturation at 94 °C (10 min), 30 cycles of 94 °C (30 s) + 55 °C (1 min) + 72 °C (1 min), and final extension at 72 °C (7 min). PCR products were analyzed by separation on 1.0% agarose gels. A PCR reaction without template DNA was used as a negative control.

The presence of genes encoding for HPV E6E7 was detected using the PCR. The PCR mixture contained 100 ng DNA template, 0.5 µL forward primer (10 µM), 0.5 µl reverse primer (10 µM), 0.5 µl dNTPs (10 mM) (Thermo Scientific, #R0182), 2.5 µL 10x NEB buffer (BioLabs, #B9004S), 1 µL NEB Taq polymerase (5000 U/mL) (BioLabs, #M0267S), 1 µL MgCl₂ (25 mM) (Promega, #A351H) and the reaction was made up to 25 µL with H₂O. The PCR cycling conditions were as follows: initial denature of 5 min at 95 °C followed by 35 cycles at 95 °C for 30 s, at 55 °C for 30 s and at 72 °C for 1 min. The final extension was at 72 °C for 5 min. PCR products were directly processed using agarose gel electrophoresis. DNA fragments were separated in 1.5% agarose (Biozym, #840004) in 0.5% v/v TBE buffer supplemented with ethidium bromide solution at 120 V for 60 min. Up to 15 µL of PCR product was loaded into the agarose gel using a 6x DNA loading buffer (Fermentas, #R0611). Primers for HPV testing were described in^{46 (link)}. Following primers for GAPDH were used as controls: Forward: 5′-GGTATCGTGGAAGGACTCATGAC-3′, Reverse: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′; The primers were purchased from Sigma-Aldrich and diluted to 10 µM. See Supplementary Fig. 6 for full scan of gels.

The total RNA of A2058 in each group was extracted based on the manufacturer's protocol of RNeasy Plus Mini Kit (QIAGEN, Valencia, CA, USA). RNA concentration was detected by NanoDrop 1000TM (Thermo Fisher Scientific, Waltham, MA, USA). An equivalent amount of RNA was reverse transcribed to cDNA using the SYBR PrimeScript^™ RT Master Mix (Takara Bio, Otsu, Japan). Referring to the instructions, real‐time PCR was processed on LightCycler 480 (Hoffman‐La Roche Ltd., Basel, Switzerland), using SYBR Premix Ex Taq Kit (Takara Bio, Otsu, Shiga, Japan). Each reaction system was 20 µL, including SYBR@Premix Ex TaqII (10 µL), ROX Reference Dye (0.4 µL), Forward Primer (0.4 µL), Reverse Primer (0.4 µL), cDNA (2 µL), and DEPC‐treated water (6.8 µL) (Invitrogen^™). GAPDH was chosen as the internal reference. All reactions were run in triplicate. The primer sequences were listed in Table 2.

Primers and probes specific to the k141_29 were designed according to the TwistDX instructions and further evaluated by TwistAmp nfo kit (TwiatDx, UK). An internal probe with a FAM, THF and C3 spacer were designed and a biotin was added at the 5ʹ end of the reverse primer (Invitrogen). The primers and probes were listed in Table S5. RPA reaction was carried out using TwistAmp nfo kit (TwistDX, UK) following the manufacturer’s instructions with 50 μL reaction mixture. To analyze the results, 2 μL RPA reaction mixture was mixed with 98 μL dipstick assay buffer and 10 μL of the diluted sample was transferred to the sample pad of a Genline Hybridetect-1 lateral flow strip (Milenia Biotec, Germany). The strip was vertically placed in 200 μL of PBST buffer and incubated for 5 min at room temperature.

The detection of virulence genes was performed by using PCR. The following genes were analyzed: ptA, zapA, ucaA, ireA [30 (link)], hpmA [7 (link)], mrpA [28 (link)], pmfA [36 (link)], and atfA [35 (link)]. The bacterial DNA was extracted using the boiling method, followed by thermal shock. PCR was performed in a thermocycler containing a final volume of 25 μL, which was composed of 1.0 μL of MgCl₂ - 2 mM - (Invitrogen®), 2.5 μL of 10X buffer (Invitrogen®), 2.5 μL of dNTPs - 0.2 mM (Invitrogen®), 0.5 μL of Forward Primer - 20 pmol, and 0.5 μL of Reverse Primer - 20 pmol (Invitrogen®), 0.5 μL of Taq DNA polymerase - 1.5 U/μL (Invitrogen®), 2.0 μL of bacterial DNA, and 15.5 μL of ultrapure water. The standard P. mirabilis HI4320 strain was used as a positive control and the reaction without DNA was used as a negative control. After the PCR, the samples were subjected to electrophoresis in an agarose gel at a concentration of 1%–2%, according to the size of the fragment to be amplified, which was stained with SYBR SAFE (Invitrogen®) and emerged in Tris-Borate-EDTA buffer (TBE) (89 mM Tris base, 89 mM Boric Acid, 2 mM EDTA; pH 8.3). The molecular marker used was a 1 kb ladder (Invitrogen®). The system was subjected to a constant voltage of 70 V for 50 min. After the estimated time, the gel was observed using a transilluminator with an ultraviolet light.