The cinnamon derived type A procyanidin (TAP) was obtained from Indus Biotech Pvt. Ltd., Pune, India. Nitrofurantoin (NIT) antibiotic was purchased from Sigma Aldrich, USA (98.0–102.0% purity). The antibiotic discs were purchased from HiMedia. The stock concentration (1 mg/mL) of TAP was prepared in sterile distilled water and the stock concentration (50 mg/mL) of nitrofurantoin was prepared in DMSO according to CLSI guidelines. The concentration of DMSO was maintained at >0.5% for all assays.

A total of 13 UPEC clinical isolates (QSLUPEC1–QSLUPEC13) were obtained from the microbial repository of JSS medical college, Mysore. These strains were collected at different time points from the patients reported to have urinary tract infections. The isolates were confirmed to be E. coli by standard microbiological screening methods. They were also checked for their expression of the fimX gene, which is type 1 pili regulator of E. coli (Bateman et al., 2013). The strains were maintained as glycerol stock at −80°C. Cation adjusted Muller Hilton Broth (CAMHB) was used for the determination of the antibacterial activity and synergy studies. The biofilm formation and inhibitory effect was done in the Luria Broth media (LB).

To screen the MDR strains, the clinical isolates were screened for resistance to the antibiotics. The following nine antibiotics which belong to different classes were tested for resistance: Co-Trimoxazole (COT), Trimethoprim (TMP), Ampicillin (AMP), Nalidixic acid (NAL), Streptomycin (STS), Cefuroxime (CXM), Cefotaxime (CTX), Norfloxacin (NOR), and Ciprofloxacin (CIP).

A standard disc diffusion assay was carried out for screening the resistant strains. Briefly, the bacterial suspensions were prepared from 16 h old plate. The turbidity was set equivalent to 0.5 McFarland standard (OD₅₉₅ = 0.08–0.1). The OD adjusted bacterial suspensions were swabbed onto Mueller-Hinton agar plates, incubated for 24 h at 37°C. The zone of inhibition was measured and interpreted with CLSI guidelines [CLSI M100–ED30: (Clinical and Laboratory Standards Institute (CLSI), 2020)]. According to the standard definition by Magiorakos et al. (2012), the strains resistant to three and more than three antimicrobial classes were defined as MDR.

The biofilm-forming capability of the 13 clinical isolates was assessed semi-quantitatively using the standard Tissue Culture Plate (TCP) method and qualitatively using Congo Red Agar (CRA) method (Hassan et al., 2011). For the tissue culture plate method, the overnight plate cultures of each of the clinical isolates were adjusted to 0.5 McFarland units (~1.5 × 10⁸ CFU/mL) with saline media. The prepared suspensions were added to 96 well microtiter plates containing LB media (1:10 dilution). After 24 h incubation at 37°C, absorbance was measured at OD₆₅₅ using ELISA plate reader (BioRad i-Mark, Japan). Then, the planktonic cells were removed by gently tapping the plate and subsequent washing with water twice. The wells were stained with 100 μL of 0.2% crystal violet stain and incubated for 20 min. After air-drying the plate for 30 min, the bound crystal violet was suspended in 33% acetic acid. The optical density was measured at the wavelength of 595 nm. The specific biofilm formation index was calculated as follows:

where SBF denotes Specific Biofilm formation index, AB denotes OD₅₉₅ of the stained attached bacteria, CW denotes OD₅₉₅ of the well containing LB media (blank), and G denotes OD₆₅₅ of the cell growth in the suspended culture (Naves et al., 2008).

Qualitatively, biofilm formation was assessed using Congo Red Agar (CRA) method. The congo red agar medium was prepared as described previously (Hassan et al., 2011). The bacterial suspensions were streaked in the CRA media and incubated for 24 h at 37°C. The colonies were visualized, and the strains with black colonies and dry consistency were interpreted as biofilm producers.

From the above assays, the strain that has the dual strength of being a strong biofilm producer and multidrug-resistant was chosen to understand the biological activity of TAP.

The biofilm inhibitory efficacy of the TAP was tested at different concentrations (128–2 μg/ml). The bacterial cultures inoculated in the above-mentioned conditions were incubated at 37°C for 24 h without agitation. The bacterial inoculated broth was taken as control and the uninoculated broth was taken as blank. The biofilm was processed using the crystal violet assay. Briefly, the planktonic cells were removed, and the adhered cells were fixed with methanol. The adhered biofilm cells were incubated with 0.2% crystal violet for 30 min at room temperature. The excess stain was washed, and biofilm stained crystal violet was eluted using 33% acetic acid. The optical density was measured at the wavelength of 595 nm. The treated biofilm at different concentrations formed is compared to the untreated culture. The minimum biofilm inhibitory concentration –MBIC₅₀ and MBIC₉₀ are the lowest concentrations at which the compound inhibits 50 and 90% biofilm as compared to the untreated control, respectively.

Confocal microscopy imaging was used to examine the biofilm inhibition of TAP. The bacterial suspensions were prepared as described above. The biofilm was allowed to form in clean, sterile coverslips in the presence and absence of the above-mentioned treatment. After 24 h incubation, the planktonic cells from coverslips were removed by rinsing with sterile water and stained with BacLight Bacterial Viability Kit (L7012) as per the kit protocol. A 40X objective lens was used to capture two and three-dimensional images using a confocal laser scanning microscope (Olympus FLUOVIEW, FV1000).

The double-dose response of TAP and nitrofurantoin was determined using checkerboard analysis. The overnight plate culture of the MDR strain was adjusted to 0.5 McFarland units (~1.5 × 10⁸ CFU/mL) with saline media. The prepared suspensions were added to 96 well microtiter plates containing CAMHB media (1:10 dilution). The different dilutions were prepared by dissolving TAP in the bacterial growth media (CAMHB). The concentration of TAP and nitrofurantoin for synergy studies were from 64 to 1 μg/ml: 2-fold dilution. The plates were incubated at 37°C for 24 h. After the incubation, the growth inhibition was measured by comparing the OD₅₉₅ of control and the plant polyphenols treated cells.

The synergism was evaluated using the BI (Bliss independence model) and FIC (Fractional Inhibitory Concentration) index. The calculations are similar to the previous reports (Kaur et al., 2016).

The synergism kinetics at different pH (5.2, 5.8, 6.4, 7.0, and 7.6) was performed in broth microdilution method for nitrofurantoin and TAP using 96 well microtiter plate. The bacterial suspension was prepared as mentioned above and was added (1:10 dilution) to the CAMHB media containing different concentrations of the TAP and nitrofurantoin (64–1 μg/ml: 2-fold dilution) at varying pH. The plates were incubated at 37°C and absorbance was measured at 595 nm at a time interval of 1–8 h and also after 24 h.

The biofilm inhibitory effect of the synergistic combination was understood using the gene expression analysis of the biofilm regulatory genes of UPEC. The planktonic cells (log phase) of UPEC were treated with Nitrofurantoin (8 μg/mL), TAP (32 μg/mL), a combination of TAP (32 μg/mL) and Nitrofurantoin (8 μg/mL) and incubated for 24 h at 37°C. The pH of 5.8 was maintained in all the treatments. Total RNA was extracted by following the manufacturer's guidelines of RNeasy® Protect Bacteria Mini Kit (Qiagen). Standard agarose gel electrophoresis procedure was developed to verify the integrity and NanoDrop (Thermo Scientific, USA) was done to evaluate the purity of isolated RNA. From the isolated RNA, cDNA was synthesized using iScript™ cDNA Synthesis Kit (Manufacturer's protocol was followed).

The expression level of genes responsible for adhesins of UPEC was analyzed using qRT-PCR. The respective primers and the melting temperature of each of the gene used were listed in Table S1. Reference gene and negative control are 16srRNA and without cDNA were maintained, respectively. Calculations of relative gene expression were done with 2^−ΔΔCT method (Hema et al., 2017).

GraphPad Prism software version 8.0.2 (GraphPad Software Inc., San Diego, CA, United States) was used for carrying out the statistical analysis. The significance was checked with Student t-test with p set at p ≤ 0.05. All the assays were carried out in biological and technical triplicates, and the results were expressed as mean ± SD.

For the clinical isolates tested, the most common resistance was observed in nalidixic acid, ciprofloxacin and ampicillin, followed by cefuroxime, cefotaxime and norfloxacin, tetracycline, co-trimoxazole, trimethoprim, and streptomycin.

It is interesting to note that no isolate was resistant to all nine antibiotics considered. But all were resistant to at least three antibiotics. Thus, all the strains were multidrug-resistant according to the definition by Magiorakos et al. (2012), QSLUPEC1 and QSLUPEC6 were resistant to at least one agent in three antimicrobial classes whereas the majority of the strains, QSLUPEC2, QSLUPEC9, QSLUPEC10, QSLUPEC11, QSLUPEC12, and QSLUPEC13 were resistant to in four antimicrobial classes. The clinical isolates, QSLUPEC3, QSLUPEC5, QSLUPEC7, and QSLUPEC8 were resistant to at least one agent in five antibiotic categories (Table S2).

The biofilm-forming capacity of the clinical isolates was analyzed using TCP method and CRA method. The data are summarized in Table S3. According to Naves et al. (2008), the strains having SBF > 1.10 is considered to be strong biofilm producers, 0.7–1.09 are moderate biofilm producers, 0.35–0.69 are weak biofilm producers. The SBF <0.35 are not capable of biofilm formation.

Of the 13 clinical isolates, seven isolates were strong biofilm producers, five were moderate biofilm producers, and only one was a weak biofilm producer. The CRA method correlated with the TCP method except for one clinical isolate. For QSLUPEC2, which showed a moderate biofilm production in the TCP method and observed pink colonies in the case of CRA method.

The correlation between biofilm formation and antibiotic resistance were next analyzed. Out of the three classified MDR isolates, three were strong biofilm formers (QSLUPEC3, QSLUPEC5, and QSLUPEC7). QSLUPEC8 was a moderate biofilm producer. In order to validate the biofilm inhibition and synergy activity of TAP, QSLUPEC7, which is multidrug-resistant and also a strong biofilm producer, was chosen.

The antimicrobial and antibiofilm activity of TAP against the QSLUPEC7 was evaluated. TAP did not have any antimicrobial activity across the concentrations considered (Data not shown). The maximum biofilm inhibitory activity was recorded at 128 μg/mL, and a monotonic dose curve was observed (Figure 1A). The confocal micrographs also substantiated the quantitative results. There was a significant reduction in the biofilm formation with TAP treatment (~71%). The absence of the red fluorescence supported the fact that there was no cell death with the TAP treatment even at the highest concentration considered (Figure 1B).

The antibiofilm activity at a very low concentration of TAP directed toward the evaluation of potentiating activity of TAP with the known frontline antibiotic, NIT. Previous reports state that the effective pH of nitrofurantoin activity is 5.5–6.5 (Fransen et al., 2017). Thus, the synergy activity was tested in different pH (5.2, 5.8, 6.4, 7.0, and 7.6) in a time-dependent manner. Figure 2 shows the time-dependent antibacterial activity at a different pH and synergy concentration. It is observed that the antimicrobial action increases in a time-dependent manner. Thus, TAP potentiates the activity of nitrofurantoin significantly at pH 5.8 and pH 7.6, as compared to the other pH values considered. Table 1 shows the synergy scores calculated using the BLISS independence model in a pH and time-dependent manner. The Bliss score and FIC index values indicate that synergistic action of the TAP and nitrofurantoin. Taking both the synergy models into the account, a strong synergism was observed for pH 5.8, at a lower concentration of 8 μg/mL of NIT and 32 μg/mL. Even though the FIC index indicated synergy in the case of pH 5.2, the BLISS calculation indicated weak synergy. A moderate synergy was observed for pH 6.4 and pH 7.0. A stronger synergism was observed at pH 7.6 at a concentration of 16 μg/mL of NIT and 64 μg/mL.

Figure 2E shows a violin plot of the synergy activity at different pH considered in a time-dependent fashion. The maximum inhibition of 43% was recorded for pH 5.2 at 8 h and maintained till 24 h. A similar observation was made for pH 6.4 and 7.0, where the inhibition of 50–65% was maintained from 4 to 24 h. A notable observation is that the potentiation activity of TAP was enhanced at acidic of pH 5.8 as well as slightly alkaline (pH 7.6). The time scale mapping reveals that at pH 5.8 and 7.6, the antimicrobial activity is enhanced from the initial time point till 24 h. A time-dependent gradual increase in the growth inhibition was observed with >75% inhibition at pH 5.8 and 7.6.

The above studies established the anti-biofilm and potentiating activity of TAP. In order to understand the biofilm inhibitory role of TAP and the synergy combination, gene expression analysis was carried out at pH 5.8 (Figure 3). The genes that control bacterial adherence was considered as they are mainly responsible for the bacteria to adhere to the urinary tract. The combination treatment showed a ~4 log₁₀-fold reduction in the expression of focA, which encodes for the significant fimbrin subunit and belongs to the F1C fimbriae family. This was followed by P-fimbriae component, papG with a ~3.5 log₁₀-fold reduction. The type I fimbrial adhesion system, fimA and fimH were also downregulated with ~2 log₁₀-fold reduction. When compared to the other fimbriae systems considered, the S fimbriae (sfaA and sfaS) did not have a significant downregulation. There was only ~1 log₁₀-fold reduction observed. A noteworthy observation is that the adhesion systems considered were slightly upregulated with only nitrofurantoin treatment. There was a proportionate downregulation of the fimbriae genes found with the TAP treatment. The downregulation of the adhesins was enhanced in the synergy treatment as compared to the individual treatments.