Strain collection was performed from UTI cases, attending Jawaharlal Nehru Medical College & Hospital (JNMCH), Aligarh Muslim University (AMU) from October 2016 to March 2017. A total of 64 clean catch urine samples were collected from infected patient’s catheters in a wide mouth sterile container from the study subjects. Isolation was performed by a surface streak procedure on Luria Bertani (LB) broth (Himedia Labs, Mumbai, India) using calibrated loops for semiquantitative method and incubated aerobically at 37°C for 24 h, and those cultures which were negative after 24 h incubations, were further incubated for 48 h. A specimen was considered positive for UTI if a single organism was cultured at a concentration of ≥10⁵ cfu/ml. Out of 64 urine samples, 42 samples were found to be positive for UTI. Of 42, 26 samples were characterized to have E. coli isolates, confirmed through BD phoenix-100™ Automated Microbiology system using panel NMIC/ID-55.

For fimH detection a single colony on LB-plate from 26 remaining isolates were suspended in 100 µl of sterilized water and were incubated at 95°C for 10 min followed by centrifugation at 8,000 g for 10 min. Supernatant was used as template to perform colony PCR amplification under the condition, mentioned in supplementary data (Tables S1 and S2 in Supplementary Material) using appropriate primers. Among 26 isolates, 19 were reported to have fimH. Further detailed study was performed on one of the strains (AK-118) of 19 fimH positive strains.

Escherichia coli clinical strain, AK-118 was used in this study. The strain was isolated from UTI patient, admitted in JNMCH AMU, Aligarh, India. The bacteria were cultured in LB broth (Himedia labs, Mumbai, India). The plasmids pdCas9 and pgRNA used in study, already mentioned in our earlier study (16). LB media supplemented with proper antibiotic (ampicillin 100 µg/ml and chloramphenicol 25 µg/ml) and inducer [anhydrotetracycline (aTc) 2 µM] was used, wherever needed. E. coli Top10 cells were used for transformation, while the cotransformation was performed in clinical strain. The microorganism were grown without (on agar) or with (in broth) shaking at 220 rpm at 37°C, overnight. Knockdown strain was supplemented with 2 µM aTc with their respective antibiotics, to suppress gene expression.

The detailed protocol for cloning has already been mentioned in our earlier study (16). The complementary sequences, i.e., 20 bp region immediately following to 5′-CCN-3 [protospacer adjacent motif (PAM)] of fimH gene was commercially synthesized as a primers with additional 35 nt region of dCas9 handle (Table S1 in Supplementary Material). It was finally inserted through inverse PCR (Table S2 in Supplementary Material) in pgRNA (ampicillin resistant). The final PCR products were cleaned and ligated to give new sgRNA expressing plasmid pgRNA-FM to be ready for cotransformation along with pdCas9 (chloramphenicol resistant) in AK-118. The new knockdown strain was named as AK-FM1 for the record. The conditions used for PCR in this study were given in Table S2 in Supplementary Material.

Extraction of total RNA was performed by Trizol method from respective strains grown in presence of inducer (2 µM aTc) and proper antibiotic concentration till log phase. Total RNA was treated with RNase-free DNase in order to remove DNA contamination and subsequently cDNA was prepared by high capacity cDNA Reverse Transcription Kits (Applied Biosystems, USA) according to manufacturer’s instruction. Quantification of mRNA was performed through comparative Ct method using SYBR green PCR master mix, along with 150 ng of cDNA sample with appropriate primers. The cycle was carried out at 95°C for 10 min, 95°C for 15 s, 60°C for 30 s, and finally 72°C for 30 s. The standard graphs for respective transcripts were observed using 16s rRNA as endogenous gene control.

In our study, hemaggultination assay was performed on guinea pig erythrocytes with 1% mannose for type 1 fimbriae detection as described in previous study with the slight modification (18). A consent from institutional animal ethical committee of Interdisciplinary Biotechnology Unit (IBU), AMU (held on June 5, 2017, at IBU, AMU), was taken to perform this experiment. Bacteria were grown overnight in LB at 37°C for full fimbriation. The bacterial suspensions were adjusted to McFarland standard (0.5–0.6) in PBS or PBS/mannose 1% and serially diluted from 1:2 to 1:128 with same buffer in 96-well microtiter plate. 0.5% erythrocytes (in PBS or PBS/mannose 1%) was added to wells. Suspensions of bacteria and erythrocyte were incubated overnight at 4°C. All assays were conducted in triplicate. An E. coli ATCC strain (type 1 fimbriae positive) was used as a positive control.

Morphological study was performed using transmission electron microscopy using the standard TEM protocol to investigate the presence and absence of type 1 Fimbriae. Untreated and treated bacterial cultures were suspended using a centrifuge followed by washing with PBS (pH 7.4). Secondary fixation was done using gluteraldehyde (2.5%) and osmium tetroxide (1%) for 2–3 h at 4°C. Further Samples were dehydrated by ethanol and finally embedded in araldite CY212 (Taab, Aldermaston, UK) resin to make cell-pellet blocks. Ultrathin sections of cells stained with uranyl acetate and lead citrate were observed under the TEM (Model: JEM 2100, Jeol, Tokyo, Japan).

Biofilm formation was estimated by CV assay as used in our earlier study (19). Microtiter plates carrying secondary culture of treated and untreated bacterial cells were incubated at 37°C for 24 h, then medium was decanted and the planktonic cells were removed by gentle rinsing with sterile water. The adhered biofilms were stained for 15 min at room temperature with 200 µl of 0.1% CV dye and the bound dye was released from the cells by adding 98% ethanol and keeping the plates on shaker for 5 min. Biofilm quantification was done by measuring the optical density (OD) at 630 nm using a Bio-Rad iMark™ Microplate Reader, India.

To assess metabolic activity and cellular viability, XTT assay was carried out as described earlier (19). XTT (1 mg/ml) was dissolved in filter sterilized PBS solution and menadione (0.4 mM) was freshly prepared in acetone. Fresh mixture of 20:1 volume of XTT and menadione was used. After 24 h of growth, biofilms were washed with 200 µl of PBS solution followed by the addition of XTT-menadione and PBS solution to a volume of 42 and 158 µl in each well, respectively, and kept at 37°C in dark for 4 h. After 4 h, the intensity of orange color formazan compound was measured, that quantify the ability of metabolically active sessile cells to reduce tetrazolium salt (2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide), using microtiter plate reader at 490 nm.

Confocal microscopy was performed to visualize the adherence property of bacteria with the human intestinal mucus. For that a resected human intestine tissue was taken as a source of intestinal mucus, which was approved by the Institutional ethical committee of IBU, AMU held on June 3, 2017, at IBU (AMU) also an informed written consent was taken from patient which remained confidential as per the protocol, established in our institution. The healthy part of large intestine was taken from the patient of diverticulitis and processed within 20 min of sample collected on ice. Mucus was isolated from the sample after washing it with PBS containing 0.01% gelatin by gently scraping through rubber spatula and collected in HEPES-HANKS buffer (10 mmol/L HEPES; pH 7.4). The collected mucus was centrifuged (13,000 g, 10 min) to remove cell debris and bacteria and stored at −20°C for further use after measuring protein content.

As described elsewhere (20), in adhesion assay mucus was diluted to the concentration 0.5 mg/ml with HEPES-HANKS buffer and 100 µg of it was immobilized directly on confocal dishes and incubates at 4°C overnight. The overnight grown bacteria after washing with PBS and adjustment of OD 0.5 at 600 nm, were stained with DAPI (355 nm wavelength for excitation and 460 nm wavelength for emission) to a final concentration of 0.2 µg/ml (DAPI) by incubating it for 30 min on mild shaking at room temperature. Finally the bacterial cells were washed three times with PBS and added to a volume of 100 µl into confocal dishes. Bacteria were allowed to adhere at 37°C for 1 h and wells were washed with HEPES-HANKS buffer to remove non adherent bacteria. The adherent bacterial cells were observed by Zeiss LSM 780 (Germany) confocal laser scanning microscope equipped with argon and He-Ne laser.

To perform fluorescence spectroscopic studies the above mentioned adherent bacteria were released and lysed with 1% SDS-0.1 M NaOH by incubation at 60°C. The florescence quenching of released stained bacteria (DAPI stained) were determined by using Shimadzu RF-5301PC spectrofluorometer (Shimadzu Corporation, Kyoto, Japan) equipped with a thermostatically controlled cell holder. The filters used were 355 nm for excitation and 460 nm for emission.

The results were reported as mean ± SD. All experiments were conducted in triplicate. Further each experiment was compared with control for analyzing Student’s t-test, two-tailed hypothesis (*p < 0.05, t-test, two sided) (**p < 0.005, t-test, two sided).

Successful implication of CRISPRi was assayed through fimH mRNA quantification using relative qRT-PCR, taking 16s rRNA gene as endogenos control. Figure 2A corresponds to the relative qRT-PCR data of fimH which showed the downregulation of fimH gene in treated condition. Taking value of relative fold change in untreated condition as 1 ± 0.414, the value of relative fold change in treated condition was observed to be 0.1560 ± 0.419, i.e., 84.39% of downregulation was observed with P-value < 0.005. Figure 2B corresponds to relative qRT-PCR data of dCas9 nuclease in strain AK-118 and proved its successful expression in this strain under induced condition with relative fold change values of 5.2986 ± 0.099 as compare to uninduced condition values 1 ± 0.080 (p-value < 0.005).

The effect of fimH gene suppression for type 1 fimbriae expression was assayed by Mannose sensitive hemaggultination assay on guinea pig erythrocytes and TEM studies. In Figure 3, row A and row B corresponds to AK-FM1 under treated and untreated conditions, respectively. In row A, a least agglutination was observed in first two wells followed by formation of blood button in the center of the well on serial dilution, while in row B, agglutination of guinea pig erythrocytes were clearly seen in almost all wells. Figure 4 shows the TEM images of AK-FM1 single bacterium in untreated (Figure 4A) and treated (Figure 4B) conditions taken from four different visual fields. In control the fimbriae appendages were present on bacterial surface due to the expression of type 1 fimbriae, while in treated cells nearly smooth bacterial surface was observed.

The biofilm forming tendency of AK-FM1 after CRISPRi treatment was preliminary investigated by CV assay as shown in Figure 5A. The observed value of CV assay in treated sample was 0.445 ± 0.012 (p-value < 0.005) as compared to untreated sample value 0.834 ± 0.006 (p-value < 0.005). The downregulation of fimH gene in knockdown strain, AK-FM1 (treated) showed the dramatic decrease in biofilm formation as quantified by CV assay. The metabolic activity and cell viability of AK-FM1 in treated condition was recorded to be 74.06% with the values of 1.69 ± 0.040 and 2.29 ± 0.126 (p-value < 0.005) in treated and untreated conditions, respectively, as quantified by the converted amount of XTT in XTT reduction assay (Figure 5B).

The bacterial adherence level on human intestinal mucus after CRISPRi-mediated fimH gene suppression was visualized through confocal microscopy. Figures 6A,B correspond to confocal microscopy images of untreated and treated samples of AK-FM1 taken from four different visual fields, respectively. The difference in the intensity of blue color due to DAPI stained adherent bacterial cells in untreated and treated samples, indicated the noticeable reduction in the adherence property of a bacteria after CRISPRi treatment. The same result came out from fluorescence spectroscopic studies where the quenching was observed in treated samples due to decrease in number of bacterial cells adhered to human intestinal mucus (Figure 7).