Clinical isolates
A total of 93 isolates were recruited including 45 P. aeruginosa, 8 P. mirabilis, and 40 K. pneumoniae. Isolates were collected from Kasr El-Aini hospital over a period of months from November 2021 to July 2022. The clinical bacterial isolates were preidentified before their acquisition by VITEK 2 system, and subsequent verification was conducted by our side through biochemical tests prior to commencing any procedures. Stock culture of all bacterial isolates was then prepared and stored at − 80ºC in brain heart infusion (BHI) broth with 20% glycerol. The pathogenic strain of E. coli (ATCC 700728) was the indole-producing bacteria utilized in this work study. The strain was purchased from Faculty of Science, Helwan University.
Preparation of indole from E. coli culture supernatant
A preculture medium was inoculated with a single colony of E. coli and incubated overnight at 37 °C. The culture was diluted 1:100 in fresh Luria Bertani broth (Sigma Aldrich, USA) supplemented with 1% tryptophan (Biolab, Hungary) and vortexed to get a uniform suspension. The uniform suspension of bacteria was incubated again overnight under dark conditions at 37 °C in shaking condition at 120 rpm [10].
Preparation of crude indole extract
After incubation, culture supernatant (CS) was collected after centrifugation at 12,000 rpm for 15 min and was subjected to ultrasonic disintegration for 10 min. Supernatant was acidified by 1 N concentrated HCl (Chem-Lab, Belgium) to reach PH 2.5-3. To extract indole, double the ethyl acetate (LiChroslv, Germany) volume was added to acidified supernatant and shaken vigorously for 10 min in a separating funnel. The mixture was left at room temperature for 10 min to get the top layer of ethyl acetate. This layer is then used for further treatment.
In a rotary evaporator, the water bath temperature is set to 45 °C boiling temperature of ethyl acetate. The ethyl acetate layer was transferred to the round bottom flask and rotation was adjusted to avoid any bumping of the liquid sample. Upon complete evaporation of the liquid ethyl acetate, pure indole is left behind in the crystalline form attached to the bottom of the rotary flask. The crystals are re-dissolved in 20% methanol (PIOCHEM) and stored at -20 °C for future use [11].
Qualitative and quantitative analysis of indole
The indole assay kit (Sigma Aldrich, USA) is based on a modified version of Ehlrich’s and Kovac’s reagents, which reacts with indole to produce a colored compound at 565 nm. The intensity of this colored compound is directly proportional to the indole in the sample. This kit is suitable for indole determination in biological samples (indole produced by indole positive bacteria) as followed through manufacturer’s recommendations. The indole was quantified in E. coli CS and as crude extract.
Effect of indole on antibiotic tolerance
In this study, a total of five antibiotics, belonging to three different antibiotic classes, were utilized: ciprofloxacin (fluroquinolone), imipenem, ceftazidime, ceftriaxone, (Beta-lactams) and amikacin (aminoglycoside). The antibiotics were generously supplied by the Egyptian International Pharmaceutical Industries Company (EPICO).
The effect of indole on bacterial sensitivity to the antibiotics mentioned above was assessed by evaluating the alteration in the minimum inhibitory concentration (MIC) of antibiotics when combined with indole at concentrations varying from 0.07 to 0.5 mM and subsequent to the last preliminary optimization, a concentration of 0.15 mM was determined as the appropriate level to be employed for the ongoing investigation into antimicrobial susceptibility. MICs were determined using the microbroth dilution method, with each antibiotic tested at concentrations ranging from 256 to 0.0625 µg/µl, both in the absence and presence of 0.15 mM of indole crude extract added to each well following the guidelines set by the Clinical and Laboratory Standards Institute (CLSI), 2022. MIC values were recorded in absence and presence of indole with vehicle control done as well. The synergistic activity of this combination was reflected by reduction in MIC values compared to untreated isolates [12].
Effect of indole on biofilm production
All isolates were screened for their ability to form biofilms by tissue culture plate method. Biofilm production was evaluated in tryticase soy broth (Difco, Sweden) with 1% glucose. Isolates from fresh agar plates were inoculated in respective media and incubated for 18 h at 37 °C in stationary condition and diluted 1:100 with fresh medium. Individual wells of 96 well-flat bottom plates were filled with 200 uL aliquots of the diluted cultures without and with indole at concentration of 0.2 mM added to each well, the indole concentration selected for continuation in the biofilm assay was determined following the conclusion of the last round of preliminary optimization. Vehicle control and negative control with only broth to check sterility, and non-specific binding were also carried out. The plates were then incubated for 16 h, 18 h and 24 h at 37 °C. After incubation, the content of each well was gently removed by tapping the plates. The wells were washed twice with 200 uL of phosphate buffer saline (PH 7.2) to remove free-floating planktonic bacteria [13].
For quantification of biofilms formed by adherent (sessile) organisms, the plate was dried at 50 °C for 30 min and the biofilm was then stained by addition of 1% crystal violet (CV). The liquid was discarded, and unbound CV was removed by washing with distilled water until transparent liquid was visually observed. The biofilm CV was solubilized in 33% glacial acetic acid and the absorbance was measured at 540–630 nm using absorbance microplate reader. Experiments were performed in triplicates and were independently repeated 2 times. The biofilm formation level of all isolates was categorized according to the classification system as non-adherent (OD ≤ ODC), weak (ODC < OD < 2ODC), moderate (2ODC < OD < 4ODC), and strong biofilm forming (4ODC < OD), where ODC (cut-off OD) is defined as three standard deviations above the mean OD of the negative control (blank value) [13].
Efflux pump phenotypic detection
The phenotypic and qualitative detection of the efflux pump in K. pneumoniae isolates was performed by Cartwheel method. Briefly, the plates of tryptic soya agar culture media containing varying concentrations of ethidium bromide (EtBr) (1, 1.5, 2 and 2.5 mg/L) were prepared on the same day as the assay. Then the bacteria with 0.5 McFarland turbidity concentration were streaked on the plates. After 24 h of incubation at 37 °C, the plates were visualized under UV transilluminator and photographed. The isolates that had efflux pumps did not show emission of fluorescence. The capacity to efflux EtBr of each bacterial isolate was then ranked relative to the reference strain according to the following formula: Index = MCEtBr(K) – MCEtBr(REF) / MCEtBr(REF), where MCEtBr(K) and MCEtBr(REF) represent the minimum concentration of EtBr that produces fluorescence of the swabbed bacterial mass for the K. pneumoniae isolates and reference strain (E. coli ATCC 25922), respectively [14]. K. pneumoniae isolates which showed fluorescence index of ˃1.5 following a validation step using a higher EtBr concentration of 3 mg/l, were chosen for gene quantification analysis of resistance-nodulation-division (RND) efflux pumps.
Quantification of the expression of the RND efflux pump genes (acrA, acrB, oqxA and oqxB) of Gram–negative bacteria using quantitative real-time (qRT)-PCR before and after indole exposure
Extraction of total bacterial RNA of five isolates of K. pneumoniae was done as previously described with minor modification. Briefly, a total of 100 µl of each bacterial suspension was added into the 96 well plates to determine MIC of antibiotics in absence and presence of indole according to CLSI guidelines as discussed above, then incubated for 18 h at 37 °C aerobically. After incubation, total RNA was isolated from bacterial suspension using a RNeasy mini kit (Qiagen, Germany) as per manufacturer’s instructions. The concentration and purity of the RNA was determined using a Nanodrop (Thermoscientific, USA) (260/280 ratio of > 1.8). The isolated RNA was reverse transcribed into cDNA using ReverAid RT Kit (ThermoFisher Scientific, USA) according to the manufacturer’s guidelines.
qRT-PCR was used to measure the relative expression of the RND-family efflux pump genes (acrA, acrB, oqxA and oqxB) using SYBR Green qPCR Master Mix (Xpert Fast SYBR (uni, Portugal) and the Bio-Rad platform in a total volume of 20 uL consisting of 2 uL cDNA, 2 uL 0.3–0.5 μm forward and reverse primers listed in Table 1, 10 uL master mix, and 6 uL nuclease free water. After a 3 min activation of the modified Taq polymerase at 95 °C, 40 cycles of 5s at 94 °C and 30 s at 60 °C were performed. In order to normalize the transcription levels of the target genes, the cycle threshold (CT) values of these genes were compared with the CT values of rrsE as a housekeeping gene, which was chosen as an endogenous reference. The expression of efflux pump genes in the tested isolates was then determined by calculating the relative expression of indole-treated isolates compared to non-treated isolates (which served as the control) in the presence of different antibiotics. Furthermore, the specificity of the generated products was verified through melting-point analysis. The 2^−ΔΔCT method was used to calculate the relative expression level of pump genes. A significant decrease of gene expression was concluded when the corresponding ratios were < 1.0. All reactions were performed in triplicate [15].
Statistical analysis
The statistical analysis was performed using IBM SPSS Statistics software (version 22). For the comparison of means between two independent groups, the independent t-test and Mann-Whitney U test were used. The independent t-test was employed for analyzing qRT-PCR data to compare gene expression levels between groups. Additionally, Pearson correlation analysis was conducted to assess the relationship between variables of interest. All statistical tests were two-tailed, statistical significance was determined at p < 0.05.
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