Impact of short chain fatty acids (SCFAs) on antimicrobial activity of new β-lactam/β-lactamase inhibitor combinations and on virulence of Escherichia coli isolates

Bacterial isolates

In this study, 140 Escherichia coli isolates were previously obtained from the laboratory of the Gastrointestinal Surgery Center (GISC) in Mansoura, Egypt. These isolates were identified by standard microbiological methods. The isolates were stored at −20 °C in tryptone soya broth (TSB, Oxoid, UK) with 20% glycerol [20].

Susceptibility of E. coli isolates to β-Lactam/β-Lactamase inhibitor combinations

Three different β-lactam/β-lactamase inhibitor combinations were examined for their antimicrobial activity including cefoperazone/sulbactam, ceftazidime/avibactam, and cefepime/enmetazobactam. Cefoperazone/sulbactam and ceftazidime/avibactam were used at ratios of 1:2, and 4:1, respectively. A fixed concentration of enmetazobactam (8 μg ml−1) was used in cefepime/enmetazobactam combination [14]. The Clinical and Laboratory Standards Institute’s broth microdilution method was used to determine minimum inhibitory concentrations (MICs) [21].

Sigma Aldrich (Darmstadt, Germany) provided the test antibiotics: cefoperazone, ceftazidime, and cefepime. MedChemExpress MCE (Monmouth Junction, USA) provided the three β-lactamase inhibitors. All powders were kept at 4 °C in airtight containers. The lowest level of β-lactam antibiotic at which no visible bacterial growth detected was considered as the MIC. Resazurin dye (Sigma-Aldrich, Darmstadt, Germany) was used to detect bacterial growth colorimetrically producing pink, fluorescent resofurin as a byproduct [22].

Susceptibility of E. coli isolates to different SCFAs singly and in combinations

The MICs of acetic acid (Sigma Aldrich, Schnelldorf, Germany), propionic acid and butyric acid (Alfa Aesar, ThermoFisher Scientific, Massachusetts, Waltham, MA, USA) were determined by broth microdilution method according to Clinical and Laboratory Standards Institute [21].

Susceptibility of E. coli isolates for combined therapy of new β-lactam/β-lactamase inhibitor along with SCFAs

The MICs for β-lactam/β-lactamase inhibitor combinations were determined in presence of sub-MIC (½ MIC) of SCFAs at pH 6.5 using the broth microdilution method [21]. Starting with a double strength β-lactam/β-lactamase inhibitor concentration, two-fold serial dilutions were made in microtiter wells leaving one well as growth control and another as negative control. Then ½ the MIC of SCFAs was added (37.5 μl from 10 mg μl−1 stock) to each well except for the growth and the negative control (un-inoculated) wells. Finally, 100 μl of bacterial suspension (1.5 × 105 CFU ml−1) were added and plates were incubated for 24 h at 37 °C and the MIC was recorded after adding resazurin dye.

The effect of SCFAs on in vitro bacterial growth of E. coli

A 24-h well isolated E. coli colony on nutrient agar was used to inoculate 10 ml of MHB that was incubated for overnight at 37 °C. Then, one ml of the incubated culture was used to inoculate 50 ml of sterile MHB in a 100-ml conical flask. The flask was incubated at 37 °C in shaking incubator. One-ml aliquots of the culture were taken for measuring the optical density (OD) at wavelength of 600 nm and for viable counting after 2 h and every 60 min. The viable count defined as the number of colony-forming units (CFU) was determined using the standard plate counting technique [23].

To investigate the effect of SCFAs on bacterial growth, a mixture of acetic, propionic, and butyric acid (with a ratio of 60:20:20 for acetate, propionate, and butyrate; respectively) was added to bacterial suspension of E. coli isolates -that remained resistant to β-lactams even after the addition of β-lactamase inhibitors- so as to mimic both ileum and colonic conditions [16]. Similarly, OD was measured after 2 h and every 60 min. At the end, growth curves were drawn for the bacterial isolate in the presence and absence of SCFAs.

Effect of SCFAs on transcription of virulence genes by quantitative real–time polymerase chain reaction (qRT-PCR)

Fifteen-milliliter falcon tubes were filled with 10 ml MHB and 100 μl of bacterial suspension (104 CFU ml−1) from an overnight culture of the test isolate. Then various concentrations of SCFAs were made in the inoculated falcon tubes as follows; i-SCFA (12 mM at pH 7.4) and c-SCFA (60 mM and 123 mM at pH 6.5) or sterile NaCl (Control). The prepared cultures were incubated to mid log phase at 37 °C in orbital shaker then centrifuged at 2000 × g for 10 min. The supernatant was discarded, and the pellet was resuspended in 250 μl of TE buffer (10 mM Tris pH 8 and 1 mM EDTA), then 25 μl of 10% Sodium dodecyl sulfate (SDS) was added. The mixture was incubated at 65 °C for 30 min, then 1 ml of trizol was added and the sample was sonicated on ice-bath using probe sonicator (Thermofisher Scientific, CA, USA,) for 2 min [24].

Total RNA was extracted using Qiagen RNA Protect-RNeasy Kit (Qiagen, Hilden, Germany) according to manufacturer’s recommendations. Immediately, RNA concentration and purity was evaluated using Thermo Scientific™ NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermofisher Scientific, CA, USA,). RNA was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis kit according to manufacture instructions (Biogen, Munich, Germany).

Table 1 demonstrated the primers for the four tested virulence associated genes fimH, ipaH, fliC, and BssS that were analyzed using SensiFAST™ SYBR® High-ROX Kit (Bioline USA Inc., USA). The 10 μl total volume of qRT-PCR reaction was made up of 1 μl of cDNA, 0.7 μl of each forward and reverse primers (10 μM), 5 μl of 2× SYBR Green Master Mix, 1 μl of QN ROX Reference Dye and 1.6 μl of nuclease-free water. The amplification conditions were made as follows: initial denaturation at 95 °C for 20 s, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s, and a final melting curve program of 15 s at 95 °C, 60 s at 60 °C, followed by a dissociation step for 15 s at 95 °C. The reaction was carried out in StepOne™ Real-Time PCR System (Applied Biosystems™, ThermoFisher Scientific, Foster, CA, USA). The comparative quantification method (∆Ct) was used to determine the up-and down-regulated genes, where the expression level of targeted genes in comparison to the control 16 S rRNA gene (Sigma Co., USA) was calculated by using the equation RQ = 2-∆∆CT where ∆∆Ct = (Cttarget genes – Ct16srRNA)treatment − (Cttarget genes − Ct16srRNA)control, where Ct is the threshold cycle.

Table 1 Primers used in amplification of targeted virulence genes in Real-Time PCR (Sigma Co., USA)The effect of SCFAs on E. coli motility

The effect of SCFAs on the swarming motility of E. coli isolates was carried out in semisolid agar plates. E. coli isolates were grown in Luria-Bertani (LB) broth overnight at 37 °C. Soft agar plates (1% tryptone, 0.5% NaCl, 0.25% agar) were prepared the day before assay. The control plates had the same quantity of saline as the total SCFAs, and SCFAs were added to the agar medium immediately before pouring into the plate. Three μl of E. coli overnight cultures were added to the center of each plate. Finally, plates were incubated at 37 °C for 10 h. The experiment was carried out in triplicate [16].

The Effect of SCFAs on biofilm formation of E. coli isolates

The biofilm formation was performed as described by Salo et al. [25]. Briefly, 5 ml of LB broth was inoculated with a test isolate colony and incubated at 37 °C for 18–20 h. Bacterial cells were collected by centrifugation at 5000 rpm for 5 min, the cells were washed with 0.5 ml phosphate buffered saline, followed by centrifugation. The washed cells were resuspended in 1 ml of LB broth and the cell density was adjusted to 107 cells ml−1. 200 μl of each test isolate were placed into the round bottomed wells of 96-well polystyrene microtiter plate. SCFAs were added to the test isolates at concentrations of 60 mM and 123 mM and plates were incubated at 37 °C for 24 h. The bacterial suspensions were gently aspirated from the wells that were washed three times each with 200 μl of sterile PBS to remove planktonic cells. The plates were then dried for 45 min at room temperature and 150 μl of 0.4% crystal violet (CV) was added to each well and left for 45 min at room temperature. After this, 150 μl of 95% ethanol was added to each well and left for 45 min. Then, 100 μl of each well was transferred to a fresh 96-well microtiter plate to measure the absorbance at 600 nm using a Synergy HT microtiter plate reader (BioTek Instruments, Winooski, VT, USA),.

The average OD of three wells for each strain was computed (ODt), and the cut-off OD (ODc) was determined as being three standard deviations above the average OD of the negative control. The test isolates were classified as non-biofilm forming (N): ODt ≤ ODc, weak biofilm forming (W): ODc < ODt ≤ 2 × ODc, moderate biofilm forming (M): 2 × ODc < ODt ≤ 4 × ODc and strong biofilm forming isolates (S): ODt > 4 × ODc [26].

The effect of SCFAs on E. coli adhesion

Bacterial adhesion was evaluated by the same technique performed with biofilm production, but instead of incubating the 96-well plates for 24 h, plates were incubated for just 2 h and the procedure was continued as before [27].

Statistical analysis

GraphPad Prism software was utilized for the statistical analysis employing the one-way ANOVA test (version 7.0, GraphPad Software Inc., La Jolla, CA, USA). Statistics are deemed significant for P-values below 0.05. All data were expressed as mean ± SEM.

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