Isolation and preservation of P. mirabilis

Clinical isolates were collected from patients admitted to different departments of Tanta University Hospitals over a period from November 2020 to September 2021. The identification of the isolates was conducted through microscopical and routine biochemical techniques [11]. Proteus mirabilis ATCC 35659 was used as a reference strain. The isolates with confirmed identity were preserved at – 70 °C in a Luria–Bertani (LB) broth with 25% v/v glycerol [11].

Antimicrobial susceptibility testing

Clinical isolates susceptibility pattern to various antibiotics was determined using the Kirby-Bauer disc diffusion method [12], and the results were interpreted in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [13]. Escherichia coli ATCC 25922 was used as the control strain. The antibiotic disks used (Oxoid, UK) were gentamicin, tobramycin, piperacillin/tazobactam, imipenem, cephazolin, cefoxitin, cefotaxime, cefepime, ciprofloxacin, trimethoprim/sulfamethoxazole, aztreonam, ampicillin, amoxycillin/clavulanic acid, and doxycycline.

Phenotypic screening of virulence factors

Virulence determinants of isolates were screened for the identification of the most virulent strains showing strong biofilm formation and positive for urease, protease, and hemolysin production as follows:

Biofilm production

The isolates were cultured in 96-well plates containing tryptic soy broth (TSB) and 1% glucose for 24 h at 37 °C. A negative control containing TSB only was also performed. The plates were washed twice with phosphate-buffered saline (PBS) after discarding the media. The resulting biofilms were fixed with 150 μl of methanol for 20 min, then stained for 15 min with 0.1% crystal violet, followed by rinsing with PBS. The residual biofilms were solubilized using 33% (v/v) glacial acetic acid. The optical density (OD) was measured at 570 nm by a microplate reader (Sunrise Tecan, Grodig, Austria) [14]. Isolates were then classified as negative, weak, moderate, or strong biofilm producers [15].

Enzymes and toxin production

The isolates were inoculated into urea broth for urease detection and incubated at 37 °C for 24 h until a color change to magenta (pink) could be observed [16]. Protease was detected on Mueller–Hinton agar containing 3% skimmed milk. After 48 h of incubation at 37 °C, plates were examined to evaluate the formation of lysis zones around the inoculated bacteria [16]. Hemolysin was detected on Columbia blood agar plates. After incubation at 37 °C for 48 h, the zone of hemolysis could be observed [16].

Determination of minimum inhibitory concentration (MIC)

The agar dilution assay based on CLSI guidelines [12, 13] was used to determine the MIC values of ciprofloxacin (CIP) against P. mirabilis isolates. E. coli ATCC 25922 was used as the control strain.

Growth curve assay

The sub-MICs of CIP for all further experiments were selected by plotting the growth curve using a spectrophotometric method [17]. Briefly, isolates were cultured in the absence or presence of CIP (at 1/2, 1/4, or 1/8 MICs) at 37 °C. At specific time intervals, an aliquot was collected from each culture, and the absorbance was read at 600 nm.

Biofilm assay

The impact of CIP was evaluated against biofilm-producing P. mirabilis isolates using a crystal violet assay, as described previously [14]. CIP was introduced to TSB containing bacteria of 106 CFU/ml at sub-MICs (1/4 or 1/8 MICs), and culture without adding any antibiotics was used as a control [18]. The formula used to determine the percent reduction in biofilm formation was:

$$\left[(}_}} - }_}})/}_}}\right] \times 100.$$

Motility assay

For the swarming assay, a 0.5 McFarland solution of P. mirabilis overnight culture was centrally inoculated on dried LB agar (1.5%) plates in the absence or presence of CIP (at 1/4 or 1/8 MICs). The plates were incubated overnight at 37 °C, and the swarming zones were measured in mm [19]. Similarily, the isolates were stabbed into the center of the dried LB agar (0.4%) plates with or without CIP (at 1/4 or 1/8 MICs). The plates were incubated overnight at 37 °C, and the swimming zones were measured in mm [19].

Urease assay

The isolates were cultured in LB broth overnight at 37 °C with or without CIP (at 1/4 or 1/8 MICs). Following that, the isolates were centrifuged, re-suspended in PBS, and adjusted to 1.0 at OD600. Then, 100 µl of each bacterial suspension was inoculated into a test tube containing 9.5 ml of Christensen’s medium in liquid form and 0.5 ml of urea (at 40%) and incubated at 37 °C for 3 h. After incubation, bacterial cells were centrifuged, and the change in the color of the supernatant was measured at 570 nm [20]. The percent reduction in enzyme production was calculated as previously mentioned.

Protease assay

The isolates were cultured overnight at 37 °C in LB broth with or without CIP (at 1/4 or 1/8 MICs). The culture supernatant (1 ml) was mixed with an equal volume of 1% (w/v) casein in 0.1 M sodium phosphate buffer (pH 7.0). The mixture was then incubated for 10 min at 30 °C before being terminated with the addition of 2 ml of 0.4 M trichloroacetic (TCA) acid. The mixture was then incubated at room temperature for 30 min before being centrifuged at 10,000 rpm for 5 min. The supernatant (1 ml) was then mixed with 5 ml of 0.4 M Na2CO3 for 10 min, and then 1 ml of Folin’s reagent mixed with 3 ml of distilled water (1:3 v/v) was added. The mixture was left to stand at 30 °C for 30 min before the OD at 660 nm was measured [21]. The percent reduction in enzyme production was calculated as previously mentioned.

Hemolysin assay

Isolates were grown overnight at 37 °C in LB broth with or without CIP (at 1/4 or 1/8 MICs). Following centrifugation and filtration of the supernatant, 600 µl was combined with 600 µl of a 2% suspension of red blood cells (RBCs) in saline and incubated at 37 °C for 2 h. The suspension was then centrifuged for 8 min at 4 °C at 10,000 rpm, and the released hemoglobin was determined by measuring absorbance at 540 nm. In the same conditions, a negative control of erythrocytes in LB broth and a positive control of totally hemolyzed erythrocytes by adding sodium dodecyl sulfate (0.1%) were employed [22]. The percentage of hemolysis inhibition was calculated as follows:

$$\left[(}-})/ (}-})\right] \times 100.$$

Fine and ultrastructure investigation

A representative isolate (code P17) was cultured overnight at 37 °C in LB broth with and without a sub-MIC of CIP. Then, the solutions (treated and control) were washed with PBS three times, and the final pellets were processed for further examination. The final pellets were resuspended, fixed with 2.5% glutaraldehyde in PBS buffer (pH 7.4) for 2 h at room temperature, and then postfixed with 1% OsO4 in PBS buffer (pH 7.4) for 1 h at 4 °C. A droplet of each bacterial suspension was placed on the microscope slide, then dehydrated with ethanol, and air-dried. The slides were then mounted on metal stubs and sputter-coated with gold, then examined using a scanning electron microscope (SEM) (Akashi Seisakusho, Japan) [23]. The final pellets were fixed for 24 h in 2.5% glutaraldehyde in PBS (pH 7.4), rinsed with the same buffer, and then postfixed for 2 h in 1% OsO4 in PBS (pH 7.4). The samples were then dehydrated with ethanol and embedded in Epon 812 epoxy resin. Ultrathin sections were cut on an ultramicrotome, which were then double stained with uranyl acetate and Reynolds lead citrate, then examined using a transmission electron microscope (TEM) (Jeol-1200 ECII, Japan) [23].

Relative gene expression analysis

The total RNA was extracted from untreated and ciprofloxacin-subMIC-treated P. mirabilis isolates using the PureLink® RNA Mini Kit (Thermo Scientific, USA). The yield and purity of the RNA extracted were measured using a NanoDrop spectrophotometer (ThermoFisher Scientific, USA). Then, RNA was reverse transcribed into cDNA using power cDNA synthesis kit (iNtRON Biotechnology, Korea). To analyze the expression pattern of selected genes in the absence and presence of sub-MIC of ciprofloxacin, qPCR was carried out using Power SYBR® Green PCR Master Mix (ThermoFisher Scientific, USA) in Rotor-Gene Q (Qiagen, USA). The expression of selected genes using specific primers (Table 1) was normalized by the housekeeping 16s rRNA gene, and the relative gene expression was determined by the 2−ΔΔCt method [24]. A PCR mixture without a template was used as a negative control. Only genes with a relative 2−ΔΔCt value above 1.0 or below 1.0 were considered significant [25].

Table 1 Primers used in real-time PCRIn-vivo wound infection model

The Research Ethics Committee (Faculty of Pharmacy, Tanta University, Egypt) approved the following protocols while they followed the standard rules for handling and caring for laboratory animals (TP/RE/2/24 p-01). The procedures were established as previously performed [30]. Male BALB/c mice (n = 30) were obtained from the Cairo University College of Veterinary Medicine's animal house (Cairo, Egypt). Mice weighed 120–150 g and were 6–8 weeks old at the time of the study. Following the creation of the wound infection, all mice were housed as individuals in a ventilated cage to prevent fighting and cross-contamination. They were supplied with free access to food and filtered water and were kept on a 12 h light/dark cycle at room temperature. Three groups of mice (10 mice each) were assigned at random. The control group, Group I, was not treated with any materials. Groups II and III were given bacteria and treated with 1/4 or 1/8 MICs of CIP, respectively. Under xylazine (5 mg/kg) and ketamine (40 mg/kg) anaesthesia, the backs of all mice were shaved and sterilized with 10% povidone-iodine. A nearly 10-mm circular incision was made on the dorsal inter-scapular region of each animal. The wounds were infected with 10 μl of the bacterial suspension in PBS (106 CFU). Following 30 min of inoculation, 20 μl of the vehicle (PBS) was injected subcutaneously in the control group I, and CIP (at 1/4 or 1/8 MICs) was injected subcutaneously in the treated groups II and III, respectively.

On the sixth day of the experiment, mice were anesthetized with isoflurane and were euthanized by cervical dislocation (CD) according to the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020 Edition). Hematoxylin–eosin (H&E) staining was employed for histopathological examination of the skin lesions.

Statistical analysis

A one-way ANOVA was employed for comparison. A p-value < 0.05 was considered statistically significant. GraphPad Prism version 5 software was used to create graphs and run statistical tests. All experiments were done in triplicate, and the results were expressed as mean ± SD.