Partially Purified Hirudin from Hirudo medicinalis as a Sustainable Natural Alternative for Controlling Biofilm Formation in blaCTX-M-Producing Citrobacter freundii from Burn and Wound Infections

2.1 Sample isolation and diagnosis

Seventeen isolates of Citrobacter freundii were obtained from burn and wound patients in various hospitals in Baghdad, Iraq. Most of the isolates were collected from 150 skin swabs taken from both sexes of different ages between December 2022 and February 2024. Morphological, biochemical, and IMViC tests, Triple Sugar Iron agar test, as well as the API 20E system, were performed on the isolates.

Medical samples were cultured on brain heart infusion broth, nutrient agar, blood agar, Salmonella-Shigella (SS) agar, MacConkey agar and Mueller-Hinton agar according to needs, and the colonies were diagnosed using microbiological techniques (morphology and biochemistry) [15].

2.2 Genetic detection of the blaCTX-M gene using polymerase chain reaction

A single-molecular polymerase chain reaction (PCR) assay was performed to amplify fragments of the blaCTX-M gene in all Citrobacter freundii isolate strains under study.

2.2.1 DNA extraction

DNA was extracted by suspending three colonies of each Citrobacter freundii isolate on MacConkey agar plates in 500 μL of nuclease-free water (Bromage, USA) and heating them for 10 minutes at 90 ℃ using a water bath. The samples were then centrifuged for 10 minutes at 10,000 rpm and used as a template for bacterial DNA analysis using PCR.

2.2.2 Primers and amplification reactions used

The primers and amplification reactions used in this study were as follows: the forward primer sequence of the blaCTX-M gene (5-TTTGCGATGTGCAGCACCA GTAA-3) and the reverse primer sequence of the blaCTX-M gene (5-CGATATCGTTGGTG GTGCCATA-3) (Alpha DNA, Canada) [16, 17].

Amplification reactions (25 μL per sample) were performed using Mastermix 2X mix (Kappa Corporation, India) (12.5 μL), forward initiator (1.5 μL), reverse initiator (1.5 μL), nuclease-free water (4.5 μL), and DNA sample (5 μL). The thermal cycling conditions included an initial denaturation process at 95 ℃ for 5 minutes, followed by 30 cycles at 94 ℃ for 30 seconds, then annealing at 55 ℃ for 40 seconds, and then elongation at 72 ℃ for 50 seconds. The cycle concluded with a final elongation at 72 ℃ for 10 minutes.

2.2.3 Agarose gel electrophoresis

Gel electrophoresis was used to detect the PCR products, which were imaged using ethidium bromide and a UV light documentation system [17].

2.3 Antibiotic susceptibility testing

Clinical bacterial isolates from burns and wounds were cultured on Mueller-Hinton agar using the standard Kirby-Bauer disc diffusion method, according to the 2020 CLSI [16]. The antibiotic discs used in this study included Amoxicillin (25 µg), Aztreonam (15 µg), Ciprofloxacin (10 µg), o-Trimoxazole (25 µg), Gentamicin (10 µg), Imipenem (10 µg), Nitrofurantoin (300 mcg), Ofloxacin (5 µg), Penicillin G (10 U), Piperacillin (100 mcg), Tobramycin (10 µg), Amikacin (10 µg), Ampicillin (10 µg), Ceftriaxone (30 µg), Cephalexin (30 µg), Piperacillin/Tazobactam (100/10 µg M-H agar plates were prepared and coated with a 0.5 M McFarland swab-modified bacterial suspension, followed by antibiotic tablets. The plates were incubated for 24 hours at 37 ℃. The diameter of the inhibition zone was measured and calculated to determine antibiotic resistance using CLSI criteria [16].

The result was that the bacteria were MDR, meaning they were resistant to one or more of three agents [12].

2.4 Examination of biofilm-producing organisms

Screening for biofilm production for all isolates was done using the tube method (TM), Congo red agar method (CRAM), and tissue culture plate method (TCPM).

2.4.1 Phenotypic features of biofilm production in Congo Red agar

The isolates were grown on CRA plates, previously prepared by adding 0.8 g/L of Congo red and 50 g/L of sucrose (from British Drug Houses/England) and 1 L of brain heart infusion agar (Himedia - India). The plates were then incubated for one day at 37 ℃ and 8 hours at room temperature. This allowed for the observation of black, rough colonies produced by the slime-producing strains, distinguishing them from non- biofilm producing isolates (red smooth colonies), and the results appeared as in Figure 1.

Figure 1. Congo red agar (CRA) assay for slime production. (A) Red, smooth colonies indicating non‑slime‑producing isolates, (B) black colonies indicating slime‑producing (biofilm‑producing) isolates

2.4.2 Phenotypic features of biofilm creation by the Tube Method test

The TM was used to establish biofilms. The vaccine was placed on 5 mL of heart and brain extract broth with a ring of bacterial culture prepared overnight in a glass tube and then incubated at 37 ℃ for 24 hours. The tubes were then washed with P-buffered saline and air-dried. The dry tubes were stained for 15 minutes with 0.1% C.V. dye, then washed with deionized H₂O to eliminate additional dye. The tubes were allowed to dry, and biofilm formation was investigated. Most tubes showed observable biofilm along the bottom and the wall, indicating positive biofilm formation. Some tubes also showed a colored disc at the air-liquid border, indicating negative biofilm formation [15, 18].

The optical absorption (ODc) values were calculated separately for each experimental group as the average of three empty control wells containing only sterile heart and brain broth. The isolates were ranked according to their ability to form biofilms (S = strong biofilm formation, M = moderate biofilm formation, W = weak biofilm formation, N = no biofilm formation) according to the equation shown in Table 1.

Table 1. The mean optical density (OD) value obtained for all isolates and the strength of biofilm formation [19, 20]

2.4.3 Phenotypic features of biofilm formation by microtiter plate method or tissue culture plate method

Tissue culture was performed on plates. Five milliliters of heart and brain extract broth was inoculated with a ring of freshly cultured isolates into 96 wells, and the plates were incubated under ideal growth conditions. The bacterial suspension was diluted 1:4. A sterile polystyrene plate containing wells, each holding 200 µL of the prepared bacterial suspension, was used. Fresh culture medium was placed in the wells. The plates were incubated at 37 ℃ for 24 h. The plates were gently shaken to eliminate the contents of the wells and washed repeatedly with 200 µL of phosphate-buffered saline solution. They were then incubated at 50 ℃ for one hour to fix the biofilms formed by the adhering bacteria. The stable biofilms were stained with C.V. (0.1%) for half an hour, and the stain was removed with deionized water. They were air-dried, and the results were read using ELISA (600 nm wavelength) to determine the optical density of the stained bacterial biofilms [15-18].

2.5 Extraction and partial purification of Hirudin from Hirudo medicinalis

2.5.1 Preparation of Hirudo medicinalis crude extract

Fourteen 10 cm long Hirudo medicinalis leeches were prepared to destroy bacteria. The leeches were cut into pieces and then ground in a homogenizer with the addition of 5 mL of normal saline solution, and put in the shearer at 25 ℃ for 2 days, obtain a homogeneous suspension, centrifuge for 20 minutes at 3000 g, filter the liquid through a Millipore 0.45 mm filter, and keep the extract at 4 ℃ to use [21], as shown in Figure 2.

Figure 2. Hirudo medicinalis

2.5.2 Partial purification of Hirudin

The crude extracts obtained from Hirudo medicinalis leeches were centrifuged at a speed of 10,000× for 20 minutes at a temperature of 4 ℃ to remove undissolved material and cell remnants. The filtrate was then filtered through a 0.22 μm membrane filter. The filtrate was used in a DEAE-Sepharose ion-exchange chromatography column pre-neutralized with 20 mM Tris-HCl buffer (pH 8.0). The associated proteins were separated using a linear gradient of sodium chloride (0-1.0 M), and the fragments that showed antithrombin activity were collected. The active fractions were concentrated using ultrafiltration through a membrane with a molecular weight cutoff of 3 kDa.

Based on the sequential purification steps, the resulting fractions were considered hirudin-rich. Protein analysis was performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which revealed a dominant protein band corresponding to the predicted molecular weight of hirudin, indicating partial purification. The biological activity of the enriched fractions was confirmed using an antithrombin activity assay. The partially purified protein was obtained as a 2 mg/mL basal solution. A 1 mg/mL dilute solution was prepared, and 100 μL of the dilute protein was mixed with an equal volume (100 μL) of bacterial culture in a 96-well microplate, resulting in a final protein concentration of 0.5 mg/mL in contact with the bacterial cells [22-24].

2.5.3 In vitro inhibitory effect of partially purified Hirudin on isolates producing biofilm

To study the inhibitory effect of partially purified hirudin from Hirudo medicinalis on biofilm of isolates, the highest biofilm-producing isolates of Citrobacter freundii were selected to be tested.

A modified spectrophotometric method was used to detect biofilm inhibition [25]. Biofilm inhibition was performed using 96-well microplates. In vitro, bacteria were cultured in heart and brain extract (BHI) broth and incubated at 37 ℃ for 18 h. The bacterial culture was diluted in BHI broth, then its concentration was adjusted using a 0.5 McFarland tube. The partially purified protein was obtained as a stock solution with a concentration of 2 mg/mL. A diluted solution was prepared at a concentration of 1 mg/mL, and 100 microliters of the diluted protein were mixed with a similar volume (100 microliters) of bacterial culture in a 96-well microplate, resulting in a final protein concentration of 0.5 mg/mL in contact with the bacterial cells. The plates were incubated for 24 h at 37 ℃. The wells were then washed in sterile physiological saline to remove any adhering bacteria and stained using the same procedure as above to detect biofilm formation.

2.6 Ethical considerations

Approval was obtained from the Ethics Committee at the College of Science, Mustansiriyah University (Approval No. BSCMU/12022/00092M, November 1, 2022). The procedures followed the ethical guidelines outlined in the Declaration of Helsinki, and written informed consent was obtained from all individual participants included in the study.

These results can be explained by the mechanism proposed by research [16]. In general, Imipenem, Ciprofloxacin, and Gentamicin proved most effective against C. freundii isolates. However, isolates were highly resistant to Piperacillin/Tazobactam, Nitrofurantoin, Amikacin, Amoxicillin, Aztreonam, Ampicillin, Ceftriaxone, Ofloxacin, Penicillin G, Co-trimoxazole, Cephalexin, and Tobramycin.

Some researchers have shown that the medicinal leech (Hirudo medicinalis) typically carries a pure culture of Aeromonas veronii bv. sobria bacteria in its digestive tract. This is unusual in digestive tracts that are usually home to multiple microbes. Other types of bacteria may proliferate or survive in the medicinal leech's digestive tract. Using a colonization assay, researchers were able to compare the ability of clinical isolates and a symbiotic strain to colonize medicinal leeches experimentally. The symbiotic strain A. veronii bv. sobria proliferated well and persisted for at least seven days within the digestive tract. It was able to inhibit the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus within the animal compared to their growth in the laboratory control sample, resulting in altered ingested blood within the digestive tract. Although both strains were able to persist in the digestive tract for a week, the number of viable Escherichia coli cells decreased approximately a thousandfold within 42 hours. By inhibiting the activation of the membrane-attack complex in the complement system present in the blood, it is possible to prevent this decrease in the number of viable E. coli. This indicates that the membrane-attack complex remains active within H. medicinalis and prevents the proliferation of susceptible bacteria. Thus, the antimicrobial properties of the blood of ingested vertebrates contribute to the specificity of the symbiosis between A. veronii and H. medicinalis, and to the changes that occur in the blood within the digestive tract of H. medicinalis [26].

The study [27] did not agree with our results, as it showed that 41.17% of bacterial isolates were sensitive to ciprofloxacin since it inhibits bacterial DNA gyrase, preventing supercoiling of DNA, which is required for chromosome compaction into the bacterial cell.

Cephalexin, one of the first-generation cephalosporins, was investigated in the current findings, in which 100% of the isolates were cephalexin-resistant. Ceftriaxone, one of the third-generation cephalosporins, was investigated in the current findings, in which 100% of the isolates were ceftriaxone-resistant. Resistance mechanisms, particularly the synthesis of large-scale beta-lactamase and other enzymes, may play a role in resistance to first-generation cephalosporins [28].

Citrobacter freundii isolates were tested as resistant to the class aminoglycoside Tobramycin (100%) and Amikacin (100%). Gentamicin's mode of action has long been assumed to involve the irreversible binding of the aminoglycoside to the 30S component of the bacterial ribosome. Inhibiting bacterial protein production [29].

Antibiotic resistance is a global problem. The C. freundii isolates in this study demonstrated resistance to multiple antibiotics. C. freundii is a major bacterial pathogen contributing significantly to hospital-acquired infections, a serious problem requiring urgent attention [14]. The significant prevalence of multidrug-resistant biofilm-forming organisms in our healthcare facility indicates a potential threat in our region. Therefore, regular monitoring of biofilms and antibiotic resistance among bacteria in clinical laboratories is recommended, along with the strict application of infection control and prevention measures.

This result is consistent with research [30], which stated that leech saliva extract had an inhibitory influence on some of the laboratory bacteria in its study. This result contradicts the report [31], and this may be attributed to the low concentration of bioactive molecules in the extract.

Leech saliva extracts demonstrated antibacterial activity against the microorganisms used in our study. Two studies [32, 33] explained that, like antimicrobial proteins and peptides, 4-bromobutyric acid, 6,17-octadine-1-ol acetate, and octadro-1,4,9,9-tetramethyl contribute to the efficacy of the leech saliva extract. The bioactive substances in the silver-treated leech saliva extract matrix may inhibit microbial growth by interacting with the thiol group in certain enzymes or respiratory enzymes, causing cell death or removal of reactive oxygen species that attack or accumulate within microbial cells. Medicinal leeches are reported to secrete saliva comprising approximately 60 diverse proteins [33, 34]. Leech secretions serve a variety of purposes beneficial to the leech during feeding, including increasing blood flow to the affected region. Many of these concealed proteins act as anticoagulants (such as hirudin), platelet accumulation inhibitors (commonly aperase, collagenase, and calin), vasodilators, and protease inhibitors [35]. In classical medicine, it was employed to treat many diseases, including middle ear infections, arthritis, inflammation of burns and wounds, and diabetic foot injuries. From the above results, it can be concluded that Citrobacter freundii clinical isolates was appearance MDR-phenotype and premium ability to produce biofilm, and clinical Citrobacter freundii isolates exhibited high resistance to commonly used antimicrobials. The TCPM is more sensitive than the CRA and TM in diagnosing Citrobacter freundii biofilm-producing strains. The CRAM is preferred as a complementary test due to its higher specificity compared to TCP. All three methods have proven effective in detecting biofilm-producing Citrobacter freundii isolates, and the three traditional tests confidently detected biofilm-producing isolates due to their appropriate sensitivity and specificity. This study showed a high prevalence of C. freundii isolates producing broad-spectrum beta-lactamase enzymes, as 10 (58.8%) of 17 C. freundii isolates were positive for producing broad-spectrum beta-lactamase enzymes by phenotypic detection, and the molecular investigation resulted in the presence of the blaCTX-M gene in 10 (58.8%) of 17 C. freundii strains. Also, Hirudo medicinalis extract shows high activity in preventing the formation of biofilm by Citrobacter freundii isolates.

This study is able to manage the treatment failure of antimicrobial drugs and avoid persistent and chronic infections by using Hirudo medicinalis extract as an alternative treatment.