Detection of Biofilm Producing Bacteria causing Co-infections with Leishmania Ulcers and their Relation with Matrix metalloproteinase-13 Enzyme

Background: Protozoan parasite causes Cutaneous Leishmaniosis (CL), dermatological disorder. Infected female phlebotomus sand flies spread these parasites. Secondary bacterial infection can worsen tissue damage and scarring. Objectives: In order to accurately ascertain the incidence of secondary bacterial infection caused biofilm formation in cutaneous leishmaniosis, it is imperative to investigate the significant immunological alterations by quantifying the levels of human Matrix metalloproteinase -13 (MMP-13). Materials and Methods : This study included 90 samples (20 controls and 70 CL sufferers). Study participants ranged in age from 3 months to 65 years, including both genders. Human Matrix metalloproteinase -13 measured by ELISA technique. Ulcer exudates were collected with sterile swabs after non-ulcerated lesions scraped. The specimens were cultured on Blood Agar, MacConkey Agar, Mannitol Salt Agar,and Hi Chrom Acinito. Laboratory methods were identified biofilm producing microorganisms. Results: Biofilms-forming secondary bacterial infections were found in 30 (42.8%) of 70 cutaneous Leishmaniosis patients. Bacterial from lesions Acinetobacter baumanni (8) cases (26.6 %), Acinetobacter lwoffi, Crobactor, Klebsiella, Proteus mirabilis, Proteus vulgaris,


Introduction:
Leishmaniasis is a vector-borne infection caused by the protozoan parasite of the genus Leishmania.The vectors are female sandfies (Phlebotomus and Lutzomyia). (1).The disease is distinguished by a range of clinical manifestations are mostly determined by the species of parasite and the immunological response of the host.Based on recent estimations, an annual incidence of 1.5 million new cases of cutaneous leishmaniasis (CL) has been reported.Over 90% of cases occur in five Old World countries.Afghanistan, Algeria.Iran, Iraq, and Saudi Arabia.Additionally, two nations in the New World, Brazil and Peru, also experience a significant number of cases (2) .The lesions induced by L. major exhibit a tendency to undergo spontaneous healing within a timeframe of roughly 18 weeks (3) .Local care, in conjunction with antileishmanial therapy, is a crucial component of the treatment for (CL).The management of secondary bacterial infection is crucial for the process of wound healing (4) .Conversely, the secondary bacterial infection of the cutaneous Lesion would augment the extent of tissue damage and subsequently contribute to scar formation (5) .Human Matrix metalloproteinase-13 (MMP-13) is a category of enzymes that rely on zinc for their catalytic activity.The enzymes are accountable for the process of protein break down within the extra cellular matrix.MMP-13 production is elevated in mucosal leishmaniasis, the more severe from L. braziliensis infection (6) .Therefore, the aim of this study was to accurately ascertain the incidence of secondary bacterial infections that are caused as a results of cutaneous leishmaniosis and their relation with immunological levels of (MMP-13).

Materials and methods
The study has been conducted from September 2022 to March 2023.The study included 90 samples (70 patients and 20 controls) were collected from both genders of studied participants, their ages ranging from 3 months to 65 years.The patients were referred to dermatology department in the Al Balad General Hospital, Samarra Hospital, Al Dhuluiya General Hospital in Salah Al-Din Governorate.For secondary bacterial isolation, 70 samples were taken from patients infected with cutaneous leishmaniasis.The parasite was diagnosed clinically by a dermatologist.A data collection sheet was applied to all individuals enrolled in this study in order to obtain Information including the location of the lesion, the number of lesion, their size, shape and texture.Inclusion criteria involve any patient with confirm diagnosis of cutaneous leishmaniasis.Exclusion criteria involve any patient with diabetes mellitus, hypertension, renal disease, and recent use of antibiotics.Collecting blood samples: Five ml of venous blood were taken using a sterile and dry medical syringe, then the blood sample was placed in a gel tube, and the tubes were left at room temperature for 15-20 minutes then placed in a centrifuge at 3000 revolutions per minute for 10 minutes to extract the serum, then transfer the acquired serum into Eppendorf tubes and store it at -20 0 C until used to the time of the immunological test of MMP-13 that was measured by using Sandwich-ELISA (7) (Sunlong, China).All samples have been brought to room temperature before 30 minute.Examine all samples at one time to avoid repeated freezing and thawing, which may affect the quality of results.

Swap from lesion for bacterial diagnosis:
The skin around the lesions of infected participants was disinfected thoroughly with alcoholic iodine-soaked cotton wool, then carefully scraping non-ulcerated lesions, finally the swap was taken (8) .All samples were cultured on blood agar MacConkey Agar, Mannitol Salt Agar then tested biochemically such as catalase, oxidase, Methyl red, Triple Sugar Iron (TSI) agar, Dnase, coagulase, and Cetrimide test were used to identify and categorize the bacteria.Finally diagnosed by the VITEK ® 2 compact system.

Microscopic examination by direct smear:
The infection was diagnosed using direct smear according to the method described (9) .whereby a sample was obtained from the edge of an ulcer for detecting amastigote stages of the parasite in fixed phagocytic cells in skin tissue.The smear method was carried out as follows: The skin ulcer was cleaned with ethyl alcohol 70%.The dermal fluid sample was drawn from the edge of the ulcer with a medical syringe with a capacity of 1 milliliter, then it was placed on the tip of a clean glass slide for the purpose of making a thin swab, and the swab was left to dry at room temperature.The dermal fluid smear was stained using Giemsa stain, as the slides were immersed in 70% methyl alcohol for 3 minutes, left to dry at room temperature, then dipped in Giemsa dye and left for 30 minutes.The slides were transferred to tap water to wash off the excess layer and shorten it, with the latter being replaced three to four times.Slides were left to dry at room temperature.The slides were examined by a light microscope and by using an oil lens at a magnification of 1000 to detect the amastigote stage of the parasite, and the sample was considered negative if no such stage was found.It must be pointed out that he had done three replicates for one sample.

Antibiotic Susceptibility Test
Preparation of Culture Media: Muller-Hinton agar medium was employed for this experiment.The medium was autoclaved , cooled to 45-50 0 C and poured in the petri dishes.When the medium solidified, the petri dishes were incubated at 37 0 C for evaporated the excess moisture.

Cong red agar:
The plates of Cong red agar prepared then inoculated with the bacterial isolates, and the plates were incubated in the incubator at a temperature of 37 0 C for 24-48 hours.After incubation, the color of the developing bacterial colonies was observed, as black colonies indicate that they are productive for the biofilm.While red colonies do not produce biofilm (8) .

Microtiter Plate Titer (MPT):
The microtiter plate (96-well plate) experiment for biofilm formation observes bacterial adhesion to an abiotic surface.This test uses vinyl (U)-Bottom or other 96-well microtiter plates to incubate microorganism.After incubation, planktonic bacteria are rinsed away and biofilms are stained with stained with crystal violet to visualize them.

Ethical approval
This study was conducted based on the ethical standards stipulated in the Declaration of Helsinki No. 12948/63/7.Before taking the sample, the patient's informed written and verbal agreement was obtained, after review and approval of the study protocol and subjects information by the local ethics committee.

Statistical Analysis
SPSS was used for statistical analysis.The continuous variable format was means ± Stander Error (SE).The link between categorical factors was examined Pvalues under or equal 0.05 were significant.

Microscopic examination
The investigation found Amastigotes, small, spherical entities (2-4 mm in diameter) with indistinct cytoplasm, nucleus, and rod-shaped kinetoplast as shown in (Fig. 1).Large nucleus, conspicuous kinetoplast and short axoneme rarely observable by light microscopy.The organism live in host macrophages throughout the body.

Bacteriological results
The results of the biochemical tests (Figure 2-7) were compared with the characteristics of secondary bacterial infection documented by others.Methyl red test is used to ascertain the specific fermentation pathway employed for glucose utilization.In the event that the culture lacks acetone, it will exhibit a color transition from brownish-green to yellow, as depicted figure 2. Klebsilla pneumoniae exhibits the phenotypic characteristics of being Methicillin-resistant (MR) and Voges-Proskauer (VP) tests positive (VP+).Pseudomonas aeruginosa is classified as a glucose non-fermenting bacterium, resulting in a negative reaction for both the methyl red (Fig. 2) and Voges-proskauer (VP) tests.Mannitol Salt Agar favors salttolerant Staphylococcus aureus.S.aureus ferments mannitol to produce acid and the production of acid lowers the PH of the medium, causing the phenol red indicator to turn from red to yellow (Fig. 3).Triple sugar iron (TSI) is most often used to identify Enterobacteriaceae, although it can also identify other Gram negative bacteria.Klebsilla spp.produce acid\acid reactions with or without gas.Acinetobacter and \Pseudomonas produce alkaline \ alkaline or K\NC.Citrobacter spp.and Proteus mirabilsi produce H 2 S-alkaline\Acid.Proteus mirabilis, and Proteus vulgaris produce acid\acid with H 2 S (Fig. 4).Dnase positive organisms like Staphylococcus aureus and Serratia spp.(Fig. 5).Coagulase clots plasma detects Coagulase positive Staphylococcus aureus in Gram positive, catalasepositive organisms.Pseudomonas aeruginosa, exhibits a positive oxidase reaction (Fig. 7).When cultivated on Cetrimide agar, Pseudomonas aeruginosa will yield blue fluorescent colonies on this agar.

Biofilm formation in patients with CL and secondary bacterial infection from clinical samples:
Approximately 30 (42.8%) patients exhibited positive cultures indicating the presence of secondary bacterial infection biofilm formation.Bacterial isolates from the lesion using the Congo red methods and micro titer plate in the gram positive and negative bacteria were as follows: Acineto baumanni (8) cases or 26.6%, Acinetobacter lwoffi, Klebsiella, Crobactor, proteus vulgaris, proteus mirabilis, pseudomonas-luteeola, sphingomonas, Raoultella, and Serratia, (1) case or 3.3% for each spp.Pantoea (2) cases or 6.6%.pseudomonas fluorescence (3) cases or 10%, and S.aureus with S.sciuri (4) cases or 13.3% for each spp, samples were strong for biofilm producer as done by Congo red agar and MTP method as seen in figure 7-A.

Micro titer Plate
The biofilm forming potential of 60 bacterial isolates was measured using crystal violet staining in 96 well Micro titer plates that observed bacterial adhesion to an abiotic surface as shown in (Figure 7-B).

Antimicrobial susceptibility testing
The antibiotic sensitivity of the bacterial isolates was assessed using the disk diffusion methods, as outlined by the(9).The test organisms were suspended in sterile normal saline solution in order to achieve a turbidity Serum Human Matrix metalloproteinase -13 level in patients with CL and control groups: The study observed no differences in the average concentration of MMP-13 in infected patients with CL 5.7±0.2(Pg/ml), compared to control group (6.9±0.6 Pg/ml) as shown in Table2.
The study observed that no significant differences in serum MMP-13 level in patients with CL and secondary bacterial infection was observed (p-value = 0.302) (Fig 9).

Discussion
Cutaneous Leishmaniasis is a prevalent global health issue that exerts a substantial influence on body mortality and morbidity rates.The clinical phenomenology of CL is thought to be influenced by innate immune mediators, which can either restrict or enhance the spread of the parasite (1,2) .The occurrence of secondary bacterial infection is recognized as a complication associated with CL (11) .The presence of secondary bacterial infection has the potential to worsen the condition and contribute to the formation of the permanent scar due to its ability to intensify tissue death and necrosis the manifestation of painful ulcers accompanied by purulent discharges and inflammation in the surrounding area may be observed.Furthermore, the length of the sickness would be extended (12) .In these instance, the proper utilization of antibiotics might lead to a reduction in the subsequent occurrence of illness.The current study conducted in Iraq has revealed that some patients exhibited positive cultures for secondary bacterial infection and biofilm formation, and several bacteria isolated.Other study identify S.sciuri and S.aureus the causative agent that isolated from CL ulcers (14) .Chicleros Ulcer, a type of CL by Leishmania mexicano, were found to include pathogenic microorganism in Yucatan , Mexico.These patients exhibited resistant to antimonial treatment, also the study indicates the necessity of eradicating bacterial infections through antibiotics therapy prior to initiating antimonial administration (15) .The current study observed no significant differences in the average concentration of MMP-13 in infected patients with CL, compared to control group.The entire lesions that have secondary bacterial infection

95% CI for the Mean
The pooled standard deviation was used to calculate the intervals.

P-value
Control (N=20) Patients(N=70) MMP-13(Pg/ml) ns 0.111 6.9±0.6 5.7±0.2Mean ±S.E were ulcerated.There were bacterial isolates from the lesions that were ulcerated; also the study concluded that destruction of the epidermis in the ulcerated lesion had predisposed the patients to the secondary bacterial infection (16) .High levels of MMP-13 can be considered a biomarker of poor prognosis indicating a high risk of parasite dissemination in the chronic phase of visceral leishmaniasis infection.MMP-13 expression has not been detected in normally healing human skin ulcers but has been detected in abundance.By fibroblasts in chronic skin ulcers, results indicate low levels of the enzyme during normal wound healing and increase in chronic wounds (17) .The topical antiseptic solution need for ulcerated lesions of the CL to prevent secondary bacterial infection that may be accelerated tissue destruction (18) .This study disagree with (19) , that show high level of MMP-13 in patients with CL which suggested the patients that infected with CL, increased macrophage that led to increase MMP-13 secretion because it is a type IV component of collagen, then the basement membrane of the skin increase, and causes excessive tissue damage through the migration of inflammatory cells to the site of injury (19) .The increased and unregulated activity of the enzymes contributed to chronic inflammation and increase tissue destruction that may help pathogen to enter the blood stream and invade different tissue.In addition the result of increase MMP-13 was due to increase enzyme secretion and inhibition gastric protein secretion (20) .Conclusions: Biofilm forming secondary bacterial infections were presented in some of patients that were infected with CL.Regarding MMP-13, there were no differences in patients with CL and those with secondary bacterial infection due to leishmaniasis.