Biofilm Formation, Pyocyanin Production, and Antibiotic Resistance Profile of Pseudomonas aeruginosa Isolates from Wounds

Pseudomonas aeruginosa is one of the most frequently resistant and dangerous bacteria isolated from infected wounds of patients. This study aimed to determine the prevalence of P. aeruginosa from infected wounds of patients in the Dschang District Hospital to evaluate their antibiotic susceptibility profiles and their ability to swarm and swim and correlate pyocyanin production with biofilm formation. Wound swab samples were collected and the identification of P. aeruginosa was performed using microbiological and biochemical tests. Their antimicrobial susceptibility was determined by the broth microdilution method. Swarming and swimming were determined by measuring the diameters of motility in semisolid/low-viscosity media. Furthermore, pyocyanin production and biofilm formation were evaluated spectrophotometrically using a microtiter plate. The prevalence of P. aeruginosa from infected wounds in our study population was 26%. All P. aeruginosa isolates were resistant to streptomycin and paromomycin, and the frequency of multidrug resistance (MDR) was 65.8%. All P. aeruginosa isolates showed the ability to produce biofilm and pyocyanin. Out of the 37 isolates screened, 19 including the reference strains (51.4%) were strong biofilm producers. A significant positive correlation was observed among biofilm formation, pyocyanin production, and the antibiotic resistance profile of the isolates. Findings from this study suggest that infected wounds could act as a reservoir for MDR and virulent P. aeruginosa. The presence of strong biofilm producers of P. aeruginosa in infected wounds is a serious public health concern. Therefore, surveillance programs to monitor and control MDR P. aeruginosa in these patients are required to prevent their dissemination in hospital settings.


Introduction
P. aeruginosa is the most important Gram-negative opportunistic pathogen and a major cause of nosocomial infection [1].It is a causative agent for 10% of all hospitalacquired infections, including pulmonary infections (tracheobronchitis and necrotizing bronchopneumonia), urinary tract infections, bacteremia, endocarditis, and skin and soft-tissue infections such as surgical and burns wounds site infections [2,3].
Wounds provide a suitable site for bacterial multiplication and are persistent sources of infection [4].In infected wounds, P. aeruginosa was found to be the most common isolate (59%) [5].Other bacteria involved in the development of chronicity and delaying wound healing included species such as Staphylococcus aureus, Proteus mirabilis, Escherichia coli, and Klebsiella pneumoniae [6].In 2017, P. aeruginosa was recognized as one of the most life-threatening bacteria and listed as a priority pathogen for research and development of new antibiotics by the World Health Organization [7].A previous study showed that 61-100% of P. aeruginosa observed in intensive care units in the majority of countries in the Middle East and North Africa region are multiresistant [8].P. aeruginosa possesses numerous virulence factors and has the potential to develop resistance to antimicrobials [2].Te bacterium produces a variety of virulence factors, such as Pseudomonas quinolone signal (PQS), pyocyanin, rhamnolipids, elastase, and two endogenous siderophores, pyoverdine and pyochelin, which are involved in chronic infections [9].Pyocyanin, a blue-green pigment characteristic of P. aeruginosa is an electrochemically active metabolite, involved in various biological activities such as bioflm formation, maintaining the ftness of bacterial cells, and gene expression [10].It is a redox-active blue phenazine pigment and the redox equilibrium inside a biological system is altered by pyocyanin [11].Pyocyanin has long been suspected to play a role in the pathogenesis.Te production of this pigment enhances the expression of virulence factors and other phenotypes that converge on P. aeruginosa.It has been reported that P. aeruginosa strains that exhibit pyocyanin have a prevalence of higher multidrug resistance, as well as more virulence factors, compared to nonproducing strains [12].
Bacterial motility plays an important role in surface colonization by bacteria [13].In addition to fagellumdependent swimming in liquid-to-low viscosity environments, P. aeruginosa is capable of swarming motility, which occurs on semisolid surfaces and requires fagellar motility as well as the production of biosurfactants [14].Tese motilities contribute to the formation of structured surfaceassociated communities of bacteria called bioflms.
Bioflm is defned as an assemblage of microbial cells that are attached to a surface and enclosed in an extracellular matrix principally made of polysaccharide material [15][16][17].Bioflm is considered an important virulence factor and plays a signifcant role in antibiotic resistance [18].Bioflm formation is a strategy of microorganisms to successfully adapt and survive in hostile environments which can increase their resistance to the efects of antimicrobial agents 10-1000 times [19][20][21].Te prevalence of bioflms in chronic wounds is estimated to be approximately 60% [22].Furthermore, bioflms account for almost 80% of chronic microbial infections in the human body.Previous works have shown the involvement of pyocyanin and bioflm formation in the bacterial resistance of P. aeruginosa isolates from other clinical samples [23].To date, little or no information is known on the distribution of P. aeruginosa isolates from infected wounds, as well as the resistance phenotype of the isolates, production of pyocyanin, and bioflm formation in Cameroon, especially in the West Region of Cameroon.In this light, this study aimed to determine the prevalence of P. aeruginosa from infected wounds of patients at the Dschang District Hospital, evaluating their antibiotic resistance profle.Furthermore, we evaluated the pathogenicity of P. aeruginosa by assessing bioflm formation and pyocyanin production, as well as the swarming and swimming ability of the isolates.

Study Design and Ethical Considerations.
A crosssectional study was conducted on 142 patients with infected wounds.Tis was done by visiting the outpatient units of the Department of Wound Care for a period of 3 months from February 2022 to April 2022 at the Dschang District Hospital, Cameroon.Any patient who was immunocompromised, under treatment with antibiotics, or who had not given his consent was not included in this study.
Tis study was approved by the Institutional Ethics Committee for Research on Human Health with reference number 2961 CEI-Udo/02/2021/T.Te samples were collected following international safety rules and ethical standards.Informed and written consent and assent were obtained from the study participants before data collection.

Sample Collection, Isolation, and Identifcation of
Isolates.A total of 142 clinical samples were collected from the infected wounds of patients under aseptic conditions with the help of sterile cotton swabs.For patients with dry wounds, the swab was moistened with a sterile saline solution before swabbing.Te samples collected were then transferred to the research unit of Microbiology and Antimicrobial Substances at the Department of Biochemistry of the University of Dschang for analysis.Te samples collected on the swabs were then cultured on Mueller-Hinton agar at 37 °C for 24 h for bacterial growth and isolation.Identifcation of the Pseudomonas aeruginosa isolate was performed by standard microbiological and biochemical methods, including inoculation on a cetrimide agar medium.Pseudomonas aeruginosa isolates were characterized based on colony morphology, pigmentation on the King A agar medium, and the ability of the bacterium to produce oxidase, catalase, citrate, and urea-indole.

Antibiotic Susceptibility Test.
Te antibiotics used for susceptibility testing belonged to the following four classes: a 3 rd cephalosporin (ceftazidime), four aminoglycosides (amikacin, paromomycin, streptomycin, and neomycin), and a quinolone (norfoxacin).All were purchased from Sigma-Aldrich, China.In vitro antibacterial susceptibility testing was performed by the broth dilution method with Mueller-Hinton broth (Merck, Germany) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [24].P. aeruginosa ATCC 27853 was used as the reference strain.Te multidrugresistant P. aeruginosa (MDR-Pa) was defned as an isolate resisting more than one antimicrobial agent in three or more antimicrobial categories or classes [25].

Bioflm Formation Assay.
Bioflm formation assay was performed by the microtiter plate method described by Kirmusaoglu and Kas ¸ikçi [26] with some modifcations.In brief, 100 μL of Mueller-Hinton broth (MHB) supplemented with 1% glucose and 100 μL of inoculum 2 International Journal of Microbiology (1.5 × 10 6 CFU/mL) were inoculated into 96-well fatbottomed sterile polystyrene microplates.Microplates were then incubated at 37 °C for 24 h, 48 h, and 72 h.After each time of incubation, planktonic cells in the well of the microplate were removed.Each well was washed three times with 200 μL of sterile phosphate-bufered saline (pH 7.2).After washing, 150 μL of methanol was introduced into each well and allowed for 20 min to fx the bacteria.Te microtiter plates were then emptied by simple ficking.Te bioflm was stained for 15 min using 150 μL of safranin 1%.Subsequently, the cell-bound dye was resolubilized with 150 μL of 95% ethanol per well.Te microtiter plates were covered (to minimize evaporation) and left at room temperature for 30 min.Ten, the optical density (OD) was measured at 570 nm by a Versamax tunable microplate reader.Te study was performed in quadruplets and repeated three times.Te uninoculated wells containing sterile MHB supplemented with 1% glucose were considered as negative controls and used as blanks.Wells in which isolates had an OD greater than that of the blank were considered as bioflm producers.

Pyocyanin Quantifcation Assay.
Pyocyanin production by P. aeruginosa was determined by the method described by Alayande et al. with slight modifcations [27].In brief, the isolates were incubated in 10 mL of Luria-Bertani medium at 37 °C for 24 h, 48 h, and 72 h.After each time of incubation, the cultured cells were centrifuged at 1500 × g for 10 min to separate the supernatants from the pellet after which 3 mL of chloroform were added to 5 mL of the supernatant and the mixture was agitated vigorously using a vortex mixer.At the chloroform layer, 1 mL of 0.2 M HCl was added and then centrifuged at 1500 × g for 10 min.Te optical density of the HCl layer was measured at 520 nm using a spectrophotometer (Biobase BK-D590 Double Beam Scanning UV/Vis; China).Te HCl 0.2 M was used as a control (Blank).Te concentration of pyocyanin was obtained using the following formula: concentration of pyocyanin (µg/mL) � (DO 520nm − DO blank ) * 17.072.

Swarming and Swimming Motility Assays.
Te swarming motility test was determined following the method described by O'May and Tufenkji with a few modifcations [13].In brief, LB broth with 1% agar sterilized in the autoclave at 121 °C for 15 min was poured into 82 mm noncompartmentalized Petri dishes.A total of 2.5 μL of bacterial suspension in broth standardized at 0.5 McFarland aged 18-24 h previously cultured in the LB medium were gently deposited on the surface of the agar (at the center), allowing the visualization of the motility zone on the agar surface.Dishes were sealed and placed in incubators for 24 h, 48 h, and 72 h.At the end of each incubation period, the diameter of the motility zone was measured.
Te swimming motility test was determined according to the method described above but with some variations.Te previously sterilized LB broth with 0.5% agar was poured into the 82 mm Petri dishes.Te bacterial broth was deposited at the center of the agar (and not at the surface).Dishes were sealed and placed in incubators for 24 h, 48 h, and 72 h.At the end of each incubation period, the diameter of the motility zone was measured.

Statistical Analysis.
Te Pearson correlation test was used to evaluate the relationship between pyocyanin production and bioflm formation with antimicrobial susceptibility.Data analyses were performed using GraphPad Prism 8.0.P < 0.05 was considered statistically signifcant.

Prevalence of P. aeruginosa in Infected Wounds
. A total of 37 isolates of P. aeruginosa were identifed from 142 clinical samples collected from infected wounds at the district hospital of Dschang, resulting in a prevalence of 26.1%.

Antibiotic Susceptibility Test.
Te susceptibility of P. aeruginosa isolates to diferent antibiotics tested and the diferent phenotypes of isolates obtained are shown in Figure 1.In our study, 38 isolates of P. aeruginosa showed 100% resistance to streptomycin and paromomycin, 14 (36.8%)isolates showed resistance to amikacin, and 26 (68.4%) isolates showed resistance to ceftazidime.Te different phenotypes of the isolates are represented in Figure 1(b).Out of the 38 isolates of P. aeruginosa, 25 isolates showed multidrug resistance with a prevalence of 65.8%.

Bioflm Formation Ability of P. aeruginosa Isolates.
Te ability of diferent isolates of P. aeruginosa to form bioflms was evaluated at diferent times (24 h, 48 h, and 72 h).P. aeruginosa showed the ability to form bioflms, which was successfully quantifed by the safranin dye (Supplementary material S1).Te bioflm formation capacity increased with the incubation time up to 48 h and International Journal of Microbiology a decrease was observed after 72 h (Supplementary material S2).Te best time of bioflm formation in all isolates of P. aeruginosa isolates was observed at 48 h (Figure 2).Out of the 38 isolates, 50% (19 isolates) and 37% (14 isolates) were strong and moderate bioflm producers, respectively, with optical densities ranging from 1 to 1.7 and 0.5 to 1. Finally, 13% of the isolates (5 isolates) were classifed as weak bioflm producers.

Pyocyanin Production by P. aeruginosa Isolates.
Te capacity of isolates to produce pyocyanin is shown in Figure 3. Pyocyanin production was actively higher at 72 h of incubation (Supplementary material S3).All isolates of P. aeruginosa isolates were producers of pyocyanin with concentrations ranging from 2.1 to 4.4 μg/mL.

Swarming and Swimming Motility Ability of P. aeruginosa
Isolates.Te swarming activity of the isolates is shown in Figure 4.All isolates showed the ability to move in semisolid media.Te diameter of the displacement zones increases considerably over time until reaching 38 mm for isolate 7 and 41 mm for P. aeruginosa ATCC 27853 (Supplementary material S4).
Te swimming activity of the isolates is shown in Figure 5.All isolates also exhibited the ability to move in fuid or low-viscosity media.Te diameter of the halos increases considerably with time and reaches up to 50 mm for isolate 7 (Figure 5) (Supplementary material S5).

Correlation among the Bioflm Formation, Pyocyanin
Production, and Antibiotic Resistance.Te correlation between the antibiotic sensitivity of P. aeruginosa isolates and the concentration of pyocyanin produced is shown in Table 1.Te concentration of pyocyanin increased with bacterial resistance.Pearson's coefcient from a correlation between antibiotic resistance and the concentration of pyocyanin production demonstrated a strong correlation for amikacin (r � 0.791 and P < 0.001), neomycin (r � 0.694 and P < 0.001), ceftazidime (r � 0.846 and P < 0.001), and norfoxacin (r � 0.758 and P < 0.001).No correlation was observed with paromomycin and streptomycin.Table 2 shows the correlation between antibiotic resistance and bioflm formation.

Discussion
P. aeruginosa causes severe infections, particularly in healthcare settings.It has an outstanding capacity for being selected and for spreading antimicrobial resistance in vivo [28].Our fndings confrm that P. aeruginosa is present in chronic wounds.Te prevalence of P. aeruginosa in the wounds of patients at the Dschang District Hospital is 26.1%.P. aeruginosa is one of the most common pathogens in chronic wounds, and its ability to form resistant bioflms and several virulence factors that allow the establishment in host tissue, including pyocyanin has been documented [29].Tis result corroborates the work done by Raizman et al., who reported the presence of P. aeruginosa in an infected wound [4].However, the prevalence found during this work is lower than in the previous work done by De Oliveira et al. and Raizman et al., who found a prevalence of 72% and 92.9%, respectively, of P. aeruginosa in chronic wounds [4,5].Tis diference in prevalence would be due to some factors, such as the sample size, geographical diversity, patient demographical factors, or access and exposure to antimicrobial agents.
Although risk factors for infections with resistant P. aeruginosa strains include prior use of broad-spectrum  International Journal of Microbiology antimicrobials, Pseudomonas aeruginosa strains are also known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics.In this study, the antibiotic resistance profle of P. aeruginosa isolates was investigated towards six commonly used antibiotics indicated in the treatment of Pseudomonas infections.
Paromomycin was included since a high resistance of Pseudomonas aeruginosa to paromomycin has been previously reported [30].Given the increasing prevalence of multidrug-resistant P. aeruginosa and the paucity of useful antipseudomonal agents, the use of aminoglycosides has become increasingly important in managing P. aeruginosa International Journal of Microbiology infections.Terefore, in addition to paromomycin, we also included streptomycin as well as amikacin and neomycin.
Te results of this study showed that 65.8% of the isolates were resistant to at least one agent in three or more antimicrobial categories and, therefore, categorized as MDR.Tis percentage is lower than that obtained by Moazami Goudarzi and Eftekhar, who showed that 97.4% of P. aeruginosa on wounds were MDR [31].However, De Francesco et al. and Moazami Goudarzi and Eftekhar reported a diferent prevalence of MDR of P. aeruginosa (from 20% to 100%) [31,32].Te observed discrepancy could be because P. aeruginosa infections are complex in pathogenesis and are dependent on various virulence factors such as secretory factors, elastase, phospholipase C, alkaline protease, pyocyanin, hydrogen cyanide, pyoverdine, and rhamnolipids [33].P. aeruginosa also shows an outstanding ability to develop further antimicrobial resistance to all available antibiotics via the acquisition of chromosomal mutations [28].Bacterial motility plays a key role in the colonization of surfaces by bacteria and the subsequent formation of resistant communities of bacteria called bioflms.
In the present study, 100% of P. aeruginosa isolates from infected wounds were bioflm producers, of which 50%, 37%, and 13% were strong, moderate, and weak bioflm producers, respectively.Tese data support the roles of bioflm wound healing since bacterial bioflm has been recognized as a major factor in delayed wound healing and high levels of bioflm production have been repeatedly described in multidrug-resistant organisms [34].Tese results are similar to those of Jabalameli et al., who showed that 96% of P. aeruginosa isolated from wounds were bioflm formers of which 49.9% were strong bioflm producers, 26% were moderate bioflm producers, and 22.9% were low bioflm producers [35].Tis ability to form a bioflm would be due to a variety of components playing a role in the attachment of P. aeruginosa to surfaces.Tese components include fagella, type IV pili, Cup fmbriae, extracellular DNA (eDNA), and Psl polysaccharides [36].Tis ability to form the bioflm would also be due to the release of compounds such as iron siderophores, biosurfactants, and EPS into their surrounding environment.
In this work, all isolates of P. aeruginosa were pyocyanin producers, which corroborate the report of Al-Qaysi et al. [23].Our fnding is of great importance for the studied population since pyocyanin is a major virulence factor contributing to P. aeruginosa pathogenesis.In other studies, more than 90% of clinical P. aeruginosa isolates produce pyocyanin [37].Te correlation observed between the pyocyanin produced and the resistance of P. aeruginosa to antibiotics could be due to the important role of pyocyanin in resistance to antibiotics.Tis result is in agreement with 6 International Journal of Microbiology those obtained by Khadim and Marjani, who suggested that pyocyanin itself may play a direct role in the observed link between antimicrobial susceptibilities and the phenotype [38].Furthermore, Sweedan suggested that pyocyanin production was relatively afected by antibiotic challenges [39].In addition to the virulence factors produced by P. aeruginosa, it is also characterized by its ability to form bioflms.All P. aeruginosa isolates exhibited the ability to move through semisolid (swarming) and low-viscosity (swimming) media.Tis displacement is due to P. aeruginosa fagella which allow colonization of surrounding environments.Tis work is in agreement with that carried out by Otton et al. [40].Tis fnding is supported by a report suggesting that in Pseudomonas aeruginosa, fagellar stators play several roles in the formation of bioflms and the control of swarming motility [41].Tese data were later emphasized by those who suggested a possible structural role for fagella in a bioflm, conceivably as a scafold [42].
A correlation between pyocyanin production and bioflm formation was observed.Tis correlation corroborates the study performed by Al-Qaysi et al., who obtained the    International Journal of Microbiology correlation between pyocyanin production and bioflm formation of P. aeruginosa isolates from patients in Iraq [23].Te release of pyocyanin in P. aeruginosa clinical strains induces a signifcant increase in eDNA as a bioflm promoter, which improves bioflm formation [43].Furthermore, P. aeruginosa cells in the presence of eDNA have favorable acid-base interactions, which aid in bioflm production [44].Moreover, pyocyanin infuences essential physicochemical interactions that drive bacterial cell-to-cell interactions.Pyocyanin is essential in the ability to foster aggregation.Pyocyanin is characterized by dysregulation of the QS system, leading to increased production of exoproducts, which are essential for the bioflm formation in P. aeruginosa [45].Tis study sheds light on the bioflm-forming capabilities and pyocyanin production levels of P. aeruginosa isolates, a bacterium notorious for its association with chronic wound infections.Te results ofer valuable insights into the virulence factors associated with Pseudomonas aeruginosa infections and contribute substantially to our knowledge of the mechanisms employed by P. aeruginosa in evading host defenses, providing a foundation for the development of targeted therapeutic interventions, improving the efcacy of treatments for patients in Dschang Hospital, and potentially infuencing wound care practices worldwide.Furthermore, the assessment of antibiotic resistance profles in these isolates is of critical importance in the era of escalating antibiotic resistance.By determining the resistance patterns of P. aeruginosa in the specifc context of wounds at Dschang Hospital, the study provides essential information for local healthcare practitioners.Tis insight allows for more informed antibiotic prescribing practices, promoting efective treatment strategies tailored to the unique resistance profles observed in the region.
Overall, fnding from this study not only contribute signifcantly to the understanding of P. aeruginosa infections in the local context of Dschang Hospital but also ofer insights that have broader implications for global infectious disease management.Te study's outcomes pave the way for more targeted and efective treatment approaches, reinforcing the importance of locally driven research in shaping the future of healthcare practices worldwide.

Conclusion
Findings from this study suggest that infected wounds could act as a reservoir for MDR and virulent P. aeruginosa.Te presence of strong bioflm producers of P. aeruginosa in infected wounds is a serious public health concern.Terefore, surveillance programs to monitor and control MDR P. aeruginosa in these patients are required to improve patients' quality of life.In addition, bioflm formation and pyocyanin production could also be regarded as potential targets for the development of a new therapeutic agent against P. aeruginosa infections.Further studies are needed to investigate the presence of antibiotic resistance and bioflm formation genes as well as the genetic diversity of the isolates.

Figure 1 :
Figure 1: Antibiotic susceptibility (a) and resistance phenotype (b) patterns of P. aeruginosa isolates recovered from wounds.

Figure 4 :
Figure 4: Swarming motility of P. aeruginosa.2.5 μL of bacterial suspension was dropped at the center surface of the agar plate and incubated for 24 h, 48 h, and 72 h at 37 °C.In this fgure are photographs of representative swarming motility at diferent time intervals.Te diameter of the swarming motility zone increased with incubation time.

Figure 5 :
Figure 5: Swimming motility of P. aeruginosa.2.5 μL of bacterial suspension was dropped at the center surface of the agar plate and incubated for 24 h, 48 h, and 72 h at 37 °C.In this fgure are photographs of representative swimming motility at diferent time intervals.Te diameter of the swimming motility zone increased with incubation time.

Table 1 :
Correlation between antibiotic resistance and pyocyanin production.