Diabetic Foot Ulcer Infections and Pseudomonas aeruginosa Biofilm Production During the COvid-19 Pandemic

During the different waves of the coronavirus (COVID-19) pandemic, there has been an increased incidence of diabetes mellitus and diabetic foot infections. Among gram-negative bacteria, Pseudomonas aerugino sa is the predominant causative agent for diabetic foot ulcer infections in low-resource countries. P. aeruginosa possesses a variety of virulence factors, including biofilm formation. Biofilm formation is an important benchmark characteristic in the pathophysiology of diabetic foot ulceration. The main objective of the current study was to identify the most commonly isolated organisms and their antibiotic susceptibility patterns in diabetic foot patients during the COVID-19 pandemic. We also determined the genes associated with bacterial persistence and biofilm formation in the predominantly isolated organism. Accordingly, 100 wound swab samples were collected from diabetic foot patients from different hospitals in Alexandria, Egypt. Through phenotypic detection of biofilm formation, 93% (40) of the 43 P. aeruginosa isolates examined were categorized as biofilm producers. Molecular detection of the biofilm-encoding genes among the 43 P. aeruginosa isolates was as follows: algD (100%), pelF (88%) and pslD (49.7%), and this highlights a need for biofilm formation inhibitors to prevent the persistence of bacterial pathogens, and thus achieve better clinical outcomes in diabetic foot ulcer infections.


INTRODUCTION
2][3][4] A study in Wuhan reported that diabetic patients constituted 2-20% of all positive cases, and accounted for 7.1% of intensive care unit admissions. 5,6Another study in New York reported that 33.8% of COVID-19 positive patients were diabetic.Accordingly, several reports have attempted to determine the reason for the correlation between COVID-19 and DM.8][9] In addition, the use of corticosteroids in COVID-19 patients increases blood glucose levels in both diabetic and non-diabetic individuals. 5,10Additionally, diabetic patients are more vulnerable to viral and bacterial infections. 5,11 he COVID-19 pandemic has posed many challenges for the diabetic community, such as lack of sufficient resources, overworked health care workers, and scarcity of proper care for diabetic patients to avoid the development of infections. 12,13 abetic foot ulcer (DFU) is a common complication of DM, with an increasing prevalence worldwide. 14Thirty-three percent of all diabetic patients are expected to be diagnosed with DFUs at least once during their lifetime.Untreated DFUs can result in leg amputations, permanent disability, and increased mortality rates in DM patients. 13][17][18][19][20][21][22] Microbiological studies have shown that diabetic foot ulcers generally have polymicrobial etiologies.The etiological agent may differ from one individual to another, and from country to country.Aerobic gram-positive bacteria that are frequently isolated in DFUs include Staphylococcus spp.betahemolytic Streptococcus and Enterococcus spp.Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae are among the gram-negative bacteria that are most commonly isolated in DFUs. 23][17][18][19][20][21][22] Pseudomonas spp.are generally encountered in immunocompromised patients due to their high pathogenicity and variety of virulence factors, including biofilm formation. 24Virulence factors and biofilm formation are the benchmark characteristics in the pathophysiology of DFUs. 25 The formation of biofilms by bacteria is considered to be the cornerstone that provides bacteria protection against several environmental factors, mediates persistence in medical devices, facilitates immune system evasion, and contributes to the development of antimicrobial resistance. 15,21,22ntibiotic resistance is a global public health concern, especially in patients with diabetic foot infections (DFIs).Multidrug resistance results in poor clinical outcomes, financial burden, and increased morbidity and mortality in DFU patients. 23he main objective of the current study was to identify the commonly isolated organisms and their antibiotic susceptibility patterns in DFU patients during the COVID-19 pandemic.In addition to phenotypic detection of biofilms, we also determined genes encoding biofilm formation in the predominantly isolated organism.

MAteRiAls ANd MethOds
One hundred wound swab samples were collected from DFU patients at the Vascular Surgery and Diabetic Foot Unit of Abou Hommos Central Hospital, Alexandria Main University Hospital, Mowasat Hospital, Abou Qir Central Hospital, and Medical Research Institute, in Alexandria, Egypt, between January 2020 and January 2021.The study was approved by the Ethics Committee in Pharos University in Alexandria, and all procedures were performed according to Helsinki ethical standards.Specimens were subjected to Gram staining, and were cultured in mannitol salt agar, MacConkey agar, blood agar, and Sabouraud dextrose agar (Oxoid, Cambridge, UK).Isolates were identified using standard biochemical methods, 26 and P. aeruginosa isolates were confirmed with MALDI-TOF/MS (Bruker, Billerica, MA, USA).The identified stock cultures were preserved at −80 °C in 15% glycerol.

Statistical analysis
Statistical analysis of the data was performed using IBM SPSS software version 20.0.(IBM Corp, Armonk, NY, USA).The chi-square test and Fisher's exact test were used.Statistical significance was set at a p-value of 5% or lower.

RESUlTS
The present study included swabs from 100 DFU patients (75 male and 25 female) admitted to the Vascular Surgery and Diabetic Foot Unit of Abou Hommos Central Hospital, Alexandria Main University Hospital, Mowasat Hospital, Abou Qir Central Hospital, and Medical Research Institute, Alexandria, Egypt, between January 2020 and January 2021.The ages ranged from 44 to 76 years.
Of the five S. aureus isolates, 96% were resistant to tetracycline and methicillin and with varying degree of resistance to other antibiotics.Of the two Enterococcus spp.isolates, 50% were resistant to cefoxitin, as shown in Table 4.

Molecular detection of Biofilm encoding genes in P. aeruginosa
The following genes encoding biofilm exopolysaccharides were identified in the 43 P. aeruginosa isolates: algD (100%), pelF (88%) and pslD (49.7%).The presence of algD, pslD, and pelF genes was noted in a large proportion of the 43 P. aeruginosa isolates.Our findings revealed that 82.6 % of the 23 strong biofilm producers had the genotypic pattern algD +/pslD +/pelF +, while the rest were algD −/pslD −/pelF −.On the other hand, 66.6% of the three non-biofilm producers carried the biofilm encoding genes, as shown in Table 5.

DISCUSSION
DFU is a debilitating consequence of DM with an increasing prevalence worldwide. 14uring the different waves of the COVID-19 pandemic, DM was increasingly diagnosed worldwide.8][9] Other reported reasons include the rise in blood glucose levels noted in patients receiving corticosteroids. 5,10Additionally, diabetic patients are more vulnerable to viral and bacterial infections. 5,11Other challenges contributing to the increased incidence of DFIs include a lack of sufficient resources, overworked health care workers, and a scarcity of proper care for diabetic patients to avoid the development of infections. 13he present study included swab samples from 100 patients with DFUs (75 male and 25 female).Other studies have also supported the male predominance of foot ulceration and its associated complications. 32,33Culture results of the 100 specimens showed 76% monomicrobial bacterial growth, 20% polymicrobial bacterial growth, and 4% fungal growth.Hitam et al. also reported a similar percentage (28.8%) of polymicrobial infections in DFI patients. 34dditionally, culture results showed that the majority of isolates were gram-negative (88%) bacteria, and P. aeruginosa was the predominant microorganism isolated (43%), followed by K. pneumoniae (21%), P. mirabilis (11%), E. coli (9%), and S. marcescens (4%).Among grampositive bacteria, S. aureus was the most common isolate.6][17][18][19][20][21][22] P. aeruginosa should not be regarded as a normal flora in burn wounds and diabetic foot patients.P. aeruginosa can cause extensive tissue damage in diabetic patients and result in sepsis. 24][36][37] Antibiotic susceptibility testing revealed that 100% of the forty-three P. aeruginosa isolates were resistant to Ampicillin, followed by Aztreonam (74%), Amikacin (72%), Levofloxacin, Gentamycin and Amoxicillin-clavulanate (69.7%),Cefoxitin (46.5%),Ceftazidime (41%), Imipenem (32.5%), and varied degrees of resistance to other antibiotics was observed.Multidrug resistance (MDR) was observed in 30 (69.7%) of the P. aeruginosa isolates.Sivanmaliappan et al. 24 reported that 55.5% of P. aeruginosa were multidrug resistant (MDR); 100% were resistant to ampicillin, 83.3% to piperacillin, and 66.6% to ceftazidime, gentamycin and imipenem.However, our results show that ceftazidime, imipenem, and piperacillin/tazobactam combination display higher activity as antipseudomonal agents.Banar et al. 30 also stated that ceftazidime displayed high activity in P. aeruginosa isolates.In a study on DFI in Tanzania, resistance was noted for all commonly used antibiotics, except imipenem (100% sensitivity).This can be attributed to the fact that imipenem is expensive in low-resource countries. 38Previous reports described the increased efficacy of piperacillin-tazobactam against several virulence traits, such as adhesion, biofilm production, and flagellin production. 39he increased prevalence of MDR has been noted in different studies worldwide. 30,31,40,41This can be attributed to extensive use of antibiotics, which gives a selective advantage for survival of pathogenic bacterial strains.MDR P. aeruginosa guarded by biofilms that are difficult to penetrate can survive and develop more resistance. 41,42iofilm production is the benchmark characteristic for the development of DFIs, and provides a balance between colonization and infection. 25Bacteria within biofilms produce their own matrix of extracellular polymeric substances (EPS).EPS contains glycoproteins and polysaccharides that provide protection against several environmental factors, mediate persistence in medical devices, facilitate immune system evasion, and contribute to the development of antimicrobial resistance. 15,21,22There are three major exopolysaccharides that significantly contribute to the formation and stabilization of the biofilm matrix of P. aeruginosa.The pentasaccharide Psl is essential to promote both cell-cell and cell-surface interactions, thereby initiating biofilm formation and providing structural support to the formed biofilm.The PslD protein is encoded by the pslD gene, a part of the psl operon.The PslD protein is located in the periplasm/outer membrane and contributes to the export of essential biofilm exopolysaccharides.Alginate is another important polymer that significantly stabilizes biofilm formation and provides additional protection.The synthesis of alginate protein is mediated by the algACD operon.The algD gene controls the synthesis of the alginate proteins.The algD gene controls the production of the final precursor, GDP-mannuronic acid, one of the two monomers of alginate.The pellicle operon controls the synthesis of the third major exopolysaccharide, the Pel protein, which is responsible for pellicle formation. 30,31n this context, biofilm formation was evaluated both phenotypically using the crystal violet assay, in addition to molecular detection of genes responsible for biofilm formation, algD, pslD, and pelf .Phenotypic characterization revealed that 93% (40) of the 43 P. aeruginosa isolates examined were biofilm producers; 53.4% (23) of P. aeruginosa isolates were strong biofilm producers, 23.3% (10) were moderate biofilm producers, 16.3% (7) were weak biofilm producers, and only 6.9% (3) were non-biofilm producers.Kamali et al. reported that 83.75 % of their P. aeruginosa isolates were biofilm producers with variable degrees of biofilm production. 31Banar et al. 30 reported that out of 57 P. aeruginosa isolates tested 55 (96.5%) isolates were biofilm producers with variable degrees of biofilm production, and only 2 (3.5%) isolates were regarded as non-biofilm producers.

CONClUSION
During the different waves of the COVID-19 pandemic, there has been an increased incidence of DM and DFIs.P. aeruginosa is the predominant etiological agent for DFIs.In the present study, the majority of P. aeruginosa isolates were MDR and biofilm producers.A high prevalence of biofilm-encoding genes were identified in this study, highlighting a need for inhibitors of biofilm formation to prevents the persistence of bacterial pathogens, and thereby achieve better clinical outcomes.

Table 1 .
Primers used for detection of genes involved in Biofilm formation by Pseudomonas aeruginosa

Table 2 .
Culture results of 100 diabetic foot ulcers

Table 3 .
Frequency of microbial isolates from 100 diabetic foot ulcers

Table 4 .
Resistance profile of the tested isolates to different antimicrobial agents

Table 5 .
Relationship between phenotypic biofilm characteristic and genotypic biofilm characteristic among P. aeruginosa isolates (Manual)