Study of Biofilm Formation and Antibiotic Resistance Pattern of Bacteria Isolated from Diabetic Foot Ulcers in Hôpital de Référence Saint Joseph, Kinshasa, Democratic Republic of Congo

Foot infections resulting from biofilm producers and multi-drug resistant organisms is one of the most important complications of diabetes mellitus, as it can impede the wound healing process. This study was carried out in order to determine the antibiotic resistance pattern and the biofilm production in diabetic foot ulcers isolates. Clinical samples were collected from patients suffering from diabetic foot ulcers by using sterile swabs. Antibiotic susceptibility test was done using disk diffusion method on Mueller Hinton Agar. Biofilm formation was assessed by Crystal Violet Staining Method. Staphylococcus aureus isolates were resistant to ofloxacin (83.3%), ciprofloxacin (75.0%), trimethoprim-sulphamethoxazole termining the drugs for the treatment of diabetic ulcers.


Introduction
Diabetic foot ulcers (DFUs) are a prevalent complication of diabetes mellitus (DM) and lead significant morbidity, mortality, and healthcare expenditures [1]. Africa is estimated to have 15.9 million adults living with DM making a regional prevalence of 3.1%. The annual incidence of diabetic foot ulcer worldwide is between 9.1 to 26.1 million [2].
The African continent has the greatest proportion of people with undiagnosed DM and global projections show that the continent will face even a greater burden of DM of about 156% by 2045 [3]. Around 15% to 25% of patients with DM will develop a diabetic foot ulcer during their lifetime [4]. DFU are among the most common complications for patients who have insufficiently controlled DM. It is one of the common causes for osteomyelitis of the foot and amputation of lower extremities [5]. These ulcers are usually in the areas of the foot which encounters repetitive trauma and pressure sensations [6]. When an ulcer is present, there is a clear entrance for invading bacteria. Infection can range from local infection of the ulcer to wet gangrene. Only half of infection episodes show signs of infection. In the presence of neuropathy and ischaemia, the inflammatory response is impaired and early signs of infection may be subtle. Deep swab and tissue samples (not surface callus) should be sent for culture without delay and wide spectrum antibiotics given to cover Gram positive, Gram negative, and anaerobic bacteria. Urgent surgical operation is needed in certain circumstances [7]. The ulcers often become chronic and infected with bacterial biofilm [8]. Systemic antibiotics are prescribed when the ulcer shows clinical signs of infection [9] [10] [11]. Resolution of infection after treatment of Diabetic Foot Infection (DFI) with systemic antibiotics varies widely with values ranging from 5.6% to 77.8% [12]. At a high bacterial load, the biofilm is likely to be very well established and highly tolerant to antibiotics [13] [14]. DFIs are typically colonized by bacteria similar to the surrounding skin and become more complex in microbial diversity over time and with progression of the ulcer [15] [16] [17] [18]. The common organisms seen in a DFU are Gram positive organisms such as Staphylococcus, Enterococcus and Streptococcus, Gram negative organisms such as Enterobacteriaceae and Pseudomonas sp, and anaerobes [19]. The biofilms are the management of wound infections in diabetics who, in spite of repeated antibiotic treatment, fail to respond to treatment because biofilms are not being tested for routinely [20]. The aim of this study was to evaluate the antibiotic susceptibility pattern and the biofilm formation by Gram-positive and Gram-negative organisms isolated from DFUs in Hôpital de Référence Saint Joseph, Kinshasa.

Origin of the Strains and Laboratory Procedures
The clinical samples were collected for diagnostic purposes in 2016 by the bacteriology laboratories of Hôpital de Référence Saint Joseph in Limete, Kinshasa, and were from wound secretions of DFU. Infected sites were aseptically cleaned using normal saline and sterile gauzes. Then a wound swab from each patient was collected using sterile cotton swabs. Isolated bacteria on Trypticase soy agar medium (Liofilchen, Roseto degli Abruzzi, Italy) were received in the Laboratory of Experimental and Pharmaceutical Microbiology at the Faculty of Pharmaceutical Sciences of the University of Kinshasa for biofilm formation studies. Antibiotic susceptibility tests were done to confirm the results from hospital. Pathogens studied are presented in Table 1 below.

Isolation and Identification of Bacteria
Wound swabs were inoculated into mannitol-salt and Mac Conkey agars (Liofilchen, Roseto degli Abbruzzi, Italy) and incubated at 37˚C for 24 hours. Staphylococcus sp. were identified by standard microbiological methods such as Gram staining, catalase tests. S. aureus suggestive colonies were confirmed by coagulase and DNase testing. Gram-negative bacilli were identified using microbiological conventional methods including Gram staining, oxidase tests, indole and urease production, citrate utilization, hydrogen sulphide, gas production and fermentation of sugars, phenylalanine deaminase, lysine decarboxylase (L.D.C.), ornithine decarboxylase (O.D.C.), arginine dihydrolase (A.D.H.) tests, and methyl red reaction [21]. In our laboratory Gram negative bacilli were confirmed as Enterobacteriaceae species using the same tests. Pseudomonas aeruginosa were confirmed after 24 hours incubation time into Cetrimide agar.

Antibiotic Susceptibility Testing
Antibiograms of each isolated Staphylococcus sp. strains using the diffusion method on Mueller Hinton Agar were realized with the following antibiotic disks

Biofilm Formation Assay
In the present study, we screened all the isolates for their ability to form biofilm by Crystal Violet Staining Method (CVSM). The S. aureus and Enterobacteriaceae isolates were analyzed as described previously by Stepanovic et al., [23] and Ramos-Vivas et al., [24] respectively with minor modifications. Vivas et al., [24] was used: OD ≤ 0.05, non-biofilm producer; OD > 0.05 -0.1 weak biofilm producer; OD > 0.1 -0.3 moderate biofilm producer; and OD > 0.3 strong biofilm producer.

Statistical Analyses
GraphPad software package was used to calculate mean and standard deviation.

Staphylococcus Isolates
The results of the antibiotic susceptibility tests of Staphylococcus and Gramnegative organisms are shown in tables below. Among S. aureus strains studied, the highest resistance rates were observed for ofloxacin (83.3%), followed by trimethoprim-sulphamethoxazole and ciprofloxacin with a resistance rate of 75.0%, respectively. These strains were more sensitive to imipenem and vancomycin (91.7% respectively) and oxacillin (100%). The other antibiotics showed a resistance rate of 58.3% (Table 2).

Gram Negative Isolates
The highest rates of resistance (greater than 80.0%) against Pseudomonas sp.

Discussion
DFUs can become chronic and non-healing despite systemic antibiotic treatment. The penetration of systematically-administered antibiotics to the site of infection is uncertain, as is the effectiveness of such levels against polymicrobial biofilm [25]. Regarding the antibiograms performed, the results obtained ( Table   2, Table 3(a) and Table 3(b)) showed that the strains of staphylococci studied were resistant to the majority of the antibiotics tested, with the exception for oxacillin, vancomycin, and imipenem. Methicillin-resistant S. aureus (MRSA) was not observed. Our results are consistent with a report from Kenya in which S. aureus was highly resistant to trimethoprim-sulphamethoxazole but sensitive to oxacillin and vancomycin [26]. P. aeruginosa and other Enterobacteriaceae strains were highly resistant to the majority of antibiotic tested, with the exception for amikacin. This is not in the line with report from Kenya in which P. aeruginosa was sensitive to ampicillin, ceftazidime, and trimethoprim-sulphamethoxazole  [32]. A non-healing wound is an indicator of the presence of biofilm [33]. Biofilms cause a delay in healing by initiating an immune response leading to chronic inflammatory cycle and tissue damage due to high levels of proteases and reactive oxygen species [34] [35]. In this study, CVSM was used to evaluate the capability of bacteria from DFUs to produce a biofilm.
The results have showed that the majority of Staphylococcus strains have produced a biofilm. A recent study has demonstrated that the staphylococcal isolates are able to form biofilm [36]. Several virulence genes are implicated in biofilm formation, like icaA and icaD, responsible for the biosynthesis of polysaccharide intercellular adhesion (PIA) molecules, containing N-acetylglucosamine, the main constituent of the biofilm matrix in the accumulation phase [37]. Staphylococcal strains studied were resistant to the majority of the antibiotics tested.
Indeed, biofilms exhibit enhanced tolerance to antibiotics compared to free-living bacteria, which makes treatment of wound infections challenging [32]. A retrospective study has demonstrated that Gram-negative from DFI were found to be biofilm producers [38]. The results of the present study demonstrated that all P. aeruginosa and Enterobacteriaceae strains produced biofilms. Two P. aeruginosa isolates 4 and 8 ( Figure 2) and one K. pneumoniae ( Figure 3) strain produced strong biofilms. P. aeruginosa plays an important role in diabetic foot infections. As a Gram-negative opportunistic pathogen, P. aeruginosa causes recurrent and refractory infections that are characterized by biofilm formation [39]. Extracellular matrices (ECMs) of biofilms usually consist of exopolysaccharide (EPS), extracellular DNA (eDNA), and proteins, which act as a matrix, adhesive material, and protective barrier [40] [41]. There are three identified EPSs in P. aeruginosa which are involved in biofilm formation: Psl (polysaccharide synthesis locus), Pel (a glucose-rich polysaccharide polymer), and alginate [42]. Quorum Sensing (QS) plays also an important role in P. aeruginosa biofilm formation.
Indeed, QS systems not only sense population density, but also regulate a variety of traits, such as bacterial phenotype, spatial differentiation in biofilms, motility, and biofilm formation [43]. But recent data demonstrated that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its Type III Secretion System (T3SS). The T3SS virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers [44]. Staphylococcus, P. aeruginosa and Enterobacteriaceae strains studied were highly resistant to the majority of antibiotic tested as demonstrated in previous studies. Results obtained by other authors have shown that multidrug resistant organisms isolated from DFU were biofilm formers [20] [45] [46]. The ineffectiveness of traditional antibiotics-based treatment of biofilm has been attributed to a combination of different factors. The multilayered defense against antibiotics includes poor penetration into biofilms, adaptive stress responses, and metabolic inactivation due to nutrient and gas limitation [47]. A negatively charged biofilm membrane may limit the penetration of positively charged antibiotics through the biofilm [48]. Even if the antibiotic molecule enters the biofilm, it has to diffuse through the aqueous matrix in order to reach the bacterial cells. Aminoglycosides and beta-lactams may be inactivated or sequestered by binding to any solutes present in the matrix, making it impossible for them to diffuse to the depths of the biofilm [49] [50], also referred to as mass transport limitation.

Conclusion
The present study showed that multidrug-resistant pathogens in DFUs were biofilm producers. As biofilms infections are difficult to eradicate using conventional antibiotics, it is necessary to determine the antibiotic susceptibility pattern of the biofilm producers among clinical pathogens prior to the treatment of DFI.