التوصيف الجزيئي للبكتريا السائدة المكونة للغشاء الحيوي و تحديد بعض العوامل الكيموحيوية و المناعية المرافقة لاصابة القدم السكري

Molecular Characterization of Predominant Biofilm Forming Bacteria and Identification of Some Biochemical and Immunological Parameters Associated with Diabetic Foot Infection

ABSTRACT

This study involved the isolation and identification of the most common species of bacteria responsible for diabetic foot infection along with their antibiotic susceptibility patterns and investigation of biofilm production of these bacteria as well as molecular detection of some virulence and antibiotic resistance genes .The current study, additionally, accompanied to find differences in the levels of some inflammatory markers including Toll like Receptor-2 (TLR-2), Interleukin-17A (IL-17A) and C-reactive protein (CRP) along with some antioxidants such as Superoxide dismutase (SOD) and glutathione (GSH) among type 2 diabetic subjects with and without diabetic foot ulcers and in healthy subjects.
A total of 142 swab samples were taken from diabetic ulcer patients checked in Al-Imam Al-Hasan center for endocrinology and diabetes and bacteriological examination was performed while 120 subjects enrolled in the current study for determination of biochemical and immunological markers divided into three groups :40 participants were type 2 diabetes mellitus patients with diabetic foot ulcer, 40 participants were type 2 diabetes mellitus patients without diabetic foot ulcer, and 40 participants were healthy control subject.
Cultivation of swab results in 23/142 negative culture and obtaining of high rate of poly-microbial infection reached to 82% while mono-microbial rates were restricted in 18% of which, 63/142 ( 44 %) isolates were Gram positive and 56/142 ( 39.4 %) were Gram negative bacteria. Out of 142 included subjects, 74/119 (62.18%) were male and 45/119 (37.18%) were female and most subjects that have the highest number of isolates were aged (55-64) years 33(27.7%) while subjects aged ≥ 75 years have the least number of isolates 18 (15 %). The biochemical and microscopical tests were applied and results in obtaining of Staphylococcus aureus 25 (21%) isolates, Staphylococcus epidermidis 18 (15%), Streptococcus agalactiae 15 (13%) and Enterococcus faecalis 5 (4%), while of Gram negative bacteria, Klebsiella pneumoniae isolates were 25(21%) , Pseudomonas aeroginosa 13 (11%) , Proteus mirabilis10 (8%) and Escherichia coli 8 (7 %) . Performance of susceptibility tests of S.aurues results in all isolated Staphylococcus aureus was maximum sensitive (84%) to Levofloxacin, while the lowest sensitivity (36%) was toward Azithromycin and Erythromycin , 40% of the sensitivity were toward Penicillin, Amoxicillin-Calvulonic Acid, Ceftazidime, Cefepime Cefotaxim, Ceftriaxone, Imipenem, Meropenem, Aztronem and Oxacillin, while the sensitivity of both Rifampin and Vancomycin were 52% and Ciprofloxacin was 48%. On the other hand, Klebsiella pneumoniae exhibited the highest resistance 100% toward Gentamycin and Levofloxacin followed by 96% resistance toward Ciprofloxacin and 92% resistance toward Amikacin. With respect to Pipracillin-Tazobactam, Cefotaxim, Ceftriaxone and Tetracyclin, resistance percent was 88% while Amoxicillin-Clavulanic acid, Cetazidime, and Cefepime were 84% resistant. Furthermore, K.pneumoniae has 76% resistance toward Imipenem and Meropenem and 68% toward Doxycycline and 56% with respect to Rifampin.
According to biofilm formation, tube method showed that S.aureus and K. pneumoniae were biofilm producers while microtiter plate method showed that 60% of S.aureus and K. pneumoniae obtained from Diabetic foot infection (DFI) in the current study were strong producers for biofilm while 40% of them were moderate producers.
PCR technique was used to detect that 60% of S.aureus contained mecA gene, 64% contained (icaA, sea and ermC) genes. Finally, 80% contained icaD gene. However, 88% of K.pneumoniae contained mrkD gene , 100% contained Cps gene, 12% contained K1 gene and 60% contained k2A gene.
Concerning biochemical markers, mean Toll-like receptor 2 (TLR-2) levels in DFI patients was (7.36±1.85) ng/ml which was significantly higher than in DM patients and control groups (p ≤ 0.001) while in interleukin-17A (IL-17A), the mean of levels in DFI patients were (123.7±33.52) ng/L which was significantly higher than DM and control groups (P ≤ 0.001) and C-reactive protein (CRP) showed a higher rate in DFI patients (92.9±78.26) mg/L which was significantly higher than DM and control groups (p ≤ 0.001).
Concerning the antioxidants, superoxide dismutase (SOD) activity in DFI group patients exhibited decreased levels significantly (241.4±35.61)(U / L) (p≤0.001) compared to DM and healthy individuals as well as glutathione (GSH) whereas the mean levels of this marker in DFI group patients decreased significantly (26.586±2.77) µmole/ml (p≤0.001) compared to DM and healthy individuals
The current study concluded that S.aureus is the most predominant of Gram positive bacteria in DFI while K.pneumoniae is the most prevalent of Gram negative bacteria and both of which are biofilm forming and high resistant for common antibiotics.