Molecular Detection of Intercellular adhesion Genes and Biofilm Formation among Orthopedic Prosthetic Joint Infection

Abstract

Background: this study carried out to detect a adhesion genes (icaA and icaD) among orthopedic patients showing prosthetic joint infection in Helmia Armed Force Hospital. The hip and knee aspirate samples (49) were collected from (patients showing infected prosthesis) under stander conditions and transfer rapidly to the lab: (5) samples were negative control so the total number of samples were (54) samples. The aim of this study to detect biofilm formation and also to detect some genes (icaA, icaD) which are responsible for biofilm formation and gene expression. Material and methods: The samples were cultured on blood Agar medium then the isolates were cultured on Conog Red Agar medium to detect biofilm formation. Molecular detection carried out: firstly DNA was extracted from bacterial isolates; RNA was also extracted and was used as template for real time PCR (q RCR) by light cycler (Roch) PCR, sybr green as fluorescent dye. Reverse transcriptase was done, specific primers were used for icaA and icaD to transfer RNA into cDNA genes 16rSRNA was used as reference control. Results: The results showed that 67 .5% of the samples (hip and knee aspirates) were biofilm formation with black colonies (+ve) biofilm while red colonies (-ve) biofilm formation. The results showed that in the hip aspirate samples) (4) out of (28) 14.8% were positive for both (icaA, icaD)genes and positive for biofilm formation. In knee aspirate samples (5) out of (21) 28.5% were positive for both (icaA and icaD)genes and positive for biofilm formation . The phenotype (biofilm formation) don't correlate with the presence of icaA, icaD. The gene expression was evaluated with the presence of mRNA (icaA, icaD) for example, in current study (hip aspirate samples) (4) out of (28) 14.2% and negative for biofilm formation (phenotype). In knee isolates samples (3) out of (21) were negative for icaA and icaD but positive phenotype. In the current study the icaA gene expression was 4.98while icaD gene expression was 0.88 i:e the icaA gene expression was 2.4 fold change icaD gene expression Conclusion: Biofilm bacteria are difficult to eradicate and cause catastrophic problems for infected prosthetic orthopedic patients. There are some factors lead to the biofilm formation like presence of ica genes basically and the environmental conditions such as the surface of biomaterial. In addition to the presence and the concentration of some substances like: glucose, iron, phenols, limited oxygen. The gene expression of icaA, icaD is proportional to the amount of mRNA of these genes.