Asian Research Thesis Index

Abstract

Summary Biomedical devices are indispensable for modern health care and therapeutic industry which revolutionized the treatment strategies and outcomes. Biomedical indwelling devices are prone to the contamination and colonization of endogenous or exogenous microbes. The consequences of this colonization are severe including failure of indwelling devices, treatment failure, burden to the healthcare facility, prolonged and extensive treatment and sometime death of the patient. Staphylococcus aureus is ubiquitous pathogen that harbors battery of enzymes, toxins, adhesion proteins and variety of pathogenic strategy. It is responsible for minor skin infection to life threatening infection and capable of causing disease in hospitals and community. Biofilm formation potential is another pathogenic strategy of Staphylococcus aureus which helps it to colonize on biomedical devices and results in worse consequences for patient and healthcare industry. This study was conducted on 6,424 biomedical indwelling devices which were supposed to be contaminated or infected by endogenous or exogenous pathogens. Of these biomedical devices, 4.420 (69%) were colonized by bacterial of fungal pathogens. Escherichia coli was most common pathogen, followed by Staphylococcus aureus, Acinetobacter species, Coagulase Negative Staphylococcus aureus (CONS), Klebsiella pneumoniea, Pseudomonas species, Streptococcus species and Candida species. Methicillin resistance Staphylococcus aureus (MRSA) was isolated form 626 biomedical indwelling devices which were subjected to antibiotic susceptibility testing and biofilm formation studies. Standard microbiological and molecular methods were used for identification and confirmation of pathogens involved in biomedical device related infections. Antibiotic susceptibility testing was performed following the Clinical and Laboratory Standard Institute (CLSI) guidelines. These 626 strains of MRSA subjected to SCCmec tying, agr characterization and restriction fragment length polymorphism. Phylogenetic analysis was performed based on 16S rRNA sequences of 16 selected strains of MRSA. ZS35C (MG757682), ZS39H (MG757684), ZS41C (MG757686) and ZS43C (MG757688) were subjected to internalization by osteoblasts to understand the role of bbp and cna genes in development of osteomyelitis. Of 4,420 infected biomedical devices, 28.1% (1242/4420) were urinary catheters followed by 19.9% (882/4420) central venous catheters (CVC), 16.6% (736/4420) orthopedic implants, 12.2% (539/4420) Ventriculo-peritoneal/Ventriculoatrial (VP/VA), 12% (531/4420) endotracheal tube (ETT), 11% (490/4420) peritoneal dialysis catheters (PD). Of 626 isolates of MRSA, 23% (203/882) were isolated from CVC, 14% (69/490) PD, 7% Summary _________________________________________________________________________ xi (37/531) ETT, 30% (221/736) orthopedic implants, 5% (62/1242) urinary catheters and 6% (34/539) were isolated from VP/VA shunts. MRSA was isolated from 14% (626/4420) of infected biomedical devices and catheters. MRSA was confirmed by cefoxitin disc method and amplification of mecA gene. Antibiotic sensitivity testing revealed that all MRSA isolated from biomedical devices were sensitive to vancomycin and linezolid. Of 626 MRSA, 79% were resistant to amikacin, 92% gentamycin, 96% tobramycin, 87% azithromycin, 46% doxycycline, 96% ciprofloxacin, 94% ofloxacin, 85% trimethoprim/sulfamethoxazole, 72% clindamycin and 25% chloramphenicol. A correlation was established between community acquired MRSA (CAMRSA) and hospital acquired MRSA (HA-MRSA) for antibiotic resistance profile and no significant difference (p>0.05) between these two types of MRSA was observed. All strains of MRSA were subjected to SCCmec typing and 20% were found belonging to SCCmec II, 17% SCCmec III, 33% SCCmec IV, and 8% SCCmec V, respectively. None of the isolates of MRSA belonged to SCCmec I and 22% were not classified by this method. All strains typed by SCCmec were moderate biofilm producer except 20% of SCCmec IV which were strong biofilm producers. SCCmec typed strains were subjected to agr characterization and found 52% belonged to agr I, 12% agr II, 8% agr III and 14% agr IV. MRSA belonged to agr II were strong biofilm producers, all others were moderate biofilm producers. These characterized strains were subjected to RFLP and sixteen unique DNA band patterns were obtained after gel analysis. Sixteen strains were selected, one from each RFLP group for 16S rRNA ribotyping and subjected to phylogenetic analysis. Adhesion genes were detected in 48 selected strain of MRSA and found 65% harbored clfA, 100% clfB, 85% fnbA, 60% fnbB, 83% eno, 92% fib, 60% cna, 60% sdrD, 69% sdrE, 93% icaA and 77% harbored icaD. High prevalence of adhesion genes showed these are indispensable for biofilm formation but participation of single gene couldn’t be determined accurately. SCCmec typing, agr characterization and adhesion genes detection was also conducted on 221 strains of MRSA isolated from bone and orthopedic implant related infections. Four strains were selected for adhesion and internalization by osteoblast, which is crucial for pathogenesis of osteomyelitis by MRSA. This study revealed ZS35C was internalized rapidly because it harbored bbp and cna genes which facilitated osteomyelitis. ZS41C was internalized at lower rate because it only contained bbp. Association of ZS43C to osteoblasts suggested that cna gene have role association of MRSA to osteoblast. Expression studies showed that bbp and cna were upregulated during internalization; bbp is complimentary for internalization and cna promoted association. Summary _________________________________________________________________________ xii Thymoquinone is biologically active component of Nigella sativa and showed excellent inhibitory effect against planktonic and biofilm form of MRSA, not only free living but established biofilm of MRSA was also eradicated by thymoquinone. This study concluded that CA-MRSA is emerging pathogen for biomedical device related infection. CA-MRSA is rapidly gaining resistance towards available antibiotic and will impose major threat to healthcare industry. There is no significant difference between CAMRSA and HA-MRSA regarding antibiotic resistance profile and adhesion genes frequency. This study also concluded that there is no significant relationship between antibiotic resistance profile, biofilm formation and adhesion genes in MRSA isolated from biomedical device related infections. For osteomyelitis bbp has major role and cna supports the development of osteomyelitis by MRSA. This investigation will help to understand the molecular pathogenesis of osteomyelitis and support us to manage the biomedical related infections especially orthopedic implant related infections. Thymoquinone is an active component of Nigella sativa and showed excellent antibacterial activity against planktonic and biofilm form of MRSA. Antibacterial and anti-biofilm activity of TQ was enhanced when used with antibiotics.

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