I= TRENDS IN ANTIMICROBIAL RESISTANCE AND OCCURRENCE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA): MOLECULAR BASIS AND CLINICAL IMPLICATIONS.
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Title of Thesis
TRENDS IN ANTIMICROBIAL RESISTANCE AND OCCURRENCE OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA): MOLECULAR BASIS AND CLINICAL IMPLICATIONS.

Author(s)
Syed Zahid Hussain Bukhari
Institute/University/Department Details
Department of Microbiology/ Quaid-i-Azam University Islamabad
Session
2006
Subject
Microbiology
Number of Pages
140
Keywords (Extracted from title, table of contents and abstract of thesis)
antimicrobial resistance, methicillin-resistant staphylococcus aureus, hospital-acquired infections, mrsa, staphylococcal infections, cephalothin, clindamycin, rifampicin, ciprofloxacin, gentamicin, trimathoprim, sulfamethoxazole, tetracycline, penicillin, ampicillin, erythromycin, vancomycin

Abstract
The global problem of increasing trend in antimicrobial resistance is particularly pressing in the developing countries, where the methicillin-resistant Staphylococcus aureus (MRSA) is often the severe casual agent in hospital-acquired infections. There is now an increase in difficulties to treat such patients because of emergence of resistance to all current antibiotic classes. A significant increase in methicillin-resistant S. aureus in hospitals of Pakistan has been reported in the last decade. However, regional differences in the epidemiologic factors of MRSA appear to be significant. Rapid and reliable detection of MRSA is essential in order to secure the optimal treatment of patients with Staphylococcal infections, as well as for infection control procedures. Phenotypic susceptibility testing often takes 2-3 days and sometimes even longer before a definitive result is obtained. In the present study S. aureus clinical isolates were collected from four busy hospitals (Mayo, Jinnah, Services and Shaikh Zayed) of Lahore the capital city of the province Punjab, Pakistan from February 2003 to March 2005. The specimens were processed by the standard microbiology technique. The isolates were identified as S. aureus on the basis of colony morphology, Gram's staining, catalase, coagulase and DNase tests. All confirmed S. aureus isolates were processed for antimicrobial susceptibility testing, performed by the modified Kirby-Bauer technique and results were interpreted according to the National Committee for Clinical Laboratory Standards (NCCLS) criteria. The following antibiotics were tested; cephalothin, clindamycin, rifampicin, ciprofloxacin, gentamicin, trimathoprim/ sulfamethoxazole (TMP/ SMZ), tetracycline, penicillin, ampicillin, erythromycin, and vancomycin. Methicillin resistance was tested by using 1 µg oxacillin discs on Mueller-Hinton agar containing 4% sodium chloride. Antimicrobial susceptibility by MIC method was done by PhoenixTM system, BD Biosciences, MD, USA. Detection of mecA gene was performed by Polymerase chain reaction (PCR) as gold standard. Center for Diseases Control & Prevention (CD C) definition was employed for case finding of hospital acquisition of MRSA. The distribution or total 1102 S. aureus isolates among hospitals was as follows; Mayo hospital 432 (39.2%), Services hospital 185 (16.79%), Jinnah hospital 273 (24.78%) and Shaikh Zayed hospital 212 (19.23%). The unit wise distribution was as follows; Medical and Surgical Intensive Care units (MSICU) 162 (14.7%), Medical units 225 (20.4%), Surgical units 239 (21.7%), Cardiac units 44 (4%), Obstetrics & Gynecology 118 (10.7%), Pediatrics 136 (12.34%), Nursery & Neonatal Intensive Care units (NICU) 78 (7.1%) and OPD 100 (9.1%). The organisms were most prevalent in respiratory samples followed by swabs taken from skin and soft tissue lesions. Highest number of S. aureus isolates belonged to Mayo Hospital and Jinnah Hospital. On antimicrobial susceptibility by disc diffusion method, 462 (41.9%) isolates were found methicillin-resistant S. aureus (MRSA). The antimicrobial resistance of other antibiotics among such isolates was as follows; clindamycin 745 (67.6%), ciprofloxacin 513 (46.6%), trimethoprim-sulphamethoxazole -TMP/SMZ 699 (63.6%) resistant; penicillin 1056 (95.8%); gentamicin 665 (60.3%); rifampicin 40 (3.6%); tetracycline 725 (65.8%); ampicillin 1023 (92.8%), cephalothin 584 (53.0%). No resistance was found with vancomycin. Therefore vancomycin and rifampicin were found most effective drugs against S. aureus. Highest resistance was found for penicillin, ampicillin, cephalothin, and erythromycin. An increasing trend of antimicrobial resistance was found in the present study. The main reason of such resistance was due to misuse of antibiotics in critical areas of the hospitals. The factors behind the misuse of antibiotics were; misdiagnosis of multi-resistant organisms; giving antibiotics to the colonized patients associated with risk factors in high risk areas, and treating mild skin infections with systemic antibiotics.

Out of 462 resistant strains on disc diffusion; 420 had MIC's to oxacillin of < 4 mg/L on BD PhoenixTM system. The S. aureus strains showed borderline resistance to oxacillin with MICs between 4 and 16 mg/L and later found mecA negative were not included in statistical analysis. There was a significant correlation between MRSA identified on disc diffusion and minimum inhibitory concentration (p value < 0.05). The mecA gene was detected in 307 strains from all MRSA isolated on MIC. The prevalence of MRSA strains among S. aureus isolates was 41.9%, 38.1% and 27.9% on disc diffusion, MIC, and mecA gene detection respectively. MRSA status was lost in 155 isolates on disc diffusion and 113 on MIC. The sensitivity and specificity rates of disc diffusion test were 100% and 83.7% while MIC 96.2% and 93.3% respectively.

Overall 307 (27.9%) isolates were found MRSA from the following clinical units; ICD 65 (21.2%), medical 59 (19.2%), surgical 67 (21.8%), cardiac 12 (3.9%), obstetrics & gynecology 21 (6.8%), pediatrics 42 (13.7%), neonatology 25 (8.1%) and OPD 16 (5.2%). Medical records of all patients who grew MRSA were reviewed and clinically classified as blood stream infections 65 (21.2%), respiratory tract infection 114 (37.1%), urinary tract infection 12 (3.9%), skin & soft tissue infection 99 (32.3%), genital tract infection 4 (1.3%), ENT & eye 5 (1.6%), other body fluids 0 (0.0%) and miscellaneous 8 (2.6%). The efficacy rate of MIC was 89.7% which was higher than disc diffusion (85.9%). The positive predictive values of the tests performed for the defining MRSA on MIC (73.1%) was higher than disc diffusion (66.5%). It was concluded that oxacillin resistance on MIC was the more reliable than disc diffusion for defining MRSA isolates. Both phenotypic tests of methicillin resistance in S. aureus strains created false-susceptible results. These problems can be avoided using a mecA gene-based detection system, as the presence of the mecA gene was proved the hallmark for identification of MRSA strains.

On the basis of current data; establishment of molecular diagnostic laboratory in secondary and tertiary units is urgently required. Although it is an expensive technique but it is cost effective. By the application of the rapid and reliable detection method of methicillin resistance can decrease unnecessary use of toxic and expensive drug vancomycin, reduces the cost of care in isolation precautions. It also provides a tool to control the emergence and spread of multi-resistant organism. The risk factors for the emergence and transmission found were; no prior endorsement while shifting or transferring of patients infected or colonized with multi-resistant organisms; elderly patients (above 70 Years); prolonged stay in the hospital especially in intensive care unit and high risk nursery (more than 3 months); co-morbidity associated with MRSA infection like diabetes mellitus and malignancies; device association like central line, ventilator, urinary catheter; misuse of antibiotics as treating colonized patients; chronically ill with multiples sepsis; lack of isolation rooms with negative pressure in the hospitals and lack of orientation of updated infection control program among health care workers. Hospital acquisition (68%) of MRSA found higher than the community association (32%). Hospital acquired-MRSA was almost multi drug resistant while community acquired-MRSA showed susceptibility to clindamycin (63%), ciprofloxacin (24.2%) and SMZ/TMP (3.9%). Among device associated hospital acquired infections; ventilator associated pneumonia was found in most of the patients have MRSA infection followed by central line associated blood stream infection and folly's catheter associated urinary tract infection. It is recommended to establish surveillance of nosocomial infection protocol recommended by the Center for the Diseases Control (CDC) and each hospital compares its nosocomial infection rates with other organizations through comparative database. Device associated nosocomial infection rates of medical and surgical intensive care units should be compared with National Nosocomial Infection Surveillance (NNIS) benchmark. The data presented in current study was unable to find a significant difference of multi-resistant organisms on geographical location of the patients attending the four hospitals located wide apart. Male preponderance and elder age were important demographic findings. MRSA flagging system is recommended to those hospitals that already developed computer network system in the hospital.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
280.23 KB
2 1 Introduction 1
382.39 KB
3 2 Review Of Literature 14
634.07 KB
4 3 Materials & Methods 33
255.18 KB
5 4 Results 43
501.95 KB
6 5 Discussion 73
1658.12 KB
  5.1 Conclusion 93
  5.2 Recommendations 96
  5.3 References 105
  5.4 Appendices 132