PREVALENCE OF CEFTRIAXONE-RESISTANCE ESCHERICHIA COLI AND BETA LACTAMASE GENE PROFILE AT A HOSPITAL IN BANDUNG

• Main Author: Wikaningtyas, Pratiwi
• Format: Dissertations
• Language: Indonesia
• Subjects: Farmakologi dan terapeutik
• Online Access: https://digilib.itb.ac.id/gdl/view/32841
• Institution: Institut Teknologi Bandung

Bacterial resistance has reached critical point worldwide and mortality due to bacterial resistance is predicted to increase in the coming years. Escherichia coli was reported as the most highly infectious bacteria and exhibits a high rate of resistance to various antibiotics resulting in increased morbidity, mortality and the cost of treatment. Ceftriaxone is the most widely prescribed antibiotics as both empirical and definitive therapies to patients with E. coli. The study was conducted to implement the antibiotic resistance stewardship program initiated by World Health Organization by providing the bacteria resistance pattern periodically and conducting the early test at the gene level to identify the gene related to antibiotic resistance. Therefore, the early detection of those genes is urgently needed to monitor the infection events, prevent the spreading of bacterial resistance and to select the appropriate antibiotic to improve the antibiotic use. There were three steps used in this study. The design of study of all steps were as follows: the first step was initiated with the preliminary study conducted at five hospitals to provide the highest patients infected-bacterial resistance and the antibiotic usage profile by those patients during 2012. Further three hospitals became the research focus on the second steps which selected hospitals based on the similar pattern of bacterial resistance and antibiotic usage. The second steps was conducted at three hospitals in 2012 and 2013 used retrospective and concurrent observational study respectively by data collection of resistance pattern of the most antibiotic usage derived from first steps named ceftriaxone, patient demography and the antibiotic usage profile. The third step determined the beta lactam resistance mechanism through detection of beta lactamase genes including blaSHV, blaCTX-M, blaTEM, and blaCMY which were suspected to be the cause of ceftriaxone-resistant E. coli. The isolates derived from various biological specimens of the patient such as urine, feces, blood, sputum ant the other body fluid. The amount of isolates was determined by the Slovin formula. Gene detection began with preparation of the isolates into selected media contained ceftriaxone. The screening of beta lactamase was conducted using nitrocefine stick test. Positive nitrocefine isolates were amplified using Polymerase Chain Reaction (PCR) technique. The preparation should be conducted before starting the PCR such as primer design and optimization annealing temperature. PCR was performed by using the optimum annealing temperature for each primer. The PCR iv product was confirmed by two representative samples for each gene by determining the nucleotide sequence using automatic nucleotide sequencing. Since the confirmation, PCR was continued for all isolates obtained. Based on the preliminary study, E. coli was found as the most prevalent ceftriaxone-resistant bacteria at a range of 24-39% and 27-38% in 2012 and 2013, respectively. The source of clinical specimens of ceftriaxone-resistant E. coli varied at the three hospitals. In each hospital the observed predominant infected specimen were urine, sputum and feces, respectively. The most frequent prescribed antibiotic was cephalosporine class (40-42% in 2012 and 2013) and ceftriaxone was found as the most common prescribed among all cephalosporine (25-27% and 17-44% in 2012 and 2013, respectively).The demographic profile of the E. coli-infected patients from two hospitals showed that E. coli infected both adult male and female with the predominant length of stay of 4-9 days, whereas the most clinical outcome was improvement status (69-90%). There was no demographic data found in one other hospital due to limited access of medical records. There were 246 isolates obtained from Slovin formula. A total of 214 from 246 isolates of ceftriaxone-resistant E. coli were confirmed to produce beta lactamase. The isolates then tested by PCR to detect blaSHV, blaCTX-M, blaTEM, and blaCMY genes. Furthermore, the result of confirmation of nucleotide sequence in 2 samples for each beta lactamase gene showed a similarity of >94% compared to each beta lactamase gene. Of the 214 isolates, 95 isolates were detected to produce beta lactamase either singly or simultaneously in one isolate. Of the detected 95 isolates, 91 isolates obtained from one hospital (hospital A). This hospital mainly contributed to bla detection study. The detail of detected bla genes was as follows: blaSHV found in 2 isolates (2.1%); blaCTX-M found in 66 isolates (69.5%); blaTEM in 1 isolates (1.1%); blaCMY in 6 isolates (6.3%); blaSHV and blaCTX-M in 3 isolates (3.2%); blaCTX-M and blaTEM in 7 isolates (7.4%); blaCTX-M and blaCMY in 6 isolates (6.3%); blaTEM and blaCMY in 1 isolates (2.1%); blaSHV, blaCTX-M, and blaTEM in 1 isolates (1.1%), and blaCTX-M; blaTEM and blaCMY in 2 isolates (2.1%). This research findings showed 95/214 ceftriaxoneresistant E. coli harbored beta lactamase encoding gene with blaCTX-M as the most prevalent gene from 85 isolates as singly in 66 isolates (69.5%) and simultaneously in 19 isolates (16.8%).