A NEW DIAGNOSTIC METHOD TO DETECT BACTEREMIA TARGETING 23S AND 16S rRNA SEQUENCES BY STANDARD PCR

*Dr. K. Bramarambica 1 , Prof. Y. V. K. Durga Prasad 2 , B. Priyanka 3 and Prof. S. Harasreeramulu 4 . 1. Research Scholar Dept. of Biotechnology Dr.V.S.Krishna Govt. College(A), Affiliated to Andhra University. 2. Research guide & Director Centre for Research Studies.Dr.V.S.Krishna, Govt. College(A) Affiliated to Andhra University. 3. P.G. Dept of Biotechnology, Dr.V.S.KrishnaGovt.College(A). 4. Dean Biotechnology A.P.IIIT-IdupulapayaKadapa&Nuzvid. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (5), 1524-1531 1525 interaction of infection and immune response plays a decisive role in the pathogenesis of sepsis. In the process it does not matter if the infection is of bacterial, viral, fungal or parasitic origin. All pathogens can potentially trigger sepsis.It has been estimated that between 400 000 and 500, 000 patients are so affected each year in both the USA and Europe. Morbidity and mortality have remained high despite improvements in both supportive and antimicrobial therapies. Mortality rates vary from 40% for uncomplicated sepsis to 80% in those suffering from septic shock and multi-organ dysfunction 3 . More than 750,000 episodes of severe sepsis occur each year in the United States, making sepsis the tenth most common cause of death. The average cost per case for treatment of severe sepsis is approximately $22,000, and increases with the number of dysfunctional organs. ICUs play a critical role in an AMR emergency because they facilitate a high percentage of patients who are taking extended-spectrum antibiotics.

Sepsis in India:-
Severe sepsis was common in Indian ITUs (Intensive therapy units). ITU mortality was higher compared to western countries. Gram-positive infections were less common although incidences of parasitic and viral infection were higher than in the West. According to National Neonatal & Perinatal Database (2002-03) 5 , the incidence of neonatal sepsis in India was 30 per 1000 live-births; Klebsiellapneumoniae and Staphylococcus aureus were the two most common organisms isolated in sepsis cases.

Materials and Methods:-
Primers used in PCR were designed by using the target sequences from the genes encoding 16S-23S rRNA. PCR was used for the detection of different standard and clinical bacterial strains. The primers used are taken from SIGMA Company. DNA Extraction from the microbial Colonies:-Microbial colony was taken into a micro centrifuge tube which was containing 100 µl of phosphate buffer and centrifuges at 12,000 rpm for 3 minutes. The supernatant was discarded and the pellet was homogenized by finger flicking and the contents were collected into a new 1.5 ml of micro centrifuge tube, and 50 µl of 5% chelax resin & 2 µl of Proteinase K (10mg/ml) were added. They were incubated at 56 o C for 15 minutes and followed by incubation at 100 o C for 8 minutes. The tubes were centrifuged at 1200 rpm/2 min and the supernatant was collected into new micro centrifuge tube and they were stored at -80 o C for further use i.e. for setting up PCR. 2micro liter of DNA was used to setup PCR.

Figure-3a
In Fig-2a, Lane 1 is PCR subjected from human DNA that served as negative control. Lane 2 has DNA marker. 3-7 lanes show the bacterial PCR results with 896bp amplicon size.
In Fig-3a, shows 2-4 lanes positive for PCR with 896bp representing various bacteria. Lane 1 has DNA marker. In Fig-1b, lane 4 shows the DNA marker, lane 1& 2 shows 1404 bp amplicon size. Lane 3 is PCR subjected from human DNA that served as negative control.
In Fig-2b, 1-4 lanes show the bacterial PCR results with 1404bp amplicon size. In lane 5 no band is seen probably it is due to failure of DNA extraction or PCR. Lane 7 has DNA marker.

Results:-
The standardization of PCR for the detection of broad range clinical bacteria targeting 23S rRNA and 16S rRNA sequences was done. The results were tabulated depending upon the gel documentation figures, and are as follows.

Discussion:-
Severe sepsis and septic shock are important causes of death in intensive care units 3 . Although our understanding of the pathogenesisof inflammation and sepsis has improved, until recently this hasnot translated into clinical benefit. Several new treatment approacheshave given encouraging results. Evidence suggests that the wayforward is to develop pathogen specific regimens rather than assumethat one treatment fits all.
A study published by the Centers for DiseaseControl in the United States indicated that the incidence of septicaemia had increased from 73.6 per 100 000 patients in 1979 to 175.9 per 100 000 patients in 1987 4 . Recent US and European surveyshave estimated that severe sepsis accounts for 2-11% of all admissionsto hospital or intensive care units.
Although Gram negative infections were predominant in the 1960s and early 1970s, Gram positive infections have increased inthe past two decades and now account for about half of cases ofsevere sepsis. Fungal infections are also increasing in manycountries. Despite better supportive care, the hospital mortalityfrom severe sepsis and septic shock (30% and over 60%, respectively)has not changed much over recentdecades.
From the perspective of the physician, early identification of an infectious agent will allow confirmation that infection underlies an inflammatory process, allow the use of efficacious and narrow spectrum antibiotics, and may open the door to new therapies targeted at pathogen-specific inflammatory pathways.
Culture of these bacteria is considered as a gold standard with some limitations such as sensitivity, time-consuming factor and growth failure when antibiotics are used. As a consequent, DNA-based methods have increasingly developed. PCR offers a better approach for rapid detection. Among PCR based methods, multiplex PCR is a good technique which simultaneously detects many targets in a single reaction.
In Fig-3b All the colonies which were positive by broad range 16S & 23S rRNA primers were tested by biochemical assays and morphological characters of genus of the bacteria. The 10 different colonies used in the study were developed from various clinical samplesincluding urine, pus and blood. All the colonies gave correct amplicon of size 1404bp and 896bp products. Only one sample could not be amplified and was identified as E.coliby biochemical analysis.This could be due to an error in DNA extraction or could be due to presence of different bacteria which was not able to recognize by our primers. It needs further investigation to elicit the most probable reason. Excluding the sample the sensitivity of 16S & 23S rRNA broad range PCR is 100%. Including this sample, the sensitivity of 23S rRNA is 100% and 16S rRNA is 90%. However, the PCR was done from colonies of bacteria which have got million copies of bacteria. When this procedure is directly applied to the clinical sample the sensitivity might reduce due to low copy no. of bacteria in blood or any other clinical specimens. This procedure further needs to be optimized, for applications in clinical samples against gold standard cultures.

Conclusion:-
Polymerase chain reaction for the detection of broad range clinical bacteria targeting 23S rRNA and 16S rRNA sequences was standardized, as correct size of amplicon 896bp and 1404bp was obtained. All the clinical isolates including Staphylococcus aureus, Pseudomonas aeroginosa, Klebsiella pneumoniae and E.coli which are characterized by morphology and biochemical parameters were detected by broad range PCR with 100% sensitivity with 23S rRNA and 90% sensitivity with16S rRNA The 100% sensitivity of bacterial isolates detection by 23S rRNA PCR, relative to 90% sensitivity achieved with 16S rRNA Targeted PCR (details elsewhere) reiterates the more conserved nature 23S rRNA sequences. Probably 23S rRNA sequences are better for detection of bacterial presence and the heterogeneity sequence nature of 16S rRNA can be exploited for typing of the bacteria.