MOLECULR CHARACTERIZATION OF ANTIBIOTIC RESISTANT GENE OF STAPHYLOCOCCUS AUREUS ISOLATED FROM HOSPITALS

JASIM MOHAMMED SADEQ *1 and HUSSEIN HAMEED ABBAS 2
1Department of Biotechnology, Acharya Nagarjuna University, Guntur, India.
2Department of Biotechnology, Acharya Nagarjuna University, Guntur, India.
MOLECULR CHARACTERIZATION OF ANTIBIOTIC RESISTANT GENE OF STAPHYLOCOCCUS AUREUS ISOLATED FROM HOSPITALS

ABSTRACT
Bacterial infections are common for everyone. However, these kinds of infections are especially problematic for people with type 2 Diabetes.  In the present study the organism isolated from freshly discarded dressing material from wounds in hospitalized diabetic patients was identified as Staphylococcus  aureus from Morphological And Biochemical Tests according to Bergey’s Manual. The study was extended to identify the same organism by Molecular Methods. DNA was extracted from the Isolated Organism by the Phenol-Chloroform method  and used for amplification and detection of the Pathogen by Polymerase Chain Reaction (PCR) using suitable Primers.
Agarose Gel Electrophoresis and subsequent staining with Ethidium bromide indicated that the size of the PCR product was 1.5 KB by comparison with marker DNA. The results would help in early detection of causative organisms and treatment regimens in diabetic patients with infected  wounds.
KEYWORDS:Staphylococcus Aureus, Pcr, Molecular Characterization, Diabetic Patient.
INTRODUCTION:
Hospital environment offers an increased rate of infection of burn and general wounds. Thus the outbreaks of enteric diarrhea and food borne diseases, variety of respiratory tract infections and the infectious fevers of childhood may occur from time to time.Hospital waste is the wastes generated during diagnosis, treatment or immunization of human beings in research activities or in the production or testing of biological products, including categories like discarded medicines.
A hospital infection shadows the patient in the hospital and the healthy individuals in and around hospitals by its waste and proves to be the final cause of various diseases and death or a major factor in infectious and fatal outcome.High risk of infection may be due to the poor resistance to disease, inadequate nutrition due to lack of normal food, frequent use of invasive devices and procedures-exogenous and endogenous flora. The lack of barrier nursing, immunosuppressive drugs, use of broad spectral antimicrobials, breakdown of sepsis during emergency, excessive pressure of work, the above criteria individually or in a combination are elucidating the risk factors which are predisposing any one for many infections due to hospital wastes.
Hospital wastes may be contaminated by various organisms, including the enteric gram negative bacilli- E. Coli, Klebsiella, Enterobacter, Proteus, Salmonella, Shigella, Pseudomonas aeruginosa, and Gram positive cocci like staphylococcus aureus,  streptococcus species etc.The presence of such highly pathogenic and opportunistic bacteria in hospital wastes, may lead to infection for both healthy individuals and patients in and around the hospital environment, as primary or secondary infections. Apart from hospital areas, surrounding areas may also get infected, for example, by the side of the hospital waste disposal site; there is a primary school with children who may be affected, not only during epidemiological outbreaks but also in normal conditions.
Every year, nearly 2 million patients in the US get an infection as a result of receiving health care in a hospital. These hospital-acquired infections are often difficult to treat because the bacteria and the other microorganisms that causes them frequently are resistant to antimicrobial drugs. Bacteria and fungi and even viruses can become resistant to drugs. But bacteria were known to cause most problems because once a particular type of bacteria developed resistance to a drug, it can pass on this resistance to other types of bacteria.Nosocomial infections continue to remain an important cause of morbidity and mortality in the neonatal period.Nosocomial infection is a significant epidemiological problem. It can result in the prolongation of hospital stay, increased mortality and morbidity and add to the cost of health care.
Aerobic bacteria are the aetiological agents in most of the hospital acquired infections, Outnumbering anaerobes in the ratio of 84 percent to 2 percent respectively. Only, in surgical wards the most common pathogen is E coli followed by Klebsiella, Enterobacter, Salmonella, Shigella, Pseudomonas aeruginosa, staphylococcus aureus, are prominent casual agent in urinary tract, lower respiratory tract and intra abdominal infections. Staphylococcus aureus is the second most commonly isolated organisms in lower urinary tract infection, whereas it is most common organisms responsible for primary bacteriaemia. As nosocomial infections are one of the major hazards of proper hospital waste management. It is playing an important role in infecting the individuals. Hospital infection is considered to be nosocomial, if there is no evidence that infection is present  at the time of admission to hospital .
In the last decade gram positive bacteria became major pathogen associated with nosocomial infections . Antibiotic resistance is a major contributor to the disease, death and costs resulting from hospital- acquired infections. Clonal dissemination of resistant strains are a major determinant of the increasing incidence of nosocomial infections caused by  multiresistant strains of staphylococcus aureus and Klebsiella pneumonia.  Staphylococcus aureus is a gram-positive cocci, which remains an important pathogen of nosocomial infections. It exhibits extra ordinary adaptive capabilities and is able to overcome a variety of environmental adversities). It is a hardy, ubiquitous organism commonly colonizing the skin and nails. Several international studies have been carried out for the dissemination of staphylococcal infection both nosocomially as well as into the community. The emergence of increasingly resistant strains with high epidemic potential and virulence is of particular concern in the control of nosocomial staphylococcus aureus infections.
MOLECULAR DIAGNOSIS OF MEDICALLY IMPORTANT BACTERIAL INFECTIONS
Infectious diseases are common diseases all over the world. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood. It is reported that infectious diseases are responsible for more than 17 million deaths worldwide each year, most of which are associated with bacterial infections. In patients with severe burns over more than 40% of the total body surface area (TBSA), 75% of all deaths are currently related to sepsis from burn wound infection or other infection complications and/or inhalation(Atiyeh,et al,2005).
The ability to control such bacterial infections is largely dependent on the ability to detect these etiological agents in the clinical microbiology laboratory. Diagnostic medical bacteriology consists of two main components namely identification and typing. Molecular biology has the potential to revolutionize the way in which diagnostic tests are delivered in order to optimize care of the infected patient, since the discovery of PCR in the late 1980s, there has been an enormous amount of research performed which has enabled the introduction of molecular tests to several areas of routine Clinical Microbiology. Molecular biology techniques continue to evolve rapidly, so it has been problematic for many laboratories to decide upon which test to introduce before that technology becomes outdated.
Improved outcomes for severely burned patients have been attributed to medical advances including early identification if the infecting organism, fluid resuscietation, nutritional support, pulmonary and burn wound care, and infection control practices.
Presently Molecular Biology offers a wide repertoire of techniques and permutations of these analytical tools.  The last ten years of the twentieth century allowed for an exponential increase in the knowledge of techniques in molecular biology, following the cellular and protein era of the 1970s and 1980s. Molecular bacteriologists are now beginning to adopt general molecular biology techniques to support their particular area of interest.

1.      SAMPLE COLLECTION.
2.      MORPHOLOGICAL CHARACTERIZATION:
2-1STAING METHODS:
  2-2GRAMS STAINING  , ENDOSPORE STAINING ,  CAPSULE STAINING ,  MOTILITY TEST: (Hanging drop method)

 BIOCHEMICAL TESTS

1-       .IMVIC TESTS:
A-INDOLE PRODUCTION TEST    B-METHYL RED TEST      C-VOGES-PROSKAUER TEST   D- CITRATE UTLIZATION TEST
2-      HYDROGEN SULFIDE (H2S) PRODUCTION TEST
3-      NITRATE REDUCTION TEST
4-      CATALASE TEST
5-      UREASE TEST
MOLECULAR TESTS :

1-  ISOLATION OF BACTERIAL GENOMIC DNA

2-  THE EXPERIMENT:
The full compliment of DNA present in the genome of the cell or organ is genomic DNA. Most methods of DNA isolation involve the breakage or lysis of the cells to release nuclei and further breakage of nuclei to release the chromatin.  DNA cells exist as nucleoprotein complexes and therefore isolation of DNA involves removal of proteins and carbohydrates associated with it. Finally  the polymeric nature of DNA is utilized to precipitate it and make it free of small molecular contamination
MATERIALS REQUIRED:
50mM tris (pH=8.0), 50mM EDTA, 0.5% SDS , 50mM tris (pH=7.5) , 0.4M EDTA , 1mg|/ml proteinase K , 1mM EDTA , 200µg/ml RNase , Chloroform , 0.1 vol of 3M sodium acetate , 2 vol of 100% ethanol (or) , 0.6 vol of isopropanol (360 micro liters) , 20-50 micro liter of TE buffer [20 milli micro –tris  (PH –8.0) 1 milli micro –EDTA]


PROCEDURE:
·        Take overnight culture grown in 50ml of LB broth into 1.5ul vials.
·        Centrifuge the vials at 10,000rpm for 5mins by using cooling -microfuge.
·        Then add 500µl of 50mM tris (pH 8.0) and 50mM EDTA.
·        To the above mixture add 100µl of 0.5% SDS, 50mM tris (pH 7.5), 0.4M EDTA and 1mg/ml proteinase K.
·        Place the appendorf  tubes in water bath at 50-550C for 1 hour.
·        Add 600µl of tris saturated phenol.
·        Then centrifuge at 10,000rpm for 5mins.
·        Take upper aqueous phase, to that add 500µl of 50mM tris(pH 7.5, 1mM EDTA and 200µg/ml RNase(20µl).
·        Incubate for an hour at 370C (room temperature).
·        To that add equal volume of chloroform and mix it by inverting the tube.
·        Then centrifuge at 10,000rpm for 5 mins.
·        Later transfer the top layer to the new tube.
·        To that add 0.1vol of 3M Sodium Acetate nearly 60µl and mix gently by inverting.
·        Then add 2vol of 100% ethanol or 0.6vol of Isopropanol (360µl) and mix by inverting.
·        Centrifuge at 10,000rpm for 5mins.
·        Then discard the supernatant and dissolve the pellet in 20-50µl of TE buffer [20mM tris pH 8 and 1mM EDTA].
·        Later check the purity by spectrophotometer and gel electrophoresis.
·        Take 36µl of the freshly isolated DNA along with 5µl of gel loading dye. Mix and load into the gel. Take 10 micro liter of control DNA and RUN electrophoresis along with the isolated samples in 1 % agarose gel.
PREPARATION OF 1% AGAROSE GEL AND ELECTROPHORESIS:
·        Prepare 1XTAE by diluting approximately amount of 50XTAE buffer with distilled water.
·        Take 50 ml of 1XTAE in a 250 ml conical flask and add 0.5g of agarose. Boil to dissolve agarose (till clear solution results). Allow it to cool.
·        Mean while adjust the combs in the electrophoresis set in such a way that the combs are on the left side is about 2 cm from the cathode.
·        When the gel temperature is around 600c (add ethidium bromide to view DNA under transilluminator) pour the gel slowly in to the gel tank without creating bubbles. Keep the set undisturbed till the agarose solidifies.
·        Once the gel has solidified pour 1XTAE buffer slowly into the gel till the buffer level stands at 0.5-0.8 cm above the gel surface.
·        Gently lift the combs to avoid damage of the wells. The gel is now ready for loading.
·        Make prior connection of the electrode to the power supply, the red cord connecting to the black cord to the black electrode. Before loading make sure to immerse the gel in 1X TAE buffer.
·        Connect the cords of the Electrophoresis set and the power supply, before loading the samples. After loading start the power connection and adjust the knob at 50 V. Run till the second dye (BLUE DYE) from the well has reached ¾th of the gel (1 hour approx).
MATERIALS REQUIRED:
The following reagents are enough to perform 10 amplifications. All the reagents carefully stored as per the storage temperature indicated on the label.
TAQ DNA POLYMERASE: Supplied 30units as 3units/µl. 1µl is recommended per reaction store at –200C or freezer compartment of fridge.
DEOXYNUCLEOTIDE TRIPHOSPHATES: 30µl dNTP mix and recommended use in 3µl/reaction. This mixture has a final concentration of 2.5mM of each dNTP and a 10mM concentration of total mix store at –200C  or freezer compartment of refrigerator.
ASSAY BUFFER:100µl 10X Taq polymerase assay buffer with MgCl2 . Composition: 100mM tris HCl (pH 9.0), 500mM KCl, 15mM MgCl2 and 0.1% Gelatin (store at –200C or freezer compartment of refrigerator.)
DNA TEMPLATE: This is a genomic DNA (10µl) purified from Serratia- marcescens  of approximate concentration to be estimated .Use 1µl/reaction
PRIMERS:
FORWARD PRIMER:
·         It is an 18 bases long oligomer  (10µl of 250ng/µl of  Concentration). Use 1µl/reaction.
REVERSE PRIMER:
·        It is 28 bases long  oligomer  ((10µl of 250ng/µl of Concentration). Use 1µl/reaction.
·        Store at –200C  or freezer compartment of refrigerator.
NUCLEASE FREE H2O – 1ml store at 40C.
·        Mineral Oil: 0.5ml,store at RT.
·        Agarose : 5g Store at RT.
·        Gel loading dye: 100µl. Store at 40C.
·        PCR tubes –12 no.
CONTROL – DNA MARKER - 5µg.
50X TAE  - 40ml

PROTOCOL FOR DNA AMPLIFICATION:

PRIMER SEQUENCES:
F –- 5' – GTA GGT GGC AAG CGT TAT CC – 3'
R –- 5' – CGC ACA TCA GCG TCA G – 3'
For the reaction add the following reagents to a PCR tube
10X assay Taq Pol Assay buffer 15 mM MgCl2
5µl
DNTP Mix
3µl
Template DNA (ng/µl)
1µl
Forward primer(250ng/µl)
1µl
Reverse primer (250ng/µl)
1µl
Taq  DNA Polymerase (3 µ/µl
1µl
Sterile water
38µl

Total reaction volume

50µl

Mix the contents gently and layer the reaction mix with 50 µl of mineral oil. Carry out the amplification using following the reaction condition for 30 cycles.

PCR steps

Temperature

Time

Initial denaturation
940C
1minute
Denaturation
940C
30 secs
Annealing
480C
30 secs
Extension
720C
1minute
Final extension
720C
2 minutes

After the reaction is over 10µl of the aqueous layer was run in 1% agarose gel for 1 to 2 hours at 100 volts. along with marker and locate the amplified product by comparing with the 0.8 kb fragment of the marker.
RESULTS
1-       GRAMS STAINING:
RESULT: Violet color colonies were observed which indicates GRAM POSITIVE BACTERIA.
2-       ENDOSPORE STAINING:
RESULT:  No spore formation.     
3-       CAPSULE STAINING:.                           
RESULT: The Organism Was Found To Contain A Capsule.
4-       MOTILITY TEST: (Hanging drop method)
RESULT:  Non-Motility Of The Microorganisms Was Observed. Hence The Test Is Negative.      
5-      . BIOCHEMICAL TESTS
SUMMARY OF THE RESULTS ON BIOCHEMICAL TESTS
S.NO
TEST
RESULT
1
INDOLE PRODUCTION TEST

NEGATIVE

2
METHYL RED TEST
POSITIVE
3
VOGES-PROSKAUE
NEGATIVE
4
CITRATE UTILIZATION TEST
NEGATIVE
5

H2S PRODUCTION

NEGATIVE
6
CHO FERMENTATION
POSITIVE
7
NITRATE REDUCTION
POSITIVE
8
CATALASE TEST
POSITIVE
9
UREASE TEST
POSITIVE
The organism isolated from burn wounds was identified as Staphylococcus aureus from Morphological And Biochemical Tests according to Bergey’s Manual. 

5-  MOLECULAR TESTs
Ø    ISOLATION OF BACTERIAL GENOMIC DNA

VISUALIZING DNA:                                  

Cut the gel, lift and place on the transilluminator. DNA can be seen as orange band under UV. Wear gloves while handling ethidium bromide stained agarose gels.
RESULT AND INTERPRETATION:
The Molecular Weight of Control DNA Band is around 50 KB in size. Genomic- DNA being high molecular weight (equal or above 50Kb), should run along with the control DNA or above if shearing has occurred during isolation, one may see DNA bands below the control DNA. If RNA is present along with the isolated DNA it will be seen between the purple dyes or on the purple dye.
POLYMERASE CHAIN REACTION

RESULTS:

       Genomic DNA  16s rRNA gene ladder sample  DNA ladder  Sample        
1. KB
 
                                                                          
DNA ladder is of the size 1.0KB.
The arrow (      ) indicates 1.5 KB, which represents 16S rRNA Gene in the Microorganism.
After running the PCR, the PCR product was analyzed by1% agarose gel electrophoresis and was photographed under U.V light.
The 16srRNA was sent for sequencing to “OcimumBiosolutions”. This will be finally submitted to National Centre for Biotechnology Information (NCBI) for authentication.

s.aureus pcr prm seq.jpg
pcr s.aureus image.jpg

5. DISCUSSION AND CONCLUSIONS
The organism isolated from infected diabetic patients wounds was identified as Staphylococcus aureous from morphological and biochemical tests.
S. aureous was the second most frequent organism isolated in a eight year study of bacterial isolates from wound infections in Osmania Hospital, Hyderabad.
Studies were further extended to amplification of DNA encoding for ribosomal RNA genes ( 16S rRNA ). The 16 S sRNA genes have been the most commonly employed genes for identification purposes in pathogenic bacteria (Hill et al., 2003; Xu et al., 2003).
16S rRNA genes are highly conserved and are found in all bacteria. Highly variable zones of 16 S rRNA genes sequences provide unique signatures to any bacterium giving useful information about their identity.Molecular detection of pathogenic bacteria has several advantages for their adoption into routine clinician and diagnostic bacteriology. Although perceived to be little expensive, the overall quality of the test, and er detection leading to early institution of therapy may overweigh the cost factor.
CONCLUSIONS
Molecular Diagnostic Techniques are expected to play a significant role in clinical and diagnostic bacteriology. Although their adoption may never replace the conventional methods their efficiency, quality, quickness and their role in the detection of slow growing organisms cannot be overlooked.
Infection control programs need to document and report burn wound infections according to the recently established definitions of the classification system. Future studies of burn wound infections should use this standardized burn wound classification system so that clinical outcomes can be compared for burn patients with a specific condition (e.g., burn wound cellulitis) (273, 331). More research is required to determine the best methods for sampling excised and unexcised burn wound areas over the course of a severe deep partial-thickness and/or full-thickness injury. Reproducible standardized methods should be developed so that clinical microbiology laboratories can routinely test burn wound bacterial isolates for susceptibility to the topical antimicrobial agents on formulary at a particular burn center. A rotation program for topical antimicrobial use may also retard the development of resistance. Laboratory surveillance should include the reporting of burn unit-specific antibiograms for topical antimicrobial agents once standardized methods are available for performing susceptibility testing.
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