SUMMER TRAINING REPORT ON

STUDY OF ANTIBIOTIC SENSTIVITY OF

VARIOUS PATHOGENIC BACTERIA IN CASE OF UTI AND THROAT INFECTIONS

AMITY INSTITUE OF BIOTECHNOLOGY

Submitted to: DR. NEETA BHAGAT SENIOR LECTURER, AIB

Submitted by: ER. MISHEL ARORA ROLL NO. 1043 M.TECH (BIOTECH.)

(Formerly, Government Medical College) ROHTAK-124001, HARYANA

CERTIFICATE
It is to certify that Mishel Arora s/o Mr. Subhash Arora has successfully completed two month practical training from 13th May,2011 to 13th July,2011 from the department of Microbiology, Pt. B.D. Sharma PGIMS, Rohtak. He has completed his work sincerely.

Dr. UMA CHOUDHARY Deptt. of Microbiology, PGIMS , Rohtak

Dr.Uma Chaudhary Senior Professor and head Department of Microbiology PGIMS, Rohtak

ACKNOWLEDGEMENTS
I express my heartiest thanks to the administration of PGIMS, Rohtak for providing a research based focused environment, which encouraged me to have hands on experience of all the major and minor tasks of the lab. It is most opportune to acknowledge Dr.Uma Choudhary, HoD Department of Microbiology PGIMS, Rohtak for her excellent scientific guidance and her valuable time during the course of investigation and preparation of the manuscript. My appreciation goes to Dr. Aman Ahlawat my guide and Dr. Amit Mehta, Sr. Resident PGIMS, Rohtak for their constant encouragement and support throughout the process.

MISHEL ARORA

CULTURE AND SENSTIVITY OF PUS AND URINE SAMPLES

SAMPLES PROCESSED 1. 2. 100 pus samples, 100 urine samples.

PROCESSING OF PUS SAMPLE 1. Sample collection - sample is either collected with the sterile swab stick or sample is taken with a syringe and transferred into a sterile vial or tube and transported to the laboratory for processing. 2. Now a primary smear is prepared for Gram staining.

GRAM STAINING It divides bacteria into Gram-positive and Gram-negative. Method : Heat fix the smear, Pour crystal violet (primary stain) on the smear and keep it for one minute,

 Wash with water and pour Grams iodine over the slide for 1 min., Wash again with water and decolorize with acetone for 2-3 sec., Wash the smear again with water and counter stain with safranin or carbol fuchsin for 30 sec. Differentiation on basis of Gram-Positive : Resist decolourisation and retain the colour of primary stain and appear purple Gram-Negative: Decolourised by acetone and therefore, take counter stain and appear pink CULTURE

Specimen is inoculated on blood agar and MacConkey agar Culture plates are incubated at 370C overnight Now, bacterial colonies that grows on culture plates are observed and identified on following basis

Media Mac-Conkey Agar

Basis of identification LF or NLF

LF- Lactose fermenter (Pinkish in colour) NLF Non lactose fermenter (translucent white to gray)

Blood agar

Heamolytic or non-heamolytic

Identification of bacterial culture Medium Blood agar Probable organism Staphylococcus aureus Streptococcus pyogenes Proteus spp. (seen as swarming) Mac Conkey agar Lactose fermenters E.coli Klebssiella spp. Non-lactose fermenters] Salmonella spp. Pseudomonas aeruginosa Citrobacter spp

Growth from the plate is passed in to peptone water and we put biochemical tests and antibiotic sensitivity. And one secondary smear is also prepared and again Gram staining is performed. BIOCHEMICAL TESTS 1) Catalase Test Certain bacteria have an enzyme catalase which acts as on hydrogen peroxide (H2O2) to release oxygen. Catalase H2O2 Procedure : Pick up few colonies of test bacteria with loop and mix it in a drop of H2O2. Interpretation
Positive test Negative test : : Immediate bubbling No bubbling

H2O+[O]

Positive & Negative Bacteria Catalase positive Catalase negative Staphylococcus, Micrococcus, Bacillus Streptococcus, Clostridium

2) Oxidase Test
To determine the presence of enzyme which catalyses the oxidation of reduced cytochrome by molecular oxygen Procedure: A filter paper strip, soaked in oxidase reagent, is smeared with test organism. Interpretation Positive Deep purple within 10 sec Negative No colour change Positive & negative bacteria Oxidase positive Pseudomonas spp., Vibrio spp., Neisseria spp. Oxidase negative all members of Enterobacteriacae 3.) Coagulase test To determine the presence of enzyme coagulase which clots plasma. Procedure Pick up few colonies of test bacteria and mix it well with normal saline. Now add few drops of plasma to it and rotate it gently for a minute. Interpretation Positive Negative Agglutination occurs No change

Positive & Negative Bacteria Positive Negative Staphylococcus aureus Staphylococcus epidermidis, Staphylococcus saprophyticus,

4.) Sugar Fermentation To determine the ability of an organism to ferment a specific carbohydrate (sugar) incorporated in a medium. Glucose, lactose, sucrose and mannitol are widely used sugars. The test organism is inoculated in a sugar medium and incubated at 370C for overnight. Interpretation : Positive Negative : : Yellow (acidic) Blue-green (alkaline)

Gas production can be seen as bubbles in Durahams tube. Examples of fermentative bacteria Glucose fermenters all members of Enterobacteriaceae Glucose and lactose fermenters Escherichia coli (E.coli) and Klebsiella spp. Glucose and mannitol fermenter Salmonella spp..

5). Indole Production To determine the ability of an organism to decompose amino acid tryptophan into indole. It is detected by inoculating the test bacterium into peptone water and incubating it at 37 0C for overnight. A few drops of Kovac`s reagent are added & the peptone water is observed for change in colour Interpretation Positive Negative : : A red coloured ring near the surface of medium Yellow coloured ring near the surface of medium

Positive & Negative Bacteria Indole positive- E. coli, Proteus vulgaris, Edwadsiella spp. Indole Negative klebsiella spp., Proteus mirabilis

6.) Methyl Red (MR) Test This test detects the production of sufficient acid during fermentation of glucose by bacteria. The test organism is inoculated in glucose phosphate broth and incubated at 370C for overnight. Then 4-5 drops of methyl red solution are added.

Interpretation Positive Negative : : Red colour Yellow colour

Positive & Negative Bacteria MR positive

MR negative

E.coli
Klebsiella spp., Enterobacter spp

7). Citrate Utilisation Test It is based upon the ability of an organism to utilize citrate as the sole source of carbon for its growth. A bacterial colony is picked up by a straight wire and inoculated on the media and incubated at 370 for overnight. Interpretation Positive : Negative : Growth with an intense blue colour on slant No growth with no change in colour.

Positive & Negative Bacteria Citrate positive Klebsiella spp., citrobacter spp., Enterobacter spp. ,Salmonella spp. Citrate negative E. coli, salmonella typhi

8.) Urease Production To determine the ability of an organism to produce an enzyme urease which converts urea to ammonia. The test organism is inoculated on the entire slope of medium and incubated at 370 for overnight. Interpretation Positive Negative : : Pink colour Pale yellow colour

Positive and negative bacteria Urease positive :

Ureas negative -

Klebsiella spp., Proteus spp.

E.coli

9). Hydrogen Sulphide Production To determine whether H2S has been liberated by enzymatic action, from sulphur containing amino acids. Organisms are grown in culture tubes and a filter paper impregnated with lead acetate is inserted between the cotton plug and the tube is incubated for overnight at 370C Interpretation Positive Negative : : Blackening of filter paper No change in colour

Positive & Negative Bacteria H2S positive Proteus mirabilis, Proteus vulgaris, Salmonella spp. (With some exceptions) H2S negative Salmonella paratyphi A.

Antibiotic Sensitivity (Antibiotic sensitivity was performed in accordance with Kirby-Bauer disc diffusion method). -When the growth already passed in peptone water reaches up to .5 Mac Farland opacity standard, a lawn culture is prepared on Mueller-Hinton agar plate with the help of sterile swab stick. -Now antibiotic discs are put on the plates with the help of sterile forceps. -After placement, press the disc on the surface of medium to provide uniform contact. Do not move the disc once it comes in contact with the agar, because some of the drug diffuses almost immediately. The discs must be evenly distributed on the agar so that they are not close than 24mm centre to centre. -The plates are then incubated at 35-370C for 16-18 hrs. - Visible growth of the bacteria occurs on the surface of the agar where the concentration of the antibiotic has fallen below its inhibitory level for the test strain. Bacterial growth occurs in the form of a circle with middle of the disc forming the centre of the circle. The inhibition zones are observed and measured with the help of a vernier caliper to the nearest millimeter and then the antibiotics having clear zone of inhibition around them are given sensitive. Interpretation of zone size into susceptible, moderately susceptible or resistant is based on comparison of the measurements of the inhibition zone with the standard interpretation chart.

. URINARY TRACT INFECTIONS Urinary Tract Infection (UTI) is defined as a disease caused by microbial invasion of the genitourinary tract that extends from the renal cortex of the kidney to the urethral meatus. The presence of detectable bacteria in the urine is named as bacteriuria. Presence of pus cells in urine denotes pyuria which most often accompanies UTI. TYPES OF UTI 1) Lower UTI i) ii) iii) Urethritis Cystitis Prostatitis

Lower UTI is due to ascending infection caused by feacel coliforms. 2) Upper UTI i) ii) Acute Pyelitis Acute Pyelonephritis

Pyelonephritis is probably due to haematogeneous infection. CAUSATIVE ORGNAISMS A) Gram Negative Bacilli They are the most common infecting agents.

1)

E. coli Commonest cause of UTI. It is responsible for about 7080% acute infections and 50% hospital acquired infections.

2) 3) 4) 5) B.

Klebsiella spp. Proteus spp, especially P. mirabilis Enterobacter spp. Pseudomonas aeruginosa Gram Positive Cocci

1) Enterococcus 2) Staphylococcus. aureus 3) Staphylococcus saprophyticus C. Miscellaneous 1) Citrobacter spp 2) Myobacterium. tuberculosis 3) Salmonella spp. D) Fungus Candida albicans may cause UTI in diabetic and immunocompromised patients. LABORATORY DIAGNOSIS A) 1. Specimen Collection Midstream Urine Specimen (MSU)

It is collected prior to administration of antibiotics. Specimen is collected in a sterile tube. 2. Catheter specimen Urine is collected directly from the catheter and not from the collection bag. The catheter should not touch the container. 3. Urine specimens from infants Specimen can be collected either by suprapubic aspiration method or after cleansing of genital. B. Transport After collection, sample should reach the laboratory with minimum delay, if not possible, the specimen is to be refrigerated at 40C.

C.

Laboratory Methods Part of specimen is used for bacteriological culture and the rest is examined immediately under the microscope.

1)

Microscopy Urine is centrifuged at a speed of 2000 rpm for 2 min. Supernatant is discarded and the deposits are examined under microscope for detecting pus cells,. Epithelial cells, erythrocytes and bacteria. Casts crystals, budding yeast cells. In microscopy, presence of more than 3 pus cells per high power field is suggestive of infection.

Samples positive for Pus cells are cultured

. Uncentrifuged urine is inoculated on blood agar and MacConkey agar. And the culture plates are observed as per Kass`s criterion. .Kass (1956) gave a criterion of active bacterial infection of urinary tract according to which a count exceeding 105 organisms /ml denotes significant infection and indicates active UTI. Contamination accounts for less than 104 organisms/ml (usually less than 103/ml).

These culture plates are examined and organisms are identified by standard microbiological procedure as performed in case of pus samples.

Gram staining , Bacterial culture, Blood agar on basis of property to cause heamolysis, MacConkey agar On basis of the property to ferment lactose.

BIOCHEMICAL TESTING For LF colonies we perform Indole, Methyl red test, Sugar sets which include lactose, sucrose, maltose and glucose fermenting tests , Citrate utilization test and Mannitol motility test.

In glucose tube a paper strip dipped in Pbs (Lead sulphate) is also hanged from the mouth of tube to detect the presence of H2S. Species E. Coli Klebsiella spp. Enterobacter spp. Indole Positive Negative Negative Urease Negative Positive Positive Motility Motile Non-Motile Motile

For NLF colonies we perform Indole, Methyl red test, Sugar sets which include lactose, sucrose, maltose and glucose fermenting tests , Citrate utilization test and Mannitol motility test. Antibiotic Sensitivity (Antibiotic sensitivity was performed in accordance with Kirby-Bauer disc diffusion method). -When the growth already passed in peptone water reaches up to .5 Mac Farland opacity standard, a lawn culture is prepared on Mueller-Hinton agar plate with the help of sterile swab stick. -Now antibiotic discs are put on the plates with the help of sterile forceps. -After placement, press the disc on the surface of medium to provide uniform contact. Do not move the disc once it comes in contact with the agar, because some of the drug diffuses almost immediately. The discs must be evenly distributed on the agar so that they are not close than 24mm center to center. -The plates are then incubated at 35-370C for 16-18 hrs.

- Visible growth of the bacteria occurs on the surface of the agar where the concentration of the antibiotic has fallen below its inhibitory level for the test strain. Bacterial growth occurs in the form of a circle with middle of the disc forming the center of the circle. The inhibition zones are observed and measured with the help of a vernier caliper to the nearest millimeter and then the antibiotics having clear zone of inhibition around them are given sensitive. Interpretation of zone size into susceptible, moderately susceptible or resistant is based on comparison of the measurements of the inhibition zone with the standard interpretation chart. .TABLE-1 DISTRIBUTION OF PUS SAMPLES IN VARIOUS AGE GROUPS AND SEX

Age Group 1-10 11-20 21-30 31-40 41-50 50 & above Total

No. of Males 5 8 11 10 4 13 51

No. of Female 5 7 8 11 6 12 49

TABLE-2 TABLE SHOWING %AGE OF CULTURE POSITIVE SAMPLES Culture +ve Culture ve 58% 42%

TABLE-3 TABLE SHOWING %AGE OF VARIOUS ORGANISMS ISOLATED FORM PUS SAMPLES Organism Pseudomonas aeruginosa E.coli Staphylococcus aureus Enterobacter spp. Acitenobacter spp. Citrobacter spp. Klebsiella spp. Proteus spp. Total No. of Samples 17 12 12 9 4 3 2 1 60 %age 28.33% 20% 20% 15% 6.66% 5% 3.33% 1.66% 100%

TABLE-1
DISTRIBUTION OF URINE SAMPLES IN VARIOUS AGE GROUPS AND SEX

Age Group 1-10 11-20 21-30 31-40 41-50 50 & above Total

No. of Males 1 4 11 13 11 9 49

No. of Female 2 9 12 11 8 9 51

TABLE-2
TABLE SHOWING PUS CELL INTERPRETATION BY MICROSCOPY

Microscopy Pus cells present No. pus cells

No. of Males 49 51

%age 49% 51%

TABLE-3 TABLE SHOWING %AGE OF CULTURE POSITIVE SAMPLES Culture +ve Culture ve 29% 71%

TABLE-4 TABLE SHOWING %AGE OF VARIOUS ORGANISMS ISOLATED FORM URINE SAMPLES Organism E.coli Staphylococcus aureus Citrobacter sp. Total No. 16 7 4 27 %age 59.2% 25.9% 14.8% 100%

ANTIBIOTIC SENSITIVITY CHART

Urine (Staphylococci) Nitrofurantoin (Nf) Cotrimoxazole (Co) Doxycycline (Do) Norfloxacin (nx) Lizezolid (Lz) Amoxyclav (Ac) Oxacillin (Ox) Pristinamycin (Pm) Urine (LF/NLF) Amoxycillin (Am) Nitrofurantoin (Nf) Norfloxacin (Nx) Gatifloxacin (Gf) Cotrimoxazole ((Co) Cefotaxime (Ce) Gentamicin (G) Amoxyclav (Ac) Urine Non-urine (Pus) (Staphylococci) Cephalexin (Cp) Erythromycin (E) Doxycycline (Do) Getifloxacin (Gf) Linezoid (Lz) Amoxyclav (AC) Pristinamycin (Pm) Oxacillin (Ox) Non-Urine (Pus) (LF/NLF) Cotrimoxazole (Co) Doxycycline (Do) Ciprofloxacin (Cf) Amikacin (Ak) Amoxyclav (AC) Ceftaxidime (Ca) Meropenem (Mr) Cefbazidime + Clavolinic acid (CaC)

(Pseudomonas)
Ofloxacin (Of) Cefepime (Cpm) Amikacin (Ak) Ceftizoxime (Ck) Netilmicin (Nt) Impinem (I) Piperacillin+ Tazobactam (Pt) Aztreonam (Ao

Non-urine (Pus) (Pseudomonas) Ofloxacin (Of) Amiakcin (Ak) Ceftazidime (Ca) Ceftizoxime (ck) Netlimicin (Nt) Meropenum (Mr) Piperacillin+ Tazobactam (Pt) Aztreonam (Ao)

TABLE-5 INTEPRETATION OF URINE SAMPLES AS PER %AGE SENSITIVE AGAINST COMMONLY USED ANTIBIOTICS

Antibiotics Nitrofurantoin (Nf) Cotrimoxazole (Co) Doxycyline (Do) Norfloxacin (Nx) Linezolid (Lz) Amoxyclav (AC) Pristinamycin (Pm) Amoxycillin (Am) Gatifloxacin (Gf) Cefotaxime (Ce) Getaimicin (G) Ofloxacin (Of) Cefepime (Cpm) Amikacin (Ak) Ceftizoxime (CR) Netilmicin (Nt) (Imipenem) (I) Piperacillin + Tazobactam (Pt) Aztronam (Ao)

E. coli (16) 37.5% 62.5% 6.2% 0.0% 25% 6.2% 0.0% 18.7% -

S. aureus (7) 42.8% 14.2% 0.0% 14.2% 71.4% 14.2% 0.0% -

Citrobacter (4) 50% 50% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% -