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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: Submitted by:

DR. NEETA BHAGAT ER. MISHEL ARORA

SENIOR LECTURER, ROLL NO. 1043

AIB M.TECH (BIOTECH.)



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 SENSTIVITYOF PUS AND URINE SAMPLES

SAMPLES PROCESSED

1. 100 pus samples,

2. 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 Gram’s 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 Basis of identification

Mac-Conkey Agar – 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 Probable organism

Blood agar - 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.

CatalaseH2O2 H2O+[O]

Procedure :

Pick up few colonies of test bacteria with loop and mix it in a drop

of H2O2.

Interpretation

Positive test : Immediate bubbling

Negative test : No bubbling

Positive & Negative Bacteria

Catalase positive - Staphylococcus, Micrococcus, Bacillus

Catalase negative - 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 - Agglutination occurs

Negative - No change




Positive - Staphylococcus aureus

Negative - 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 : Yellow (acidic)

Negative : Blue-green (alkaline)

Gas production can be seen as bubbles in Duraham’s 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 370C for overnight.

A few drops of Kovac`s reagent are added & the peptone water is

observed for change in colour

Interpretation

Positive : A red coloured ring near the surface of medium

Negative : Yellow coloured ring near the surface of medium


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 : Red colour

Negative : Yellow colour


MR positive - E.coli

MR negative - 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.

InterpretationPositive : Growth with an intense blue colour on slant

Negative : No growth with no change in colour.


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 : Pink colour

Negative : Pale yellow colour

Positive and negative bacteria

Urease positive : Klebsiella spp., Proteus spp.

Ureas negative - 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 : Blackening of filter paper

Negative : No change in colour


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 discdiffusion 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) Urethritis

ii) Cystitis

iii) Prostatitis

Lower UTI is due to ascending infection caused by feacel coliforms.

2) Upper UTI

i) Acute Pyelitis

ii) 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 70-

80% acute infections and 50% hospital acquired infections.

2) Klebsiella spp.

3) Proteus spp, especially P. mirabilis

4) Enterobacter spp.

5) Pseudomonas aeruginosa

B. 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 immuno-

compromised patients.

LABORATORY DIAGNOSIS

A) Specimen Collection

1. 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 Indole Urease Motility

E. Coli Positive Negative MotileKlebsiella spp. Negative Positive Non-Motile

Enterobacter spp.

Negative Positive 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 discdiffusion 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 No. of Males No. of Female

1-10 5 5

11-20 8 7

21-30 11 8

31-40 10 11

41-50 4 6

50 & above 13 12

Total 51 49



TABLE-2TABLE SHOWING %AGE OF CULTURE POSITIVE SAMPLES

Culture +ve 58%

Culture –ve 42%

TABLE-3TABLE SHOWING %AGE OF VARIOUS ORGANISMS ISOLATED

FORM PUS SAMPLES

Organism No. of Samples %age

Pseudomonasaeruginosa

17 28.33%

E.coli 12 20%

Staphylococcusaureus

12 20%

Enterobacter spp. 9 15%

Acitenobacter spp. 4 6.66%

Citrobacter spp. 3 5%

Klebsiella spp. 2 3.33%

Proteus spp. 1 1.66%

Total 60 100%



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

SEX

Age Group No. of Males No. of Female

1-10 1 2

11-20 4 9

21-30 11 12

31-40 13 11

41-50 11 8

50 & above 9 9

Total 49 51

TABLE-2TABLE SHOWING PUS CELL INTERPRETATION BY MICROSCOPY

Microscopy No. of Males %age

Pus cells present 49 49%

No. pus cells 51 51%



TABLE-3TABLE SHOWING %AGE OF CULTURE POSITIVE SAMPLES

Culture +ve 29%

Culture –ve 71%

TABLE-4

TABLE SHOWING %AGE OF VARIOUS ORGANISMS ISOLATEDFORM URINE SAMPLES

Organism No. %age

E.coli 16 59.2%

Staphylococcus

aureus

7 25.9%

Citrobacter sp. 4 14.8%

Total 27 100%



ANTIBIOTIC SENSITIVITY CHART

Urine

(Staphylococci)Nitrofurantoin (Nf)Cotrimoxazole (Co)Doxycycline (Do)Norfloxacin (nx)Lizezolid (Lz)

Amoxyclav (Ac)Oxacillin (Ox)Pristinamycin (Pm)

Non-urine (Pus)(Staphylococci)Cephalexin (Cp)Erythromycin (E)Doxycycline (Do)Getifloxacin (Gf)Linezoid (Lz)

Amoxyclav (AC)Pristinamycin (Pm)Oxacillin (Ox)

Urine

(LF/NLF) Amoxycillin (Am)Nitrofurantoin (Nf)Norfloxacin (Nx)Gatifloxacin (Gf)Cotrimoxazole ((Co)Cefotaxime (Ce)Gentamicin (G)

Amoxyclav (Ac)

Non-Urine (Pus)

(LF/NLF)Cotrimoxazole (Co)Doxycycline (Do)Ciprofloxacin (Cf)

Amikacin (Ak) Amoxyclav (AC)Ceftaxidime (Ca)Meropenem (Mr)Cefbazidime + Clavolinic acid (CaC)

Urine

(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-5INTEPRETATION OF URINE SAMPLES AS PER %AGESENSITIVE AGAINST COMMONLY USED ANTIBIOTICS

Antibiotics E. coli (16) S. aureus (7) Citrobacter (4)

Nitrofurantoin (Nf) 37.5% 42.8% 50%

Cotrimoxazole(Co)

62.5% 14.2% 50%

Doxycyline (Do) - 0.0% -

Norfloxacin (Nx) 6.2% 14.2% 0.0%

Linezolid (Lz) - 71.4% 0.0%

Amoxyclav (AC) 0.0% 14.2% 0.0%

Pristinamycin(Pm)

- 0.0% -

Amoxycillin (Am) 25% - 0.0%

Gatifloxacin (Gf) 6.2% - 0.0%

Cefotaxime (Ce) 0.0% - 0.0%

Getaimicin (G) 18.7% - 0.0%

Ofloxacin (Of) - - -

Cefepime (Cpm) - - -

Amikacin (Ak) - - -

Ceftizoxime (CR) - - -

Netilmicin (Nt) - - -

(Imipenem) (I) - - -

Piperacillin +Tazobactam (Pt)

- - -

Aztronam (Ao) - - -

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