ISOLATION AND IDENTIFICATION OF NLF BACTERIA IN VARIOUS SAMPLES.

IDENTIFICATION AND ISOLATION OF NON-LACTOSE FEREMNTING BACTERIA IN VARIOUS CLINICAL SAMPLES IN A TERTIARY HOSPITAL IN INDIA, INCLUDE BIOCHEMICAL TEST BASE ON THEIR ENZYMATIC ACTIVITY AND GRAPHICAL PRESENTAION OF THEIR DISTRIBUTION ACCORDING TO SEX RATION , AGE GROUP, SAMPLE AND THEIR PROFILE.

1. Submitted By: Daisy Saini B.Sc. Microbiology Final Year Branch : B.Sc. Microbiology Submitted To: Dr. Seema Bhadauria (Head, Department of Microbiology) Jaipur A Project report submitted to JECRC University IDENTIFICATION AND ISOLATION OF NON-LACTOSE FERMENTING BACTERIA FROM VARIOUS CLINICAL SAMPLES. TOPIC : Under the guidance of Dr. Saroj Hooja (Professor ,Department of microbiology) S.M.S. Medical hospital,Jaipur

2. CONTENT S.No. Title 1. ABSTRACTS 1. INTRODUCTION 2. AIMS AND OBJECTIVES 3. MATERIALS AND METHODS 4. OBSERVATION 5. RESULT 6. SUMARRY

3. ABSTRACT Non-lactose fermenting gram-negative bacilli have emerged as important healthcare-associated pathogens. This study was undertaken to identify the non-lactose fermenters isolated from various clinical samples by various methods to assess their clinical significance. The Non-Lactose fermenters were identified using standard protocols .

4. INTRODUCTION Non lactose Fermenting gram-negative b acilli(NLFGNB) are a group of aerobic, non-sporing, bacilli/coccobacilli that are either do not utilize glucose as a source of energy or utilize it oxidatively.They occur as saprophytes in the environment and also found as commensals in the human gut. This group includes numerous organisms but the ones which are known to cause infections are Pseudomonas aeruginosa , Acinetobacter baumannii , Proteus vulgaris , Proteus mirabilis and Salmonella typhi and members of genus Citrobacter etc.

5. NLF Bacteria have emerged as important healthcare-associated pathogens. They have been incriminated in infections, such as, septicemia, meningitis, pneumonia, urinary tract infections (UTI), and surgical site infections (SSI). NLF Bacteria are innately resistant to many antibiotics. The NLF Bacteria were identified using a standard protocol that included tests for motility, oxidase production, catalse test , TSI(triple sugar iron test), Indole production test, Simmon’s citrate test , urease test , Phenylalanine deaminase production test and by growth on different agar media.

6. AIMS AND OBJECTIVES To isolation and identification Non-lactose fermenting (NLF) bacteria in various clinical samples. Distribution of Non-lactose fermentative bacterial infection according to different clinical samples, wards, sex and age group. Distribution of NLF bacterial infection in different clinical samples, wards, sex and age group according to their profile.

7. Materials and Method Samples were collected from urine, pus/wound, blood, ascitic/plural fluids and were analyzed for colonial morphology and routine biochemical identification. Mac- Conkey Agar is inoculated with tested organism using streak or spread plate technique then incubated at 37 ˚C for 24-48 hours and observed.

8. Pink colony appears = LF Bacterial colony Pale or colorless colony growth = NLF Bacterial colony MacConkey agar is a selective and differential(The differential ingredient is lactose) culture media commonly used for the isolation of enteric Gram-negative bacteria. Because: Bile Salts: selective agents and inhibit Gram positive organisms. Crystal Violet: Generally inhibited Gram positive bacteria. Neutral Red : pH indicator. which is red in color at pH’s below 6.8. When lactose is fermented, the pH of the medium decreases and this changing the color of neutral red to pink .

9. Identified NLF colony further goes threw Biochemical tests for their identification according to their profile. Biochemical tests are the tests used for the identification of bacteria species based on the differences in the biochemical activities of different bacteria. Biochemical tests are the quickest and easiest tests for identifying bacteria because :- Microorganism’s visual identification is not always possible because structural differences, which may differentiate one species of bacteria from the other, are not discernible sometimes even under a microscope. Genetic testing is expensive and time consuming. Each species of bacteria has a well-defined set of metabolic activities different from all other species. These biochemical fingerprints are properties controlled by the bacterial enzymes.

10. Biochemical Characterizations Simmon’s Citrate Utilization Test - Use to identify if the organism can use citrate as its sole carbon source or not by the use of enzyme citrase . Citrate positive medium will be turn into intense Prussian blue color . Principle-

11. Urease Test - Use to identify if the organism capable of hydrolyzing urea using urease or not Urease positive medium will be turn into pink color. Principle-

12. Triple Sugar iron test(TSI)- A test which has three sugar Lactose, Sucrose and Glucose in the concentration of 10:10:1. It also contain Iron(Ferrous sulfate) which is an indicator of H2S formation and Phenol red which is Indicator of acidification (It is yellow in acidic condition and red under alkaline conditions).It also contains Peptone which acts as source of nitrogen. Some organisms generate gases, which produces bubbles/cracks on the medium.TSI is a semi solid media having slant and butt. This used to identify organism’s ability to- Ferment sugars Produce hydrogen sulphide .

13. Interpretation –

14. Sulphide Indole Motility(SIM) - Use to Test the ability of organism to do- 1- Reduce Sulphur -Blackening of the media indicates hydrogen sulfide production. 2 -Produce Indole - After addition of 5-10 drops Kovács reagent, a pink ring at the top of the tube indicates a positive indole result. 3- Motility - Growth feathering away from the stab line creating a cloudy appearance in the media indicates motility. Principle of indole test-

15. Phenylalanine Deaminase Production- Use to test the ability of organism to produce the enzyme deaminase. After adding a few drops of ferric chloride in test medium a green colored compound turn the top of the medium dark green thus indicate positive result. Principle- (PPA) Amino acid Ferric chloride act as chelating agent and combined with PPA and gives dark green color.

16. Use to identify the presence of catalase enzyme by using hydrogen peroxide. The enzyme catalase mediates the breakdown of hydrogen peroxide into oxygen and water Immediate bubble formation in given by catalase positive isolate. Catalase test - Oxidase test – The oxidase test is used to identify bacteria that produce cytochrome c oxidase , an enzyme of the bacterial electron transport chain.  When present, the  cytochrome c oxidase  oxidizes the reagent  tetramethyl -p- phenylenediamine (TMPD) to indophenols thus indicated by the development of a dark purple colour

17. Observation Name of organism Pseudomonas aeruginosa Acinetobacter baumannii Proteus mirabilis Proteus vulgaris characteristics Gram staining Negative Negative Negative Negative Motility test Positive Negative Positive Positive Catalase test Positive Positive Positive Positive Oxidase test Positive Negative Negative Negative Indole test Negative Negative Negative Positive Citrate test Positive Positive Positive Positive Urease test Negative Negative Positive Positive H 2 S production Negative Negative Positive Positive Phenylalanine deaminase test Negative Negative Positive Positive

18. OBSERVATION Proteus Mirabilis Proteus Vulgaris

19. Pseudomonas Acinobacter

20. I n During 4 month period only 381 isolates were identified as non-lactose fermentative bacteria at the Bacteriology section of Department of Microbiology and Immunology, SMS Medical College, Jaipur .Then further their distribution according to patient’s age, sex, ward and clinical samples was found out. RESULT

21. Figure-1 ( Distribution of NLF Bacteria according to Wards/ICUs ) shows that, maximum distribution of Non-lactose fermenters was found in surgical ward (28.87%) while in burn ward it was slightly lower (19.16%).Surgical ICU(25.19%) and Medical ICU(26.77%) shows similar distribution.

22. Figure – 2 (Distribution of Non-lactose fermentative bacterial infection in different clinical samples) shows that highest culture positivity was seen in respiratory samples (31.49%), while blood and others samples collected have shown lower growth only 6.29% and 6.56% samples were culture positive. Pus/Wound swab(28.34%) and Urine(27.29%) shows similar distribution . *Various samples as tissue, chest drain, ET tip, body fluids etc. were included together in other samples.

23. Figure-3 (Correlation of NLF Bacteria with age of patients) shows that maximum number of non-lactose fermenters were isolated in the age group >60 years (37%) while only 14.96% were isolated in the age group 15-29 years. the number of NLF bacteria distribution was increasing with age of patients.

24. Figure-4 (Correlation of NLF Bacteria with sex of patients) shows that distribution of non-lactose fermenter in male and female patients. The male to female ratio was found to be of 2: 1. Figure-5 (Profile of non-lactose fermenting gram negative bacilli) shows that highest culture positivity was seen in Pseudomonas species(n=212) 55.64%, while only 14.69% (n=56) non-lactose fermenters belonged to Proteus species and 2 nd most common NLF found was Acinetobacter baumannii (n=113) 29.65%.

25. Figure-6 ( Distribution of NLF Bacteria in selected wards and ICUs ) shows that T he highest distribution of Pseudomonas species was found in Surgical ward (n=61, 28.77%). Highest distribution of Acinetobacter baumanni was found in Medical ICU (n=34 , 30.08%). Highest distribution of Proteus mirabilis was found in Surgical ward (n=14 , 35%). Highest distribution of Proteus vulgaris was found in Surgical ward (n=6 , 37.5%).

26. Figure-7 ( Distribution of Non-Lactose fermenters in various clinical samples ) shows that majority of NLF isolates were isolated from respiratory specimens 120(31.49%). Among these Pseudomonas species (64.16%) was the most common isolate. Least NLF were isolated from blood 24(6.29%). In blood samples the most common isolate was Acinetobacter baumnnii (41.66%) and the maximum no. of Proteus species was found in urine samples in which Proteus mirabilis is 13.46% and Proteus vulgaris is 10.57%.

27. Figure- 8 ( Distribution of Non-Lactose fermenters according to sex) shows the distribution of NLF bacteria were higher in male patients n=254 (66.66%) then female patients n=121 (33.33%). In overall samples organisms Pseudomonas species was predominent one in male it shows 53.09% distribution and in females 59.2% cases was reported. Other organisms also shows higher rate of infection in male patients.

28. Summary Identification and isolation of NLF Bacteria include some protocols that include test for motility, oxidase production, TSI(triple sugar iron test),Indole production test, citrate test, urease test, Phenylalanine deminase production test and by growth on different agar media. There are aprox 20 types of NLF Bacteria commonly found in hospital environment and infect human and the most common species are Pseudomonas aeruginosa, Acinetobacter baumanii, Proteus vulgaris, Proteus mirabilis. In which the most abundant organism is Peudomonas species .

29. Thank you