Project Report

(Training January-May 2012) On Acinetobacter Emerging as a Nosocomial pathogen Under the guidance & supervision of Dr. Ashish Khanna, Head of Department of Microbiology At

Submitted to:Dep. Of Paramedical Sciences

Submitted By:Benika Saini Regd no: 11008687 M.sc. Clinical Microbiology

Acknowledgement
I owe the great many thanks a great many people who helped me during my practical study. My deepest thanks to Dr. Ashish Khanna( Head department of microbiology) & Dr. Kanwalpreet kaur(head of lab department).these guide of the project for guiding in my study with attention & care. They has taken a pain to go through this project and make necessary co-operation in my study. I would also thank my institution and my faculty and faculty members without whom this project would have been a distant reality. I also extend my heartfelt thanks to my well-wishers.

INTRODUCTION

The Genus Acinetobacter belongs to the family Moraxellaceae, within the gamma subdivision of proteobacteria. they are gram negative bacteria, strictly aerobic, nonmotile coccobacilli that are oxidase negative and catalase positive. The bacteria are ubiquitous and can be isolated from soil and water. they are commonly associated with skin colonization of hospitalized patients and have been associated with serious infections. Over the recent decades, the acinetobacter species has increasingly been implicated in outbreaks of nosocomial infections causing Pneumonia, bacteraemia, urinary tract infections, wound infections and meningitis worldwide. There are over 20 species of Acinetobacter, through the species Acinetobacter Baumanii accounts for more than 80% of isolates causing Human disease. previously , all were named Acinetobacter Calcoaceticus.they are now divided into 7 species:A.Johnsonii,A.Junii,A.Levoffi and A.Radioresistans.the capsule that surrounds most strains may inhibit phagocytosis and has been speculated to predispose persons with selective complement component deficiencies to infections. The lack of characteristics (no color, non-motile, unable to reduce nitrates, oxidase negative and non-ferment ting) has led Acinetobacter to be constantly reclassified. First discovered in 1908, as Diplococcus mucosus, it has successively been named Micrococcus (small), mimic (mimics), Achromobacter (colorless), Acinetobacter (motionless) and anitratus (nitrate no reducing). Presently, atleast 24 genomic species have been described within the genus Acinetobacter. Strains belonging to some of the genomic species are very similar that identification by phenotypic characterization is difficult. It has been reported that most of the isolates of clinical origin are closely related to the Acinetobacter Calcoaceticus, A.Baumanii, genospecies 3, TU13,close to TU13 and between 1 and 3 with the exception of the genomic species 1, which has been regarded as an environmental species, each genomic species within the Acb complex has been shown to be associated with outbreaks of nosocomial infections A.Baumanii, particularly is commonly isolated in hospital outbreaks and has been attributed to cause approximately 45.3% mortality in patients with

bacteraemia.Aa number of A.Baumanii strains has developed resistance towards multiple antibiotics leading to its persistent presence in the hospital environment.these antibiotic resistant-strains pose a significant threat to hospital patients especially, those with immunocompromised patients.

Material and Method

Methods: perform a review of 15 patients admitted to a hospital and identified 8
patients with acinetobacter baumanii. Result:8 cases of acinetobacter were associated with trauma wounds.the median age of the patients was 28 years. The strains were origanlly isolated from different clinical and environmental specimens .eg:blood,cerebrospinal fluid,sputum,urine,soil,E-T secretion,wound swab,bronchial wash and E-T tip. They were preserved in nutrient broth at 37 degree Celsius. Most isolates have been recovered from cases of community acquired and nosocomial pneumonia. Most patients suffer from respiratory compromise patients on respirators are at risk of infections. Organism have also been recovered from cases of UTI, Septicemia, and wound contaminated by soil and water.

Characteristics
It grows well on Macconkey agar. Colonies are circular, entire, opaque and slightly smaller than standard enteric colonies. Most isolates grow on macconkey agar as colorless colonies that may turn slightly pink due to oxidation of lactose. Two species are mostly encountered in the clinical setting. A.Baumanii and A.Calcoaceticus.they can utilize citrate as a carbon source, oxidizes glucose (ofo +) growth at 42c . Culture on macconkey agar and blood agar: firstly we inoculate the sample on macconkey agar and blood agar and keep the plates in an incubator at 37 degree Celsius. Next day we observe a growth on the plates. Colorless colonies grow on the plate that means it is non lactose ferment. Microscopy: then we make a slide and observe under microscope.rod shaped coccobacilli are seen in the microscope. Composition of macconkey agar:

Nutrient agar Bile salt(sodium taurocholate) Lactose Neutral red

Colour: transparent, reddish brown or pink color.

Growth on Macconkey agar:

Acinetobacter isolates can also grow on blood and chocolate agar media for 16-48hr incubation in 5-10% CO2 at 35-37c. Composition of blood agar: Sterile defibrinated blood 5-10% Melted agar Beef extract

Peptone water Nacl

Color: opaque red.

Growth on blood agar:

Biochemical test
Oxidative-fermentative test (of test):
The oxidation-fermentation test was developed by Hugh and Leif son in 1953.during this
time microbiologist has observed that some bacteria produce acid from carbohydrates only under aerobic and anaerobic conditions. Production of acid from the metabolism of carbohydrates in aerobic and anaerobic metabolism was at this time defined as fermentation. Hugh and Leifson were the first to refer to the production of acid from carbohydrates under aerobic conditions only, as oxidative. Hugh and Leifson developed OF media to differentiate these two populations of bacteria. OF test is used to determine if gram negative bacteria metabolize carbohydrates oxidative by fermentation or are non sacchrolytic and therefore have no ability to use the carbohydrate in the media.

Composition:
Hugh and Leifson OF basal media, Peptone - 2.0 g Nacl Glucose - 5.0 - 10.0 g

Bromothymol blue - 0.03 g Agar 3.0 g

Dipottasium phosphate - 0.30 g Bring to 1 litre with Distelled water.the PH should be adjusted to 7.1 prior to autoclaved at 121c for 15 mins,a filter sterilized solution of 10% solution of carbohydrate is aseptically added to a medium to a final conc. Of 1%.the sterile medium containing carbohydrate is aliquoted aseptically into sterile test tubes and cooled unslanted as stabs.the medium is then dissolved by heating to a boil on a hot plate or by steaming for 20 mins.prior to aliquoting into test tubes.the tubed medium is then steamed for 20 mins in place of autoclaving to prevent breaking down of the carbohydrates.

Procedure:
1. Inoculation of the tubes: first take two test tubes. Stab one of the two test tubes with growth that occurs on macconkey agar with the help of a straight loop and stab the second test tube with colony and cover it with oil so that oxygen cannot pass into it. 2. Result: we observe that there is a change in color in both of the test tubes. It means test is positive and it is acinetobacter baumanii.

oxidative results..non fermenting bacteria that metabolize glucose via oxidative metabolism give an oxidative result.this result is indicated by a small amount of acid production in the open tube.the acid produced changes the PH indicator,bromthymol blue,from green to yellow.after a 24 hr incubation a change in PH is observed at the surface of the open tube where growth in the presence of oxygen is observed.

2. Result: we observe that there is a change in color in both of the test tubes. It means test is positive and it is acinetobacter baumanii.

Negative result..nonsacchrolytic bacteria give a negative OF result.the negative result is indicated by no color change in the oil covered tube and in some cases an increase in PH changing the bromthymol blue from green to blue in the top of the open tube.