Antimicrobial Susceptibility Pattern of Clinical Isolates ...

S.Meenakumari et al. / International Journal of Engineering Science and Technology (IJEST)

Antimicrobial Susceptibility Pattern of Clinical Isolates of Pseudomonas aeruginosa in an Indian Cardiac Hospital. S.Meenakumari1, Shikha Verma2, Anam Absar2, Abhishek Chaudhary2* 1

Assistant Professor, Department of Biotechnology, SRM University Kattankulathur, INDIA 2 Department of Biotechnology, SRM University Kattankulathur, INDIA * Corresponding Author: e-mail: [email protected], Tel +91-9444298417

Abstract Out of the 5933 samples collected a total of 51 isolates of pseudomonas aeruginosa were collected consecutively between 26-December-2010 to 28-February-2011 from different patients. The total of 51 positive isolates consists both of pediatric as well as adult patients. The study was therefore carried out using both manual (Kirby-Bauer method) as well as automated (Vitek2 system) method to determine the Antimicrobial susceptibility pattern of pseudomonas aeruginosa isolates from in-patients and out-patients attending the microbiology section of the hospitals. The isolation rate of Pseudomonas aeruginosa was found to be 8.5% out of the total positive samples which were analyzed. In my study, notable sensitivity (100%) to P.aeruginosa was observed against Aztreonam while it was found to be different in case of a study carried out in India earlier which observed Carbepenems with 19.40% resistance. In the study colistin showed the highest (100%) sensitivity followed by Amikacin against P. aeruginosa , which is in corroboration with an earlier report published from India. Amikacin seems to be a promising therapy for Pseudomonal infection. Hence, its use should be restricted to severe nosocomial infections, in order to avoid rapid emergence of resistant strains. The sensitivity of Pseudomonas aeruginosa towards Imipenem is found to be 35.294% and towards Meropenem is 41.176%, which is different in case of the earlier study from India. An effective national and state level antibiotic policy and draft guidelines should be introduced to preserve the effectiveness of antibiotics and for better patient management.

Keywords: antimicrobial susceptibility, pseudomonas aeruginosa, effectiveness of antibiotics, aztreonam, amikacin, imipenem, pseudomonal infection, colistin, meropenem, antibiotic sensitivity. 1. Introduction The genus Pseudomonas is free living Gram-negative, aerobic, rodshaped bacterium with unipolar motility, and is found in most moist environments. It contains more than 140 species, most of which are saprophytic and more than 25 species are associated with humans. Most Pseudomonads known to cause opportunistic infections. It is a leading gram negative pathogen that causes nosocomial infections so has received most attention. Although it causes disease in healthy individuals, it is a major threat to hospitalised and immunocompromised patients, particularly those with diseases such as cancer and burns. The high mortality associated with these infections is due to a combination of weak host defense system and bacterial resistance to antibiotics. Resistance of this notorious bacterium to commonly used antimicrobial agents is becoming an increasing clinical problem and a recognised public health threat because there are limited number of antimicrobial agents including the antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides and fluoroquinolones with reliable activity against it. It has intrinsic resistance to many antimicrobial agents and only a few antimicrobial agents show potent antibacterial activity against this bacterium. The emergence of multidrug resistance (MDR) Pseudomonas aeruginosa has became a serious problem. There are several mechanisms which may contribute to the antimicrobial resistance among Pseudomonas aeruginosa including the production of chromosomally encoded Amy C B-lactamases. Treatment of P.aeruginosa infection can be difficult. A relatively narrow spectrum of antimicrobials is effective against P.aeruginosa, including the carboxypenicillins (carbenicillin, ticarcillin), the ureidopnicillins (mezlocillins, piperacillin),the antidopseudomonal cephalosporin (ceftazidime), monobactams (aztreonam), carbapenems (imipenem, meropenem), quinolones (ciprofloxacin, levofloxacin), and aminoglycosides (gentamicin, tobramycin, amikacin). Almost all strains are resistant to other pencilins and cephosporins,

ISSN : 0975-5462

Vol. 3 No. 9 September 2011

7117


including ampicillin, cefuroxime, and cefotaxime. P.aeruginosa has the capacity to carry multiresistance plasmids, and this feature has led to the appearance of some P.aeruginosa strains that are resistant to all reliable antibiotics. 2 Methodology Out of the 5933 samples collected a total of 51 isolates of pseudomonas aeruginosa were collected consecutively between 26-December-2010 to 28-February-2011 from different patients. The total of 51 positive isolates consists both of pediatric as well as adult patients. The study was therefore carried out using both manual (Kirby-Bauer method) as well as automated (Vitek2 system) method to determine the Antimicrobial susceptibility pattern of pseudomonas aeruginosa isolates from in-patients and out-patients attending the microbiology section of the hospitals 2.1 Specimen collection : The normal flora are regularly found in specific areas of the body. This specificity is far from arbitrary and depends on environmental factor such as pH, oxygen concentration, amount of moisture present, and types of secretion associated with each anatomical site. Native microbial flora of the species belonging to Enterobacteriaceae are located as follows Urine Midstream clean catched urine in a sterile container following adequate cleansing of the external genetilia. It was imperative to culture the freshly provided, unrefrigerated sample immediately to avoid the growth of normal indigenous organisms, which may overtake the growth of the more slowly growing pathogens. Blood Before collecting the blood samples, hands were be washed properly and dried and alcohol hand rub was be used. After the vein was selected, scrubed the venipuncture site. Iodine solution (1-2% tincture of iodine) for 30 second in concentric circles away from the puncture site covering a circular area of 1-2 inches in diameter was applied. Allowed the site to air dry for 1 minute before venipuncture. A blood sample was drawn (8-10 ml for adults and 1-4 ml for pediatrics) and introduced to the appropriate blood culture bottle for aerobic and anaerobic culture. Over a period of 3 to 7 days, the cultures are observed for turbidity and gram stained smears are prepared to ascertain the presence of micro-organisms in the blood. Upon detection of microbial growth in the cultures, they were transfered onto a variety of specialized agar media prepared for the presumptive identification of the infectious agents. Throat The collection of throat sample was done by placing a tongue depressor on the extended tongue and with a sterile cotton swab. The specimen was obtained from the pharyngeal tonsil by rotating the swab variously over its surface without touching the tongue and side of the mouth. Sputum Always, first morning sputum was collected. This required the proper rinsing of the mouth of the patient, a few times and then taking a deep breath filling the lungs, followed by emptying the lungs in one breath, coughing as hard and deeply as possible, finally spitting in the container. It was taken care that the patient provided a deep cough specimen rather than saliva. A satisfactory specimen consists of 5-10 ml of purulent or mucopurulent material with minimal amount of salivaor nasal secretions present. Wound/ Pus When collecting pus from an abscess, wound or other sites, special care was taken to avoid contaminating the specimen from skin. The sample was collected before an antiseptic dressing was applied. Using a sterile technique, we collected the pus from the deeper area rather than the opening, material from the surface may yield flora from the skin or mucous membranes. Then the pus was transferred to a sterile leak-proof container. When pus is not being discharged, we used a sterile cotton wool swab to collect a sample from the infected site. Labeled the specimen and as soon as possible, transported the sample to laboratory 2.2 Media and their preparation : The sample received is primarily inoculated in Blood agar, MacConkey agar and Chocolate agar plates. Mueller-Hinton agar plates are used for antibiotic sensitivity testing. Triple-Sugar Iron agar, Simmons’s Citrate agar and Urease agar slant tubes are employed for biochemical testing for Enterobacteriaceae species. 2.3 Processing of the samples: In routine culture, a loopful of sample is inoculated on two primary petriplates blood agar and MacConkey agar. MacConkey agar and blood agar plates are incubated aerobically whereas sputum and throat samples were processed in addition on chocolate agar plates, which are incubated in the presence of carbon dioxide atmosphere.

ISSN : 0975-5462


7118


2.4 Culturing the samples: Sputum, throat swabs is cultured on blood agar, chocolate agar and MacConkey agar (by continous streaking method). Blood is cultured in blood bottles with BacT/Alert and then cultured on blood and MacConkey agar (by quadrent streaking method). Pus, chest wound swab and endotracheal secretion is also cultured on blood and MacConkey agar (by continous streaking method. Urine is cultured on blood and MacConkey agar (by quadrant streaking). 2.5 Identification and isolation of the pseudomonas species: Pseudomonas aeruginosa species were identified and a pure culture was prepared. The identification test was based on the high concentration of cytochrome oxidase present in the cells (W. L. GABY AND C. HADLEY, 1957). 2.6 Antimicrobial susceptibility test: The susceptibility tests were performed using Vitek 2 system. The Vitek 2 is a new system from biomerieux that performs bacterial identification & susceptibility testing analysis using standardized inoculum. The vitek 2 features the AES (Advanced expert system) that integrates artificial intelligence technologies in the vitek 2 instrument AES interprets the result of the Antibiotic Susceptibility Test using a highly developed knowledge database. The base contains most of the known resistance mechanisms. The Vitek 2 System (bioMerieux) incorporates several technical improvements that automates many procedures that are performed manually .The system detects bacterial growth and metabolic changes in the microwells of thin plastic cards by using a fluorescence- based technology (a colorimetric-based instrument was introduced later in 2004). The identification card for gram-negative bacilli (IDGNB card) for the VITEK 2 is a 64-well plastic card containing 41 fluorescent biochemical tests, including 18 enzymatic tests for aminopeptidases and osidases. Substrates used for detection of aminopeptidases are usually coupled with 7-amino methylcoumarin (7AMC); substrates for detection of oxidases are usually coupled with 4-methylumbelliferone (4MU). In addition there are 18 fermentation tests, 2 decarboylase tests, and 3 miscellaneous tests. There are two negative control wells, and the remaining wells are empty. Results are interpreted by the ID-GNB database after a 3-hr incubation period.

3. Results A total of 51 isolates of pseudomonas aeruginosa were collected consecutively between 26-December-2010 to 28-February-2011 from different patients The total of 51 positive isolates consists both of pediatric as well as adult patients. The pediatric isolates were 5 in number while the Adult were 46.The total numbers of positive isolates of pseudomonas aeruginosa in different specimens are as follows: Out of the total 51 samples, 5 were blood samples (9.803%), 3 were urine samples (5.882%), 27 were respiratory tract samples (52.914%), 5 were I/V line tips (9.803%), 5 were wound/pus samples (9.803%) and 6 were miscellaneous (11.764%). The study was therefore carried out using automated (Vitek2 system) method to determine the Antimicrobial susceptibility pattern of pseudomonas aeruginosa isolates from patients. The isolation rate of Pseudomonas aeruginosa was found to be 8.5% out of the total positive samples which were analyzed. In our study, notable sensitivity (100%) to P.aeruginosa was observed against Aztreonam while it was found to be different in case of a study carried out in India earlier which observed Carbepenems with 19.40% resistance. The resistance to carbapenems, especially in P.aeruginosa , results from reduced levels of drug accumulation or increased expression of pump efflux. The resistance may also be due to the production of metallo-β-lactamases (MBL), which can be chromosomally encoded or plasmid mediated. In our study colistin showed the highest (100%) sensitivity followed by Amikacin against P.aeruginosa , which is in corroboration with an earlier report published from India. Amikacin was designed as a poor substrate for the enzymes that bring about inactivation by phosphorylation, adenylation or acetylation, but some organisms have developed enzymes that inactivate this agent as well. Amikacin seems to be a promising therapy for Pseudomonal infection. Hence, its use should be restricted to severe nosocomial infections, in order to avoid rapid emergence of resistant strains. The problem of increasing resistance to P. aeruginosa has limited the use of other classes of antibiotics like the fluoroquinolones, tetracyclines, macrolides and chloramphenicol.

ISSN : 0975-5462


7119


In our study,the sensitivity of Pseudomonas aeruginosa towards Imipenem is found to be 35.294% and towards Meropenem is 41.176 ,which is different in case of the earlier study from India.

In fact, the irrational and inappropriate use of antibiotics is responsible for the development of resistance of Pseudomonas species to antibiotic monotherapy. Hence, there is a need to emphasize the rational use of antimicrobials and strictly adhere to the concept of "reserve drugs" to minimize the misuse of available antimicrobials. In addition, regular antimicrobial susceptibility surveillance is essential for area-wise monitoring of the resistance patterns. An effective national and state level antibiotic policy and draft guidelines should be introduced to preserve the effectiveness of antibiotics and for better patient management. Hence, based on the observations of present study, it is recommend to use either semi-synthetic penicillins like ticarcillin, piperacillin or third generation cephalosporins like ceftazidime along with b-lactamase inhibitors (clavulanate or sulbactum) against Pseudomonas aeruginosa infections. Further, colistin should be considered as a reserved drug for the treatment of severe nosocomial infections. The Antibiotic Sensitivity test results were interpreted as per Clinical &Laboratory Standards Institute (CLSI) guidelines-2010. 4. Tables and Figures

Figure 1 Growth Of Pseudomonas on MacConkey

Figure 2 Growth of pseudomonas on Blood Agar Plate

Figure 3 Vitek 2 system (bioMerieux)

ISSN : 0975-5462


7120


Total no. of samples

5933

Positive for pseudomonas aeruginosa

51

Figure 4 Total number of isolates in different specimen

Samples (pediatric)

5

Samples (Adult)

46

Total

51

Figure 5 Total no. of isolates positive for pseudomonas in pediatric and adult samples

Samples

Positive for pseudomonas

Percentage (%)

Respiratory Tract

27

52.941

Wound/Pus

5

9.803

Blood

5

9.803

I/V Line Tips

5

9.803

Urine

3

5.882

Miscallaneous

6

11.764

Figure 6 Sample wise distribution

ISSN : 0975-5462


7121


Antibiotics

Pseudomonas aeruginosa Sensitivity (S)

Percentage(%)

Resistant (R)

Percentage(%)

Gentamicin

19

37.254

32

62.745

Amikacin

22

43.137

29

56.862

Ciprofloxacin

17

33.333

34

66.666

Ceftazidime

18

35.294

33

64.705

Cefepime

19

37.254

32

62.745

Colistin

51

100.00

0

0

PiperacillinTazobactum

20

39.215

31

60.784

Cefoperazonesulbactum

20

39.215

31

60.784

Cotrimoxazole

1

1.960

50

98.039

Imipenem

18

35.294

33

64.705

Meropenem

21

41.176

40

78.431

Aztreonam

0

0

51

100

Norfloxacin

1

1.690

50

98.039

Figure 7 Antimicrobial Susceptibility test Results

Antibiotic sensitivity pattern of pseudomonas

60 50 40 30 20 10 0

Antibiotic sensitivity pattern of pseudomonas

TCA – Ticarcillin clavulanic acid P+Tz - Piperacillin Tazobactum Cfs –Cefoperazone sulbactum Figure 8 Graphical representation of the effect of various antibiotics on the growth of Pseudomonas aeruginosa

ISSN : 0975-5462


7122


5. Conclusions Antibiotic resistant organisms appear to be biologically fit and are capable of causing serious, life-threatening infections that are difficult to manage because treatment options are limited. This increase in the prevalence of drug resistant pathogens is occurring at a time when the discovery and development of new anti-infective agents is slowing down dramatically. In our project Colistin and Amikacin were the two antibiotics found to be the most susceptible against this pathogen. The emergence of multidrug resistant (MDR) Pseudomonas aeruginosa is a challenging clinical problem. In fact, the irrational and inappropriate use of antibiotics is responsible for the development of resistance of Pseudomonas species to antibiotic monotherapy. Hence, there is a need to emphasize the rational use of antimicrobials and strictly adhere to the concept of "reserve drugs" to minimize the misuse of available antimicrobials. In addition, regular antimicrobial susceptibility surveillance is essential for area-wise monitoring of the resistance patterns. An effective national and state level antibiotic policy and draft guidelines should be introduced to preserve the effectiveness of antibiotics and for better patient management. Hence, based on the observations of present study, it is recommend to use either semi-synthetic penicillins like ticarcillin, piperacillin or third generation cephalosporins like ceftazidime along with b-lactamase inhibitors (clavulanate or sulbactum) against Pseudomonas aeruginosa infections. Further, colistin should be considered as a reserved drug for the treatment of severe nosocomial infections. ABBREVIATIONS AST Antibiotic Sensitivity Testing ATCC American Type Culture Collection BA Blood Agar CFU Colony forming units CLSI Clinical Laboratory Standards Institute MHA Mueller Hinton Agar DCA Desoxycholate Citrate Agar CLSI Clinical & Laboratory Standards Institute

Acknowledgement We feel a dearth of words to pen down my heartfelt regards and deep sense of appreciation to eminent and major advisor Dr. Anita Arora (Principal Consultant & Lab Head, Microbiology),FEHI ,New Delhi). I am also heartily thankful to her for allowing me and giving me a hand to work in this laboratory. I owe a deep sense of gratitude and indebtness to my project Supervisor Dr. Shweta Sharma (Senior Resident, FEHI, New Delhi). Lastly I would like to thank my department at SRM University for supporting me in my work. References [1]

Comparison of the in vitro susceptibility of clinical isolates of pseudomonas aeruginosa in a local hospital setting in Karachi, Pakistan (S.G. Nadeem , S.A. Qasmi , F. Afaque, M. Saleem and S.T. Hakim) BJMP 2009:2(4) 35-39. [2] Javiya VA et al. 2008. Antibiotic susceptibility patterns of Pseudomonas aeruginosa at a tertiary care hospital in Gujarat, India. Indian J Pharmacol. 40(5): 230-234 [3] Antibiotic susceptibility profiles of clinical isolates of Pseudomonas aeruginosa from Selayang Hospital, Malaysia [4] (Fazlul MKK, Zaini MZ, Rashid MA, Nazmul MHM) Biomedical Research 2011; 22 (3): 263-266 [5] Antimicrobial susceptibility pattern of clinical isolates of Pseudomonas aeruginosa. [6] (Al-Jasser AM, Elkhizzi NA) Saudi Med J. 2004 Jun;25(6):780-4 [7] Prevalence and resistance pattern of pseudomonas aeruginosa against various antibiotics (Jamshaid Ali khan, Zafar iqbal, Saeed Ur Rahman*,Kalsoom Farzana** and Abbas Khan) pjps-21-3-08 [8] Antibiotic susceptibility pattern of clinical isolates of pseudomonas aeruginosa in a tertiary health institution in kano, nigeria (E.O.K. Nwankwo1, *, S.A. Shuaibu2) Journal of Medicine and Biomedical Sciences, ISSN: 2078-0273, November, 2010 [9] Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL(D De Vos, A Lim Jr, JP Pirnay, M Struelens, C Vandenvelde, L Duinslaeger, A Vanderkelen and P Cornelis) Journal of Clinical Microbiology, Jun 1997, 1295-1299, Vol 35, No. 6 [10] Practical laboratory test for the identification of pseudomonas aeruginosa (w. l. gaby and c. Hadley) 1957

ISSN : 0975-5462


7123


Biography Koneman’s Color Atlas and Textbook of Diagnostic Microbiology – Sixth Edition , Chapter-7, Part 2nd, Page – 316 & 945. Bailey & Scotts Diagnostic Microbiology – Twelth Edition , Part -2 , Section – 1,3, Topley & Wilson’s Microbiology & Microbial Infections , volume -2 , Chapter – 46, Page -1091. . Mackie & Mc Cartney Practical Medical Microbiology , 14th Edition , Section A , Chapter 5 & 6 , Page 95. Received xx 20xx Accepted xx 20xx Final acceptance in revised form xx 20xx

ISSN : 0975-5462


7124