Prevalence of ESBL in Surgical Wound Infections and Burns

Prevalence of ESBL in Surgical Wound Infections and Burns PREVELANCE OF EXTENDED SPECTRUM BETA LACTAMASES PRODUCERS AMONG SURGICAL WOUND INFECTIONS AND BURNS PATIENTS AT DR. SHANKARRAO CHAVAN GOVERNMENT MEDICAL COLLEGE, NANDED. *Vivek M Gujar1, Sharmila S Raut2, Sanjaykumar R More3 1. Assistant Professor, Dept. of Microbiology, Dr. S.C. Government Medical College, Nanded. 2. Professor, Dept. of Microbiology, Dr. S.C. Government Medical College, Nanded. 3. Associate Professor, Dept. of Microbiology, Dr. S.C. Government Medical College, Nanded. ABSTRACT Purpose:- The purpose of this study was to know the prevalence of Extended Spectrum beta lactamases (ESBL) among surgical wound infection and burn patients. Methods:- A total of 100 patients admitted to the surgical wards with post operative wound infections and burns from January 2014 to May 2014 were studied. A total of 137 isolates were obtained from these patients. Of these, 87 organisms (63.5% of the total isolates) were found to be Extended Spectrum beta lactamases (ESBL) producers. The commonest were Escherichia coli and Klebsiella pneumonia . They were studied for ESBL production by screening test, CLSI disc diffusion method phenotypic confirmation by disc potentiation test. Result:- Out of 100 strains, 87 (63.5%) were confirmed as ESBL producers. Among the ESBL producer all the isolates were sensitive to Imipenem. Resistance against Ampicillin (10ug) is 100%, Gentamicin (10ug) is 80.46%, Ciprofloxacin (5ug) is 74.72%, Tetracycline(30ug) is 63.22% and Amikacin (30ug) is 16.1 0.% Conclusion:- Our study shows presence of ESBL producer among surgical wound infections and burn patients and their prevalence is 63.5%. The routine antimicrobial sensitivity test may fail to detect ESBL. Detection of ESBL production should be carried out as a routine in diagnostic laboratories by disc potentiation test as it is a simple and cost effective test. Antibiotics resistance is significantly more prevalent in ESBL positive isolates as compared to ESBL negative. Key words:- Extended Spectrum Beta Lactamases, ESBL, INTRODUCTION The beta lactam antibiotics are amongst the most widely prescribed antibiotics and are an important component of empirical therapy in intensive care unit and high risk ward.1,2,3 Resistance to beta lactam antibiotics is an increasing problem worldwide.4 Increase in the prevalence of penicillin resistance in Streptococcus pneumoniae, Methicillin resistance in Staphylococcus aureus, Vancomycin resistance in Enterococci, Extended spectrum beta lactamases (ESBL) production in Enteric Gram negative bacilli and Fluroquinolone resistance in Neisseria gonorrhoea are just a few examples of the rising problem of resistance documented by both national and international surveillance system in the past few years.5 The ESBL are plasmid mediated enzymes that hydrolyze the oxyimino beta lactam (3rd generation cephalosporine) and monobactam (aztreonam), but have no effect on cephamycins (cefoxitin and cefotatan). It is situated in periplasmic space.6 Although TEM type beta lactamases are most often found in Escherichia coli and Klebsiella pneumoniae, they are also found in Enterobacter spp., Salmonella spp., Morganella morganii, Proteus mirabilis, Serratia marcescens, Pseudomanas aeruginosa, Shigella dysenteriae, Capnocytophaga ochracea and Citrobacter 7,8,9,10. However, the frequency of ESBL production in these organisms is low.11 Over 150 different ESBLs have been described as of today.12 ESBL pose a major problem for clinical therapeutic. It is necessary to identify the prevalence of these strain in hospitals and to characterise their epidemiology, control spread of these strains and to determine suitable preventive measures and treatment policies. MATERIALS AND METHODS A present study was conducted at Dr. Shankarrao Chavan Government Medical College, Nanded between January 2014 – May 2014. A total number of 100 post operative wound infections and burns patients wound swabs were processed during the study. A total of 137 isolates were obtained from these patients. COLLECTION AND IDENTIFICATION OF THE ISOLATES Using aseptic precautions, wound swabs were collected from the patients using sterile tipped swabs. The organism(s) isolated were identified based on colony morphology on blood agar, MacConkey agar and by standard biochemical tests.13,14 Strains:- Escherichia coli ATCC 25922( ESBL negative) and Klebsiella pneumoniae ATCC 700603 (ESBL positive)were used as control organism throughout the study. Antimicrobial Susceptibility testing:- The antibiotic sensitivity test was performed by Kirby Bauer disc diffusion technique with commercial available discs (HiMedia, Mumbai, India) on Muller Hinton agar plates. The discs used were Ampicillin (10ug), Amikacin (30ug), Gentamicin (10ug), Ciprofloxacin (5ug), Imipenem (10ug) and Tetracycline (30ug). The diameter of the zone of inhibition of each antibiotic was measured and interpreted as sensitive, intermediate sensitive or resistance according to CLSI criteria.15 Detection of ESBL15:- In the present study 137 isolates were tested for ESBL production by the following methods- SCREENING TESTS15:- CLSI disc diffusion method PHENOTYPIC CONFIRMATION TEST15:- Disc potentiation test CLSI ESBL Screening test:- 15 According to NCCLS 2002 for screening test to be positive or to consider an organism as probable ESBL producer the zone diameter should be- Antibiotic Zone diameter In mm or less Ceftazidime(30ug) 22 Cefotaxime (30ug) 27 Ceftriaxone (30ug) 25 Cefpodoxime(10ug) 17 Aztreonam (30ug) 27 The use of more than one antimicrobial agent suggested for screening will improve the sensitivity of ESBL detection15. Ideally the most sensitive ESBL screening agent is Cefpodoxime for Escherichia coli and Klebsiella pneumoniae.9 In the present study, ceftazidime (30ug), cefotaxime (30ug), ceftriaxone(30ug), cefpodoxime (10ug) and aztreonam (30ug) were used. These were stored in refrigerator. Before use they were taken out of refrigerator and brought to room temperature. Then they were applied on Muller Hinton agar for Antibiotic sensitivity testing. DISC POTENTIATION METHOD 15 As per CLSI guidelines disc potentiation method was used as phenotypic confirmatory test. For confirmation of ESBL production ceftazidime (30ug), ceftazidime + clavulanic acid combination disc (30/10ug) manufactured by HiMedia and cefotaxime (30ug) + cefotaxime clavulanic acid (30/10ug) prepared in laboratory were used. PREPARATION OF CLAVULANIC ACID STOCK SOLUTION For preparation of clavulanic acid stock solution Augmentin powder (gsk company) was used- 1.2gm vial of (Augmentin) contains 200mg clavulanic acid 1200 mg contains 200mg clavulanic acid Therefore, 6 mg Augmentin contains 1 mg clavulanic acid. 6 mg Augmentin is dissolved in 1 ml sterile distilled water to make a solution i.e 1ml solution contain 1 mg clavulanic acid. i.e 1000ul solution contains 1000ug clavulanic acid. PREPARATION OF CEFOTAXIME-CLAVULANIC ACID DISC15,16 Cefotaxime (30ug) discs were kept separately in a sterile petridish. 10ul of stock solution of clavulanic acid was added to each disc with a micropipette. 30 minutes were allowed for clavulanic acid to absorb and also for the disc to dry. The discs were used immediately after preparation. STORAGE OF CEFTAZIDIME+CLAVULANIC ACID DISC Clavulanic acid being labile, discs were placed in separate screw capped glass vials and stored at -200C. When antibiotics discs were required for test, they were removed from the freezer and allowed to come to room temperature before application. 17 APPLICATION OF DISCS:- After preparing the inoculum, Muller Hinton agar plates were inoculated. With the help of sterile forcep antibiotic discs containing ceftazidime and ceftazidime+clavulanic acid and cefotaxime and cefotaxime+clavulanic acid were placed on inoculated Muller Hinton agar plate at a distance of 24 mm from center to center. Plates were inverted and incubated at 370C for 16-18 hours. INTERPRETATION More than or equal to 5mm increase in a zone diameter for ceftazidime and cefotaxime tested in combination with clavulanic acid versus its zone when tested alone indicate ESBL production. ESBL POSITIVE:- If an isolate is confirmed as ESBL producer, the isolate reported as resistant to all Penicillin, Cephalosporins and Monobactam (Aztreonam). ESBL NEGATIVE:- If an isolate is not confirmed as ESBL producer, the sensitivity of the isolate was reported as per sensitivity test report. RESULT The total number of patients screened were 100 of which 64 were males and 36 females (M : F = 1.78:1). The average age was 44.72 years (Range 12-80 years). The types of wounds were post operative wounds (65.7%) and burns (34.3%). Duration of hospital stay ranged from 15 days to 3 months. Out of 137 strains, 87 (63.50%) were confirmed as ESBL producers (Table 1). Susceptibility pattern of the ESBL producers were studied. All the isolates were sensitive to Imipenem. Resistance against Ampicillin (10ug) is 100%, Gentamicin (10ug) is 80.46%, Ciprofloxacin (5ug) is 74.72%, Tetracycline(30ug) is 63.22% and Amikacin (30ug) is 16.10.% (Table 3). TABLE 1 Distribution of ESBL strains among the different organisms isolated Sr. no Organism No. of organisms Isolated No. of ESBL strains % ESBL strains 1 Escherichia coli 71 45 63.38% 2 Klebsiella pneumonia 57 36 63.15% 3 Enterobacter spp. 07 04 57.14% 4 Morganella morganii 01 01 100% 5 Providentia rettgeri 01 01 100% TOTAL 137 87 63.50% Table 2 Distribution of ESBL strains based on clinical diagnosis Sr. no Clinical diagnosis No. of organisms Isolated No. of ESBL strains % ESBL strains 1 Post operative wounds Infections 90 55 61.11% 2 Burns 47 32 68.08% Table 3 Antimicrobial susceptibility pattern of ESBL positive strains Sr. no Organism Susceptibility Category A Ak G Cf T I 1 Escherichia coli (45) S 00 37 07 10 18 45 IS 00 05 02 01 02 00 R 45 03 36 34 25 00 2 Klebsiella pneumonia (36) S 00 30 05 07 10 36 IS 00 02 02 02 01 00 R 36 04 29 27 25 00 3 Other. (06) S 04 06 05 05 04 06 IS 00 00 00 01 01 00 R 02 00 01 00 01 00 A=Ampicillin, Ak = Amikacin, Cf = Ciprofloxacin, G = Gentamicin, T = Tetracycline, I = Imepenem, R= Resistance, S = sensitive, IS = Intermediate sensitive DISCUSSION The prevalence of ESBL among clinical isolates very greatly worldwide, indifferent geographic areas and are rapidly changing overtime.18 In, 1983, Knothe et.al describe for the first time transferable resistance to the broad spectrum cephalosporins in clinical isolates of Klebsiella pneumoniae.19 The routine susceptibility test done by clinical laboratories fail to detect ESBL positive strains. The incidence of ESBL producing organisms in various studies has varied from 0-84%. In our study prevalence of ESBL producing strains is found to be 63.5%. All ESBL producers were sensitive to Imipenem. The result is in accordance with observation reported by other investigators.3,12,18,20 The new inhibitor based confirmatory test approach has been recommended by the CLSI for detection of ESBL. In the present study we found disc potentiation method to be reproducible, sensitive, easy and cost effective for use in a busy diagnostic laboratory.3,11 The use of both cefotaxime and ceftazidime with and without clavulanic acid increases the sensitivity of detection of ESBL compared to the use of only one of them. Inclusion of Cefpodoxime has been reported to further increase the sensitivity of this tests. 3,11 Among the Enterobacteriaceae, ESBL are most prevalent in Escherichia coli and Klebsiella spp. isolates. CONCLUSION The prevalence of ESBL producing strains is 63.5%. Multidrug resistance was found to be significantly higher in ESBL positive isolates as compared to ESBL negative. All the ESBL producers are sensitive to Imipenem. If an isolate is confirmed as ESBL producer, the isolate reported as resistant to all Penicillin, Cephalosporins and Monobactam (Aztreonam). Detection and reporting of beta lactamases producer is responsibility of every clinical Microbiologist. To prevent the spread of ESBLs producing organisms, infection control precautions like barrier nursing, cohorting of patients and nurses, attention to hand washing are essential. REFRENCES Chambers H F, Neu H C, Other beta lactam antibiotics In:Mandell G L, Bennetts J E, Daolin R, editors. Principles and Practice of infectious diseases 4th ed. Vol.I, New york: Churchill Livingstone;1995p.264-72. Fatima H M,, Chanawong A, Kevin G K, Birkenhead D and Hawkey P M. Detection of extended spectrum beta lactamases in members of the family Enterobacteriaceae: comparision of the MAST DD test, the double disc and Etest ESBL. J antimicrob Chemother (2000) 45: 881-885. Mathur P, Kapil A, Das B and Dhawan B. Prevalence of extended spectrum beta lactamases producing Gram negative bacteria in a tertiary care hospital. Indian J Med Res (2000) 115: 153-157. Samaha-Kfoury J N and Georges F A. Recent development in beta lactamases and extended spectrum beta lactamases. British Med J (2003) 327: 1209-1213. Tenover F C, Mohammed M J, Stelling J, O’brien T and Williams R. Ability of Laboratories To Detect Emerging Antimicrobial Resistance: Proficiency Testing and Quality Control Results from the World Health Organisation’s External Quality Assurance System for Antimicrobial Susceptibility Testing. J Clin Microbiol (2001) 39(1):241-250. Louis Rice, MD. Evolution and Clinical Importance of Extended Spectrum beta Lactamases. CHEST (2001) 119: 391S-396S. Decre D, Guchot B, Lucet C, Guillaume A, Bergogne B and Regnier B. Clinical and Bacteriologic Epidemiology of Extended Spectrum beta Lactamases Producing Strains of Klebsiella pneumoniae in a Medical Intensive Care Unit. Clin Infect Dis (1998) 27: 834-844. Thomas K S. Controversies about Extended Spectrum and AmpC beta Lactamases CDC (2001) 7(2): 1-9. Rodrigues C, Joshi P, Jani S H, Alphonse M, Radhakrishanan Ramd Mehta A, DETECTION OF BETA LACTAMASES IN NOSOCOMIAL GRAM NEGATIVE CLINICAL ISOLATES. Indian J Med Microbiol (2004) 22(4): 247-250. Tankhiwale S S, Jalgaonkar S V, Sarfraz Ahmed and Hassani U. Evaluation of extended spectrum beta lactamases in urinary isolates. Indian J Med Res (2004) 120: 553-556. Chaudhary U and Aggarwal R. EXTENDED SPECTRUM BETA LACTAMASES (ESBL) – AN EMERGING THREAT TO CLINICAL THERAPEUTICS. Indian J Med Microbiol (2004) 22(2): 75-80. Menon T, Bindu D, Kumar CPG, Nalini S and Thirunarayan M A. COMPARISON OF DOUBLE DISC AND THREE DIMENSIONAL METHODS TO SCREENING FOR ESBL PRODUCERS IN A TERTIARY CARE HOSPITAL. Indian J Med Microbiol (2006) 24: 117-120. Betty A Forbes, Daniel F Sahm, Alice S Weissfeld. Laboratory cultivation and isolation of bacteria. In: K.Fabiano, Sarahly L, Ellen Wurm, editors. Bailey and Scott’s Diagnostic Microbiology, 11th ed. Mosby Elesvier; (2002): 133-147. Koneman E W, Allen S D, Janda M W, Schreckenberger P C and Wine W C. The Enterobacteriaceae. In: Andrew A, Collins H and Deitch S editors. COLOUR ATLAS AND TEXTBOOK OF DIAGNOSTIC MICROBIOLOGY, 5th ed. Philadelphia: J b Lipincott Co. 1991: 105-184. National Committee for Clinical Laboratory Standards (NCCLS). Performance Standards for Antimicrobial Susceptibility Testing. Twelth Information Supplement 2002. M100-S12. Vol.20 No.1 2 Villanova Pa. Steward C D, Rasheed J K, Hubert S K, Biddble J W, Raney P M, Anderson G J, Williams P P, Brittain K L, Oliver A, McGowan J E and Tenover F C. Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standard Extended spectrum beta Lactamases Detection methods. J Clin Microbiol (2001) 39(8): 2864-2872. Watt C, Louie M, simor A E. Evaluation of Stability of Cefotaxime(30ug) and Ceftazidime(30ug) discs impregnated with clavulanic acid(10ug) for detection of Extended spectrum beta Lactamases. J Clin Microbiol (2000) 38(7): 2796-2797. Babypadmini S and Appalaraju B. EXTENDED SPECTRUM BETA LACTAMASES IN URINARY ISOLATES OF ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE – PREVALENCE AND SUSCEPTIBILITY PATTERN IN A TERTIARY CARE HOSPITAL. Indian J Med Microbiol (2004) 22(3): 172-174. Liu P Y, Jai-Chain T, Se-Chin Ke and Chen S L. Molecular Epidemiology of Extended Spectrum beta Lactamases producing Klebsiella pneumoniae Isolates in District Hospital in Taiwan. J Clin Microbiol (1998) 36(9): 2759-2762. Shukla I, Tiwari R and Agarwal M. PREVALENCE OF EXTENDED SPECTRUM BETA LACTAMASE PRODUCING KLEBSIELLA PNEUMONIAE IN A TERTIARY CARE HOSPITAL. Indian J Med Microbiol (2004) 22(2): 87-91.