Indian Journal of Applied Microbiology, (2006) 6(1): 99-103 ESBL Producting Enterobacteriaceae in post operative wound infection in a tertiary care hospital - Tiruchirappalli S. India * B.N. Selvakumar a, D. Samson Daniel a, T. Saravana Muthu a, Kishore a, R.E. Clement a, S.Dagnya a, P.Rajendran b a CSI Mission General Hospital, Tiruchirappalli - S. India a Department of Microbiology, University of Madras, Tharamani Campus - Chennai- 113. ABSTRACT Extended spectrum beta-lactamases (ESBL) are usually associated with multiple drug resistance, including beta-lactam and non-beta lactam antibiotics. Microorganisms producing ESBLs have been reported in many countries. But the prevalence and phenotyphic characteristics among clinical isolates may vary between geographical areas. Only a few reports are available on the prevalence of ESBL producing Enterobacteriaceae in the pus and wound swab cultures in our country so, in this study we aimed to examine (i) the prevalence of ESBL producers among the members of Enterobacteriaceae in post operative wound infection. (ii) to determine the antibiotic susceptibility pattern of all the isolates (ESBLs) and non-esbls). Keywords: ESBL, Enterobacteriaceae, Post operative wound infection. Method: Bacterial isolates from pus, wound swabs in the CSI Mission General Hospital, Tiruchirapalli S. India were identified by conventional methods. Antimicrobial susceptibility was done by Kirby-Baur disc diffusion method. Isolates resistant to any one of the cephalosporins were tested for ESBL production by double disc synergy test method. Results: A total of 247 gram negative bacteria were isolated. The common enterobacteriaceae isolated and the rates of ESBL producers in our study were as follows E.coli (n = 122), 26%; Klebsiella species (n = 31), 29%; pseudomonas species (n = 25), 10.5%, Aeromonas species (n = 28), 14.2%; Proteus species (n = 12), 9% Morganella (n = 6) ; 16.6%; Serratia (n = 8), 0%; Citrobacter (n = 13), 1%, Acinetobacter (n = 5) 0%. Conclusion: Our study shows the presence of extended spectrum beta lactamases producing Enterobacteriaceae in the post operative wound infection. Most of the isolates were resistant to ampicillin, cefuroxime, fluoroquinolones, cotrimoxazole and tetracycline. Overall amikacin (86%), ticarcilin / clavulonic acid (72%) and piperacillin / tazobactam (83%) were the most effective antibiotics. Introduction Extended spectrum betacalactamase (ESBL) is recognised world wide as a problem in hospitalised patients (1). ESBL are the plasmid coded enzymes, produced by certain bacteria, which possess the property of hydrolysing the cephalosporins of third generation (Cefotaxime, Ceftazidime, Ceftriaxone) and fourth generation (Cefepime, Cefpirome) along with aztreonam, broad spectrum penicillins and structurally related beta-lactams, but have no effect on Cephamycins (Cefoxitin and Cefotetan), Carbapenems imipenem and related compounds. In 1983, the first reported extended spectrum betalactamase (ESBL) producer occurred in K.pneumoniae (3) followed by the detection of an ESBL producer in E.coli in 1987 (4) so named for their ability to hydrolyse extended spectrum cephalosporins, these plasmid mediated beta-lactamases are mostly descendants of the TEM-1 and TEM-2 and SHV-1 enzymes (5), (6). The prevalence of ESBL among clinical isolates varies between countries and institutions. In the United States, occurrence of EBSL production in Enterobacteriaceae ranges from 0-25%, depending on the institution, with the national average being around 3% CDC national nosocomial infections surveillance. In Europe, the prevalence of ESBL production among isolates of enterobacteriaceae varies greatly from country to country. ESBL producers have been found in not only in the E.coli and Klebsiella but also in many other
100 Indian Journal of Applied Microbiology Vol. 6 No. 1 genera, including other members of the family enterobacteriaceae such as Citrobacter, Enterobacter, Morganella, Proteus, Aeromonas, Pseudomonas, Providencia, Salmonella and Serratia species (7). Only a few reports are available on the incidence of ESBL producing enterobacteriaceae in post operative wound infection in our country so the present study was carried out in the Department of Microbiology of our hospital. Materials and Methods This retrospective study included 585 patients who underwent surgical treatment (abdominal, vascular and orthopedic) during the 15 months period (March 2004 - May 2005). The samples from pus, wound swab, aspirated fluids and soft tissue were received from the patients (inpatients / outpatients) of our hospital were processed for the isolation and identification of bacterial pathogens according to the standard microbiological methods (8). Antibiotic susceptibility for these isolates was done by Kirby Bauer s Method (9) using antibiotic disks from Hi-media, Mumbai. Antibiotics used for Gram Negative bacilli were Ampicillin (10 μg), Chloramphenicol (30 μg), Gentamycin (10 μg), Norfloxacin (10μg), Ciprofloxacin (5 μg), Ceftazidime (30 μg), Amikacin (30 μg), Ofloxacin (5 μg), Cefoperazome (75 μg), Ceftizoxime (30 μg), amoxycillin - Clavulanic acid (10 μg), Piperacillin / Tazobactam (100/10 μg), Imipenem (10 μg), Ticarcillin / Clavulanic acid (75 / 10). For staphylococcus penicillin (10 U), oxacillin (1 μg), Vancomycin (30 μg), and Linezolid (30 μg) were also used. The diameter of the zones of inhibition of growth was recorded and interpreted as sensitive, Intermediate or Resistant based on the NCCLS guidelines (10). Screening for ESBL production The organism was swabbed onto a Mueller Hinton Agar Plate. Antibiotic discs of amoxycillin - clavulanic acid (20/10 μg) and cefotaxime (30 μg) were placed at a distance of 15 mm apart and incubated. Organism that showed a clear extension of cefotaxime inhibition zone towards the disc containing clavulanate was considered as ESBL producer. NCCLS Confirmatony test While performing antibiotic testing ceftazidime (30 μg) and ceftazidime / clavulanic acid (30/10 μg) were placed on Mueller Hinton agar and incubated. Organism was considered as ESBL producer if there was 5 mm increase in zone diameter of ceftazidime / clavulanate disc and that of ceftazidime disc alone. Escherichia coli ATCC 25922 (ESBL negative) Pseudomonas aeruginosa ATCC 27853 (ESBL Negative) and Klebsiella pneumoniae ATCC 700603 (ESBL positive) strains were used as control organisms. Table 1: Pathogens isolated from Pus and wound swabs Organism No isolated Percen tage Gram positive cocci Staphylococcus aureus 137 33 Coagulase negative S.aureus 8 2 MRSA 29 6.8 Sub total 174 Gram negative bacilli Escherichia coli. 122 29 Aeromonas species 28 6.7 Acinetobacter species 5 1.1 Pseudomonas species 25 6 Klebsiella pneumoniae 26 7.3 K. Oxytoca 5 Citrobacter freundii 8 3 C. diversus 5 Proteus mirabilis 8 2.8 P. vulgaris 4 Morganella margannii 6 1.4 Serratia liquefaecienes 5 1.1 Subtotal 247 Total 421 Results A total of 247 gram negative bacilli were isolated. Of the 585 clinical pus samples. 410 samples showed the growth of bacteria and no growth was seen in 175 cultures. Among the 247 gram negative bacteria, the ESBL producing phenotype was found most frequently among E.coli (n = 36), Klebsiella species
December 2006 ESBL Producting Enterobacteriaceae in post operative wound infection... 101 (K.pneumoniae (n = 10), K. Oxytoca (n = 2)), Aeromonas species (A. hydrophila (n = 8), A. Sobria (n = 2), Pseudomonas aeruginosa (n = 6), Citrobacter freundii (n = 2), Proteus mirabilis (n = 2), and Acinetobacter baumanni (n = 1). All the isolates were tested by using the combination disks, and 69 (28%) isolates were confirmed as being ESBL producers. The ESBL phenotype was least prevalent among Citrobacter, Proteus and Acinetobacter species. No ESBL production was observed in Morganella and Serratia species in our studies. It was found that gram positive organisms predominated in the etiology of the wound infection. Discussion In our study the prevalence of Gram positive and Gram negative bacteria were almost similar. The most common aerobes isolated in our study were Staphylococcus aureus, Escherichia coli, Klebsiella, Aeromonas, Pseudomonas, Acinetobacter, Citrobacter, Proteus, Morganella and Serratia species. The following observations were made from Table 2. More than 75% of the Enterobacteriaceae tested were resistant to ampicillin at the same time only a few were resistant to the combination of Amoxycillin-Clavulanic acid, Piperacillin-Tazobactam and Ticarcillin / clavulanic acid. This finding suggests that the resistance observed was due to mainly the production of beta lactamage by the organisms. All Staphylococcus aureus strains isolated from the wound infection were resistant to penicillin. This ineffectiveness of penicillin in Staphylococcus aureus has been reported by others also (16, 17, 18). The methicillin resistance was frequently high among Staphylococcus aureus isolates in both hospitalised as well as out patients. Overall 17% of MRSA was observed in our study. For all S. aureus including MRSA Linezolid (100%), Cefotaxime (96%), Amikacin (93%) and Piperacillin / tazobactam were effective drugs. ESBL detection in gram negative bacteria Identifying organisms that are ESBL producers is a major challenge for the clinical microbiology laboratory. Cefpodoxime and ceftazidime have been proposed as indicators of ESBL production as compared to cefotaxime and ceftriaxone. Hence an Table 2: Sensitivity Pattern of Common Aerobic bacterial isolates S. No. Antibiotics E.coli (n = 122) Klebsiella spp (n = 31) Aeromonas spp (n = 28) Pseudomonas spp. (n = 25) S. aureus (n = 174) 1. Ampicillin 20 (16.3%) 5 (16%) - (0%) - (0%) 20 (11.4%) 2. Amikacin 101 (83%) 25 (81%) 26 (93%) 8 (32%) 162 (93.1%) 3. Cefotaxime 84 (68%) 23 (74%) 16 (57%) 13 (52%) 168 (96.3%) 4. Ceftazidime 64 (52%) 12 (38.7%) 22 (79%) 18 (72%) 42 (24%) 5. Cefuroxime 44 (36%) 23 (74%) 8 (29%) 6 (24%) 129 (74%) 6. Clindamycin -- -- -- -- 150 (86%) 7. Cotrimoxazole 80 (66%) 20 (64.4%) 15 (54%) 8 (32%) 65 (37.3%) 8. Ciprofloxacin 85 (70%) 18 (58%) 10 (36%) 10 (40%) 138 (79%) 9. Erythromycin -- -- -- -- 72 (41.3%) 10. Gentamycin 89 (73%) 14 (45%) 12 (43%) 8 (32%) 106 (61%) 11. Norfloxacin 20 (16.3%) 8 (26%) 4 (14%) 5 (20%) 93 (53.4%) 12. Tetracycline 38 (31%) 6 (19%%) 6 (21%) 9 (36%) 83 (48%) 13. Penicillin -- -- -- -- 28 (16%) 14. Piperacillin / Tazobactam 94 (77%) 19 (68%) 19 (68%) 23 (92%) 165 (95%) 15. Cefoperazone sulbactam 74 (60%) 14 (50%) 14 (50%) 12 (48%) 148 (85%) 16. Ticarcillin / Clavulanic acid 91 (75%) 18 (64%) 18 (64%) 16 (64%) 160 (92%)
102 Indian Journal of Applied Microbiology Vol. 6 No. 1 institution where only cefotaxime and ceftriaxone are used in the routine sensitivity may have difficulty in detecting ESBLs (19). So in our institution we used all the four indicators for the detection of ESBL. Five Acinetobacter species were isolated from the pus and wound swab culture. Of these four were A. baumannii and one was A. lwoffi. All the five Acinetobacter species were multi drug resistant strains. They are recently emerged as a nosocomial pathogen and an important cause of morbidity and mortality mainly among debilitated patients (22). Anuradha D and Brook I, reported that Staphylococcus spp., group D Streptococci, Pseudomonas and other Enterobacteriaceae were recovered from pus and wound swabs (20, 21). Recently Aeromonas species has been recognised as causing infection in wounds (23, 24, 25). We isolated these bacteria from 28 patients as a sole causative agents. Of the 28 isolates 22 were A. hydrophila and 6 were A. sobria. Most of the strains were resistant to ampicillin, cefuroxime but are sensitive to amikacin, ceftazidime and piperacillin / tazobactam. Most of the P. aeruginosa isolates were resistant ciprofloxacin and other quinolones, but only a few isolates were resistant to (N = 3) netilmycin. All the isolates were susceptible to imipenem, piperacillin / tazobactam and ceftazidime. The susceptibility data in this study suggests that some antibiotics would have very limited use for the prophylaxis or empirical treatment of wound infections. For example, most of the gram negative bacteria were resistant to ampicillin, cefazolin and cefuroxime. While the majority of Staphylococcus strains were resistant to penicillin and ampicillin. In conclusion, this study demonstrated the high prevalence of ESBLs in post operative wound infection. Because these β- lactamases are shown to significantly affect therapeutic outcomes,it is critical that clinical laboratories be able to detect their occurrences and to screen various antimicrobials that are effective against them. From our data, we can suggest that Imipenem, amikacin and piperacillin / tazobactam were found to be effective drugs against these multi drug resistant β-lactamase producers. Careful monitoring and rapid detection of these β-lactamase producers are essential. References 1. Giacometti A, Cirioni, O, Schimizzi A.M., Del Prete M.S., Barchiesi F, D errico M.M., Petrelli. E., and Scalise G. Epidemiology and Microbiology of surgical wound infections. J. Clin. Microbiol. 2000; 38(2) 918-922. 2. Jacoby GA, Medeiros AA. More extended spectrum beta lactamases. Antimicrob Agents Chemother 1991; 35: 1697-1704. 3. Knothe, H, P. Shah, V.Kremery, M.Antal, and Mitsuhashi. Transferable resistance to ceftaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of K.pneumoniae and serratia marcescens. Infection 1983; 11: 315-317. 4. Bauernfeind, A., and G. Horl. Novel R. factor borne betalactamase conferring resistance to cephalosporin. Infection. 1987; 15:257-259. 5. Dubios, S.K.S., M.S. Marriott and S.G.B. Amyes. TEM and SHV-derived extended spectrum β-lactamases: relationship between selection, structure, and function. J. antimicrob. Chemother 1995; 35:7-32. 6. Nathisuwan S, Burgess DS, Lewis II JS. ESBLs: Epidemiology, Detection and Treatment. Pharmacotherapy 2001; 21(8): 920-928. 7. Bradford, P.A. Extended-spectrum Beta lactamases in the 21 st century: Characterisation, epidemiology, and detection of this important resistance threat. Clin Microbiol. Rev. 2001; 14: 933-951. 8. Koneman EW, Allen SD, Janda WM, Schreckenberger Winn WC, editors. The Enterobacteriaceae. In: Color atlas and text book of diagnostic microbiology, 5 th ed. Philadelphia: JB Lippincott Co., 1997, p.171-230. 9. Bauer AW, Kirby WMM, Sherris JC, Tuck M. Antibiotics susceptibility testing by a standardised single disc method. Am J Clin. Pathol 1966; 45:493-6. 10. National committee for clinical laboratory standards performance standards for antimicrobial disk susceptibility testing. Twelfth informational supplement (M 100-S12) 2002. Wayne. PA: NCCLS. 11. National committee for clinical laboratory standards. Performance standards for antimicrobial disk susceptibility test, 7 th edition. Approved standards. NCCLS document M2-47, Vol.20 No.1, Wayne PA 2000. 12. Jarlier V, Nicolas M. Fournier G, Philippon A, Extended broad-spectrum β-lactamases conferring transferable resistance to newer β-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility pattern. Rev. Infect. Dis. 1998: 10, 867-878. 13. Cruse PJE. Surgical wound infection. In: Gorbach SL, Barbett JG, Blacklow MR, eds. Infectious diseases. Philadelphia: WB Saunders company, 1992: 738-64. 14. Olson MM, Lee JT, Continuous 10 year wound infection surveillance results, advantages, and unanswered questions. Arch. Surg. 1990; 125: 794-803. 15. Prabhakar H, Arora S. A bacteriological study of wound infections. J. Indian Med. Association. 1979; 73:145-8.
December 2006 ESBL Producting Enterobacteriaceae in post operative wound infection... 103 16. Agarwal SL. Study of post operative wound infection Indian J surg. 1972; 34:314-20. 17. Rao AS, Harsha M. Post operative wound infections. J Indian Med Assoc. 1975; 64:90-3. 18. Anvikar AR, Deshmukh AB, Karyakarte RP, Damle AS, Patwardhan NS, Malik AK, et.al. A one year prospective study of 3,280 surgical wounds. Indian J.Med. Microbiol. 1999; 17:129-32. 19. Nathisuwan S, Burgess DS, Lewis II JS. ESBLs: Epidemiology, Detection and Treatment. Pharmacotherapy 2001; 21(8): 920-928. 20. Joshi SG, Litake GM, Niphadkar KB, Ghole VS. Multidrug resistant Acinetobacter baumannii isolates from a teaching hospital. J infect. Chemother. 2003; 9:187-90. 21. Anuradha D., Biswas J., Saraswathi K, Gogate A. Microbiological features of necrotising fascitis. Ind. J. Med. Microbiol 1999; 17(1): 18-21. 22. Brook I, Frazier E.H. Clinical and Microbiological features of necrotising fascitis. J. Clin. Microbiol. 1995; 33(9): 2382-7. 23. Moses AE, Leibergal M, Rahav G, Perouansky M, or R, Shapiro M. Aeromonas hydrophila myonecrosis accompanying mucormycosis five years after bone marrow transplantation. Eur. J. Clin. Microbiol. Infect Dis. 1995 Mar; 14(3): 237-40. 24. Skiendzielewski JJ, O Keefe KP, Wound infection due to fresh water contamination by Aeromonas hydrophila. J Emerg Med. 1990 Nov-Dec; 8(6): 701-3. 25. Petroni A., Corsa A., Melano R., Cacace M.L., Bru A.M., Rossi A., and galas M. Plasmidic extended - Spectrum Beta lactamases in vibrio cholerae OIEITor isolates in Argentina. Antimicrob. Agents Chemother. 2002; 46: 1462-1468.