Antibiogram of Aerobic Bacterial Isolates from Post-operative Wound Infections at a Tertiary Care Hospital in India

Original Article Vol. 28 No. 1 Bacteria in postoperative wound infections:- Malik S, et al. 45 Antibiogram of Aerobic Bacterial Isolates from Post-operative Wound Infections at a Tertiary Care Hospital in India Shruti Malik, MBBS 1, Alok Gupta, MBBS 2, K.P. Singh, M.D. 1, Jyotsna Agarwal, M.D. 1, Mastan Singh, M.D. 1 ABSTRACT Background: The aim of the study was to isolate aerobic bacteria from post-operative wounds and establish their antimicrobial susceptibility pattern. Methods: A total of 202 swab specimens from post-operative wounds of patients were screened for the presence of aerobic pathogens using standard bacteriological methods. The samples were collected using sterile cotton swab sticks and inoculated onto 5 percent Sheep blood Agar, MacConkey Agar and Robertson cooked meat broth. Antimicrobial susceptibility testing was performed on Muller-Hinton agar plates by Kirby-Bauer disc diffusion method according to the CLSI guidelines 2009. Results: The most common isolated organism from post operative wounds was S. aureus (61 isolates- 30.1%) followed by P. aeruginosa (36 isolates- 17.8%), E. coli (29 isolates-.4 %) and K. pneumoniae (19 isolates- 9.5 %). Thirty-four of 61 (55.7%) strains of S. aureus were found to be methicillin resistant (MRSA) and two were vancomycin resistant (VRSA). Extended spectrum beta-lactamases (ESBL) production was noted in 72 percent (21/29) of E. coli isolates, 73 percent (19/)of Klebsiella spp. isolates and 41% (5/12) of Proteus spp. isolates. An overall ESBL production rate of 67.1 percent (45/67) was found for all isolates screened. Conclusion: The incidence of multidrug resistant pathogens as a cause of post operative wound infection is rising. An institutional antimicrobial policy based on local microbiological data should be established to reduce mortality, morbidity and health care costs associated with post operative wound infections. (J Infect Dis Antimicrob Agents 20;28:45-51.) 1 Department of Microbiology, Chhattarpati Shahuji Maharaj Medical University, Lucknow, Uttar Pradesh, India. 2 Department of Medicine, Chhattarpati Shahuji Maharaj Medical University, Lucknow, Uttar Pradesh, India. Received for publication: December 28, 2010. Reprint request: Dr. Alok Gupta, PG Resident( III rd Year), Department of Medicine, CSM Medical University, Lucknow, Uttar Pradesh, India. Email: alokgupta16@yahoo.co.in Keywords: Antibiogram, Aerobic bacteria, Post operative wound infections, Antibiotic susceptibility 45

46 J INFECT DIS ANTIMICROB AGENTS Jan.-April 20 INTRODUCTION Surgical site infections are an important cause of hospital acquired infections among surgical patients. In fact they are the third most commonly reported nosocomial infection and they count for approximately a quarter of all nosocomial infections. 1 They have been responsible for the increase in cost, morbidity and mortality related to surgical operations and continue to be a major problem even in hospitals with the most modern facilities and standard protocols of preoperative preparation and antibiotic prophylaxis. 2 Surgical site infection rate has varied from a low of 2.5 percent to a high of 41.9 percent. 3-10 A nosocomial surgical wound infection lengthens the hospitalisation by an average of 7.4 days. The objective of this study was to determine the frequencies of various pathogens causing post operative wound infections in our hospital with their antibiotic sensitivity pattern. MATERIALS AND METHODS The study was conducted in the clinical bacteriology section, Post Graduate Department of Microbiology, Chattrapati Shahuji Maharaj Medical University, Lucknow, India. All specimens received from patients hospitalized from January 2009 to February 2010 were processed for isolation and identification of bacterial pathogens according to the standard microbiological techniques. A total of 202 post operative wound swabs were collected aseptically with a sterile cotton wool swab from clinically suspected infected wounds from different wards. Gram stain preparations were made from all swabs. Samples were inoculated onto 5 percent Sheep Blood agar, MacConkey agar; Robertson cooked meat broth (RCM) which were reconstituted according to the manufacturer s specifications. The plates were incubated at 37 degrees celsius for 18- hours. The cultures were read after hours but extended to 48 hours if there was no bacterial growth after hours. All the RCM were sub cultured subsequently. Isolated colonies were subjected to Gram staining and biochemical tests for identification. Identification was carried out according to the standard biochemical tests. Antimicrobial susceptibility testing was performed on Muller-Hinton agar (Hi Media, Mumbai) plates by Kirby-Bauer disc diffusion method according to the CLSI guidelines 2009. Standard stains of Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) were used as controls. E. coli, Klebsiella pneumoniae, Klebsiella oxytoca and Proteus mirabilis isolates from infected wounds were screened for ESBL production. Reduced susceptibility to cefotaxime(30 μg) and ceftriaxone(30 μg) with zone sizes 27 mm and 25 mm respectively was used as screening method for ESBL production. S. aureus strains were tested for methicillin resistance according to the CLSI guidelines 2009. Oxacillin disc (1 μg) was used to test for methicillin resistance. All the Staphylococcus aureus isolates were also subjected to disc diffusion testing using vancomycin discs 30 μg. The isolates showing no zone of inhibition around the disc were then subjected to MIC testing and isolates showing MIC values of more than 32 μg/ml were labeled as VRSA as per CLSI guidelines 2009. RESULTS A total of 202 specimens were obtained from post operative wounds of each patient hospitalized at surgical, orthopaedics and obstetrics and gynaecology wards. All the specimens were received at the Microbiology lab and cultured onto appropriate media. Of the 202 samples, 194 showed bacterial growth on culture and 8 were culture negative. Of the 194, 187 showed mono-microbial growth and 7 showed mixed infections with two bacterial isolates, thereby accounting to 201 isolates.

Vol. 28 No. 1 Bacteria in postoperative wound infections:- Malik S, et al. 47 Table 1 shows that the most common isolated organism from post operative wounds was S. aureus (61 isolates- 30.1%) followed by P. aeruginosa (36 isolates- 17.8%), E. coli (29 isolates-.4%) and K. pneumoniae.(19 isolates-9.5 %). Caesarean sections (36 cases-17.8%) followed by surgery done for diabetic foot (29 cases-.3%) and abscess drainage ( cases- 12.8%) were amongst the most common surgeries found to be associated with post operative wound infections (Table 2). Occurrence of post operative wound infections was highest in the age group of 16 to 40 years (96 cases-47.5%) followed by 44 cases (21.7%) in the age group of 41 to 60 years. 51.9 percent were males and 48.1 percent were females. All the 61 S. aureus strains from the infected wounds were resistant to penicillin. 34 of 61 (55.7%) strains of S. aureus were found to be methicillin resistant (MRSA) and two were vancomycin resistant (VRSA). ESBL production was noted in 72 percent (21/29) of E. coli isolates, 73 percent (19/) of Klebsiella spp. isolates and 41 percent (5/12) of Proteus spp. isolates. An overall ESBL production rate of 67.1 percent (45/67) was found for all isolates screened. Table 1. Microorganisms isolated from post-operative wound infections. Table 2. Number of wound swabs in relation to the type of surgery. Organism No. of Isolates Percent Surge ry No. of Swabs Percent Staphylococcus aureus 61 30. 1 Pseudomonas aeruginosa 36 17. 8 Escherichia coli 29. 4 Klebsiella pneumoniae 19 9. 5 Klebsiella ox ytoca 7 3. 3 Acinetobacter baumanii 17 8.45 Acinetobacter lwoffii 2 0.99 Proteus mirabilis 10 4. 9 Proteus vulgaris 2 0.99 Citrobacter freundii 5 2. 5 Citrobacter koseri 3 1. 5 Enterococcus faecalis 6 2. 9 Streptococcus pyogenes 4 1. 9 Total 201 100 Caesarean section 36 17. 8 Diabetic foot 29. 3 Abscess drainage 12. 8 Implants/Prosthesis. 9 Fasciotomy 12 5. 9 Intestinal obstruction 5. 5 Hysterectomy 9 4.45 Skin grafting 8 3.96 Amputation 8 3.96 Herniorraphy 7 3.46 Episiotomy 7 3.46 Mastoidectomy 6 2.97 Lipoma excision 5 2.47 Thoracotomy 5 2.47 Thyroidectomy 5 2.47 Septoplasty 2 0.99 Bone excision 2 0.99 Total 202 100 47

48 J INFECT DIS ANTIMICROB AGENTS Jan.-April 20 Table 3. Age and sex distribution of patients. Age in years No. of patie nts Percent 0-15. 9 16-40 96 47. 5 41-60 44 21. 7 > 60 38 18. 8 Sex Male 105 51. 9 Female 97 48. 1 Total 202 100 DISCUSSION A surgical wound infection is a post-operative complication that brings about embarrassment to the surgeon, considerable financial burden, undue discomfort to the patient and sometimes death. 12 The predominant bacterial isolates recovered in our study included S. aureus (61 isolates- 30.1%) followed by P. aeruginosa (36 isolates- 17.8%), E.coli (29 isolates-.4 %) and K. pneumoniae (19 isolates-9.5%). Many studies have reported S. aureus as the commonest isolate from post-operative wound infection. 1,9,,13 In the present study predominance of S. aureus in surgical site infection is consistent with reports from other studies and is however not surprising as it forms the bulk of the normal flora of skin and nails. The high incidence of gram-negative organisms in the postoperative wound infections can be attributed to be acquired from patient s normal endogenous micro flora. 15 In the present study no difference between sexes with regard to susceptibility to infection was found. A number of studies in literature indicate gradual increase in the emergence of antibiotic resistant microorganisms in surgical patients. 4,5,10, Special interest S. aureus surgical site infection is mainly due to its predominant role in hospital cross infection and emergence of virulent antibiotic resistant strains. In the present study all S. aureus strains from the infected wounds were resistant to penicillin. Ineffectiveness of penicillin in Staphylococcus aureus has been reported in other studies as well. 4,5,10, 34 of 61 (55.7%) strains of Staphylococcus aureus were found to be methicillin resistant (MRSA) and two were vancomycin resistant (VRSA). The frequency of ESBL producers of 67.1 percent in our study is comparable to previous Indian studies. 16-18 The most frequent ESBL producing isolates in our study were E. coli and Klebsiella species. It is an established fact that, ESBL producers show cross resistance to other antimicrobial agents also, thus limiting the therapeutic choice. We have noted this in our study as well. Sensitivity to imipenem was almost 91 percent and meropenem was almost 88 percent. Sensitivity to cefoperazone sulbactam (89.5%) and piperacillin tazobactam (85%) was also quite similar to that of carbapenems. Screening for ESBL production as a routine procedure in clinical laboratories gives valuable information to the clinician in appropriate selection of antimicrobial agents. Hence, we conclude based on our study that, there is a high prevalence rate of ESBL producers among Enterobacteriaciae. E. coli and Klebsiella species pose a major concern among these. Based on the prevalence rate of the ESBL producers in a healthcare facility, antibiotic policy of the institution can be tailored to achieve superior therapeutic outcome and bring about a reduction in healthcare costs. It also eliminates misuse of conventional cephalosporins in a significant proportion of patients. The highest susceptibility of P. aeruginosa was against colistin (100%), imipenem (88.8%), meropenem (86.1%) and amikacin(83.33%). Navaneeth and colleagues in their study noted a susceptibility of 88

Vol. 28 No. 1 Bacteria in postoperative wound infections:- Malik S, et al. 49 Table 4. Antibiotic sensitivity pattern of the common isolates. Organism N o. o f No. of sensitive isolate s Sm isolates PB Co Me Im G L Cp CS Cz Cf Ct PT CT Z Cm Em Gm Am Tm Va Li Ox Am c Am p Tg G m (H) (H) 61 51 25 52 42 48 55 59 61 34 4 2 Staphylococcus aureus 36 36 31 32 29 28 30 28 30 2 8 Pseudomonas aeruginosa E scherichia coli 29 28 27 25 27 21 20 23 19 1 6 K lebsiella spp. 22 23 21 20 22 17 16 21 17 23 21 1 1 49 A cinetobacter spp. 19 18 17 15 13 5 15 1 7 P roteus spp. 12 10 10 9 9 10 8 10 5 8 6 C itrobacter spp. 8 8 6 7 6 6 7 5 5 5 4 6 5 3 6 5 3 6 6 3 4 4 Enterococcus faecalis 4 3 2 3 2 4 4 2 Streptococcus pyogenes PB- Polymyxin B (300 units), Co- Colistin (10 μg), Me- meropenem(10 μg), Im- Imipenem (10 μg), G- gatifloxacin (5 g), L- levofloxacin (5 μg), Cp- Cefepime(30 μg), CS- Cefoperazone+ sulbactam (75/10 μg), Cz- Ceftazidime (30 μg), Cf- Ceftriaxone (30 μg), Ct- cefotaxime (30 μg), PT- Piperacillin+tazobactam (100/10 μg), CTZ- Cotimoxazole (1.25/23.75 μg), Cm- Clindamycin (2 μg), Em- Erythromycin (15 μg), Gm- Gentamicin (10 g), Am- Amikacin (30 μg), Tm- Tobramycin (10 μg), Va- Vancomycin (30 μg), Li- Linezolid (30 μg), Ox- oxacillin (1 μg), Amc- Amoxicillin+ clavulanate (20/10 μg), Amp- Ampicillin (10 μg), Tg- Tigecycline (15 μg), Gm[H]- Gentamicin High Level (120 μg), Sm[H]- Streptomycin High Level (300 μg)

50 J INFECT DIS ANTIMICROB AGENTS Jan.-April 20 percent each to imipenem and meropenem. 19 Among P. aeruginosa isolates, Bonfijlio and colleagues in their study summarized that meropenem was the most active compound against P. aeruginosa isolates followed by amikacin. 20 Viren and colleagues concluded that all antibacterials tested in their study did not demonstrate good antibacterial activity against P. aeruginosa isolates with the exception of imipenem and meropenem and that P. aeruginosa isolates showed a susceptibility of 80.36 percent against carbapenems. 21 Although carbapenems were among one of the most successful drugs in vitro against P. aeruginosa, there is a likelihood of resistance to even these drugs as seen in studies carried out on multidrug-resistant phenotype of P. aeruginosa. 22 Resistance to carbapenems is most likely to occur through the interplay of excess betalactamase production, impermeability via a loss of porin protein Opr D, together with the up-regulation of multidrug efflux systems, primarily Mex Amex BOpr M. 23 CONCLUSION The incidence of multidrug resistant pathogens as a cause of post operative wound infection is rising. Here lies the importance of formulating an institutional antimicrobial policy based on local microbiological data. Such a treatment policy if followed will lead to reduction in mortality, morbidity and health care cost associated with post operative wound infection. It is of utmost importance that surgeons, and other medical personnel involved in patient s care be reminded of the need to enforce aseptic measures in order to prevent postoperative wound infections. References 1. Mangram AJ, Horan TC, Pearson ML, Silver LC, Jarvis WR. Guideline for prevention of surgical site infection, 1999. Hospital Infection Control Practices Advisory Committee. Infect Control Hosp Epidemiol 1999;20:250-78. 2. Yalcin AN, Bakir M, Bakici Z, Dokmetas I, Sabir N. Postoperative wound infections. J Hosp Infect 1995;29:305-9. 3. Berard F, Gandon J. Postoperative wound infections: the influence of ultraviolet irradiation of the operating room and of various other factors. Ann Surg 1964;160:1-192. 4. Agarwal SL. Study of postoperative wound infection. Indian J Surg 1972;34:3-20. 5. Rao AS, Harsha M. Postoperative wound infections. J Indian Med Assoc 1975;64:90-3. 6. Cruse PJ, Foord R. The epidemiology of wound infection. A 10-year prospective study of 62,939 wounds. Surg Clin North Am 1980;60:27-40. 7. Tripathy BS, Roy N. Post-operative wound sepsis. Indian J Surg 1984;47:285-8. 8. Kowli SS, Nayak MH, Mehta AP, Bhalerao RA. Hospital infection. Indian J Surg 1985;48:475-86. 9. Olson MM, Lee JT Jr. Continuous, 10-year wound infection surveillance. Results, advantages, and unanswered questions. Arch Surg 1990;125:794-803. 10. Anvikar AR, Deshmukh AB, Karyakarte RP, et al. A one year prospective study of 3,280 surgical wounds. Indian J Med Microbiol 1999;17:129-32.. Lilani SP, Jangale N, Chowdhary A, Daver GB. Surgical site infection in clean and clean-contaminated cases. Indian J Med Microbiol 2005;23:9-52. 12. Masaadeh HA, Jaran AS. Incident of Pseudomonas aeruginosa in post-operative wound infection. Am J Infect Dis 2009;5:1-6. 13. Prabhakar H, Arora S. A bacteriological study of wound infections. J Indian Med Assoc 1979;73: 5-8.. Isibor JO, Oseni A, Eyaufe A, Ahmadu T. Incidence of aerobic bacteria and Candida albicans in postoperative wound infections. Afr J Microbiol Rese 2008;2:288-91.

Vol. 28 No. 1 Bacteria in postoperative wound infections:- Malik S, et al. 51 15. Nichols RL. Surgical wound infection. Am J Med 1991;91:54S-64S. 16. Singhal S, Mathur T, Khan S, et al. Evaluation of methods for AmpC beta-lactamase in gram-negative clinical isolates from tertiary care hospitals. Indian J Med Microbiol 2005;23:120-4. 17. Mohanty S, Singhal R, Sood S, Dhawan B, Das BK, Kapil A. Comparative in vitro activity of beta-lactam/ beta-lactamase inhibitor combinations against gram negative bacteria. Indian J Med Res 2005;122:425-8. 18. Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum beta lactamase producing gram-negative bacteria in a tertiary care hospital. Indian J Med Res 2002;5:153-7. 19. Navaneeth BV, Sridaran D, Sahay D, Belwadi MR. A preliminary study on metallo-beta-lactamase producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2002;6:4-7. 20. Bonfiglio G, Carciotto V, Russo G, et al. Antibiotic resistance in Pseudomonas aeruginosa: an Italian survey. J Antimicrob Chemother 1998;41:307-10. 21. Javiya VA, Ghatak SB, Patel KR, Patel JA. Antibiotic susceptibility patterns of Pseudomonas aeruginosa at a tertiary care hospital in Gujarat, India. Indian J Pharmacol 2008;40:230-4. 22. Goossens H. Susceptibility of multi-drug-resistant Pseudomonas aeruginosa in intensive care units: results from the European MYSTIC study group. Clin Microbiol Infect 2003;9:980-3. 23. Kohler T, Michea-Hamzehpour M, Epp SF, Pechere JC. Carbapenem activities against Pseudomonas aeruginosa: respective contributions of OprD and efflux systems. Antimicrob Agents Chemother 1999;43:4-7. 51

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