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Year : 2013  |  Volume : 2  |  Issue : 3  |  Page : 181-185

Antimicrobial resistance and metallo β lactamase in gram-negative isolates of hospital-acquired burn wound infections

Department of Microbiology, Prathima Institute of Sciences, Karimnagar, Andhra Pradesh, India

Date of Web Publication29-Aug-2013

Correspondence Address:
Sanjeev D Rao
Department of Microbiology, Prathima Institute of Medical Sciences, Karimnagar - 505 001, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2277-8632.117184

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Introduction: In India, accidental contact with direct flame is the most common cause of burns. More than 90% of burn wounds are II and III degree, though surface area involved is less than 40%. Burn patients are at high risk of infection as a result of the nature of injury, immunocompromising effect of burn, prolonged hospital stay, and invasive procedures. Multidrug-resistant bacterial infection is major cause of mortality in these patients.
This study was undertaken to determine the prevalence of metallo b lactamase (MBL) producing gram-negative isolates in burn wound infections, so as to guide the clinicians to select appropriate antimicrobial therapy and prevent the spread of MBL in gram-negative bacteria.
Materials and Methods: Pus sample collected from 147 patients admitted to the burn unit, of government general hospital Karimnagar were processed by standard bacteriological methods. Imipenem-resistant isolates were screened for MBL production by combined disk test.
Results: Study revealed that 40% Proteus and 19.05% Pseudomonas produce MBL and MBL producers are 100% resistant to imipenem and ceftazidime.
Conclusion: Patients of burn with MBL-positive P. aeruginosa infections will have higher morbidity and mortality. Piperacillin/tazobactam seems to be effective options for the treatment of MBL-positive P. aeruginosa infections.

Keywords: Antimicrobial resistance, burn wound infection, combined disk test, metallo β lactamase

How to cite this article:
Rao SD, Kumar E A. Antimicrobial resistance and metallo β lactamase in gram-negative isolates of hospital-acquired burn wound infections. J NTR Univ Health Sci 2013;2:181-5

How to cite this URL:
Rao SD, Kumar E A. Antimicrobial resistance and metallo β lactamase in gram-negative isolates of hospital-acquired burn wound infections. J NTR Univ Health Sci [serial online] 2013 [cited 2021 Mar 3];2:181-5. Available from: https://www.jdrntruhs.org/text.asp?2013/2/3/181/117184

  Introduction Top

Infection is an important cause of mortality in burns. It has been estimated that 75% of all deaths following thermal injuries are related to infections rather than osmotic shock and hypovolemia. [1],[2] The burn eschar, moist wound environment, and depression of immune response proportional to the extent of injury favor colonization and proliferation of microorganisms. [3] Immunosuppression, large cutaneous bacterial load, possibility of gastrointestinal bacterial translocation, devitalisation of tissue, prolonged hospitalization, and cross infection due to overcrowding in burn wards contribute to increase incidence of infections in burn patients. [4] Pseudomonas aeruginosa is one of the most important cause of serious infection in burn patients. [5] In addition, the problem of multidrug resistance in ram-negative bacilli due to extended spectrum b lactamases and metallo b lactamases (MBLs) is becoming a serious threat to the patients. The present study has been planned to determine the bacteriological profile and prevalence of gram-negative multidrug-resistant strains producing MBL enzymes in burn wound infection.

  Materials and Methods Top

This prospective study was carried out for a period of 1 year at Prathima Institute of Medical Sciences, Karimnagar after approval by the institutional ethical committee. Oral informed consent was obtained from patients or their relatives. One hundred and forty-seven samples were collected from randomly selected patients admitted in Burns Unit, Civil Hospital Karimnagar (AP) India. Wound swabs were collected in duplicate under aseptic conditions. All samples were processed and identified according to standard microbiological procedures. Antibiotic susceptibility of all isolates was performed by modified Kirby-Bauer disc diffusion method on Muller-Hinton agar plates with commercially available antibiotic discs (Hi-media, Mumbai, India). [6],[7] Staphylococcus aureus ATCC 25923,  Escherichia More Details coli ATCC 25922, and P. aeruginosa ATCC 27853 were used as control strains of Kirby-Bauer disc diffusion method.

The results were recorded and interpreted as per Clinical Laboratory Standard Institute recommendations. [8] Isolates were considered as carbapenem-resistant when the zone size around imipenem disc was ≤13 mm. MBL-producing organism was suspected when the isolate was resistant to imipenem.

MBL screening

Screening and confirmation for the MBL production was done in imipenem-resistant isolates by the following method. [9]

Combined disc test: 0.5-M ethylenediaminetetraacetic acid EDTA) solution was prepared by dissolving 186.1 g of disodium EDTA 2H 2 O in 1000 mL of distilled water. Its pH was adjusted to 8.0 by using NaOH. The mixture was sterilized by autoclaving. 0.5 McFarland standard adjusted suspension of the test organism was prepared and lawn culture of the same was made on freshly prepared Mueller-Hinton agar plate. Two imipenem (10 μg) discs and two ceftazidime (30 μg) discs were placed 25 mm apart on the inoculated plate. One imipenem and one ceftazidime disc was enriched with EDTA by pouring 5 μL of EDTA (750 μg) solution. After 16-18 h of incubation at 35°C, the zones of inhibition around imipenem disc and ceftazidime disc were compared with zone of inhibition around their respective EDTA-enriched discs. An increase in the zone size of at least 7 mm around the imipenem-EDTA disc or ceftazidime-EDTA discs compared to imipenem or ceftazidime disc was recorded as an MBL-positive strain. P. aeruginosa ATCC 27853 was used as a negative control.

  Results Top

In this study of the 147 burn cases, 81 (55%) were males and 66 (45%) were females. A total of 90% burns were accidental and 10% were suicidal. Total body surface area involved in burn was 21%-40% in 40% of patients, 0%-20% in 28% of the patients, 41%-60% in 18% of the patients. A total of 10% of the patients had 61%-80% burn injuries and only 4% patients had burns more than 80%. A total of 80% burns were 3 rd degree, 18% were of 2 nd degree, and 2% were of 1 st degree. In 1 st week, 53 (36%) patients had wound infection and 94 (64%) were noninfected. Of the infected, 40 (75.4%) had monomicrobial and 13 (24.6%) had mixed infection and S. aureus was commonest isolate. A total of 107 cases of burn continued to stay in the hospital for 2 nd week, 22 (20.56%) had monomicrobial and 61 (79.44%) had polymicrobial infection and P. aeruginosa (55.26%) was commonest isolate [Table 1]. The resistance pattern of 114 gram-negative isolates were as shown in [Table 2]. Of the 36 Imipenem resistant gram negative isolates. Eighteen (15.78%) gram-negative isolates were MBL producers. By imipenem + EDTA combined disc test, only 15 (13.15%) of the total isolates were MBL positive, whereas ceftazidime + EDTA combined disc test detected 18 (15.78%) of the total isolates [Figure 1].
Figure 1: MBL detection

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Table 1: Bacterial Infection of Burn Wound in 1st And 2nd Weeks

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Table 2: Antibiotic Resistance Pattern of Gram-Negative Isolates

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MBL production rate was highest in Proteus mirabilis (40%), followed by P. aeruginosa (19.05%). Remaining isolates were negative for M. Of the 24 imipenem-resistant P. aeruginosa 12 (50%) were MBL positive and all 6 imipenem-resistant P. mirabilis (100%) were MBL positive [Table 3]. For the MBL-positive isolates piperacillin/tazobactam and netilin were the most effective drugs (50% sensitivity). In case of MBL-negative isolates, imipenem, ceftazidime, and tetracycline were most effective [Table 4]. Out of 54 isolates of P. aeruginosa from burn word (hospital strains), two strains were of MBL positive and strain of Proteus was MBL producer from 30 P. mirabilis isolates.
Table 3: Distribution of Mbl-Producing Organisms

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Table 4: Comparison of Antibiotic Resistant Pattern of Mbl Positive and Negative Isolates

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  Discussion Top

Infection has always been the predominant determinant of wound healing, incidence of complications, and outcome of burn patients. The effective fluid resuscitation regimens and protection of organ function in burn patients has led to marked decrease in shock as a cause of death and served to accentuate the relative importance of infection. Evaluation and treatment of the bacterial flora in the burn wounds to prevent serious septic complications are challenging clinical problems. Burn injuries are more common in productive period of life (16-30 years), when people are more exposed to the hazards of fire, both at home and outside. The slight male preponderance in our study is due to the occupational accidental burns in farmers, goldsmiths, and in laborers of thermal power stations. Burns in females occur as a result of an open flame licking the loose garments (sari) of those working in the kitchen. In the present study, monomicrobial infection (75.47%) was more common in 1 st week than polymicrobial infection (24.53%). Common isolates were S. aureus (50.90%) and Klebsiella pneumoniae (31.81%). Observations were similar in studies at other centres. [2],[10] The predominance of gram-positive bacteria in the early phase switches to gram-negative species 4–10 days after injury. [5]

During the 2 nd week of admission, 26.50% were monomicrobial and 73.5% infection were polymicrobial and gram-negatives (79.17%) predominated significantly over the gram-positives (20.83%). The most common isolate was P. aeruginosa (43.75%) acquired from hospital. P. spp. were most sensitive to imipenem (62%) followed by piperacillin/tazobactam, ceftriaxone (47.62% each). The results of present study were correlating with previous studies to recommend imipenem as the best drug to treat these gram-negative isolates. [11]

The result of the studies for detection of MBL by imipenem + EDTA combined disc test and ceftazidium + EDTA combined disc test are discordant. [12],[13],[14],[15],[16] In our study, ceftazidime + EDTA combined disc test was found sensitive in the screening of MBL than imipenem + EDTA combined disc test. [12],[13],[14] This can be explained by the fact that imipenem-resistant organisms might have other mechanisms for imipenem resistance due to nonmetalloenzymes and intrinsic resistance to imipenem. With such strains, imipenem + EDTA disc will not detect MBL production. MBLwere produced by 15.78% of all gram-negative isolates. A total of 40% of P. mirabilis and 19.05% of P. aeruginosa were resistant to imipenem. All strains of P mirabilis (100%) and 50% of P. aeruginosa resistant to imipenem were MBL positive. [12],[13],[14],[17] Bandekar et al[18] and Arakawa [19] in the similar study detected 50% of P. mirabilis were imipenem-resistant and of that 25% were MBL producer. This is in contrast to Agarwal et al., [13] in which 77.7% of imipenem-resistant P. aeruginosa were MBL positive. Piperacillin/tazobactam and netilin were the most effective drugs to treat the infection with MBL-positive isolates, whereas MBL-positive isolates were completely resistant imipenem and ceftazidime (100% each). [20] The percentage of imipenem resistance was very high in MBL producers (100%) when compared to nonproducers (23.5%). It has been reported in the similar study by Hemalatha et al., [14] in which 16% isolates are resistant to imipenem and MBL positive, while remaining are sensitive to imipenem and are MBL negative.

  Conclusion Top

Patients of burn with MBL-positive P. aeruginosa infections will have higher morbidity and mortality. Early appropriate antimicrobial therapy may be the only modifiable factor able to decrease the mortality. Hospitals with high prevalence of MBL P. aeruginosa should review empirical therapeutic approaches in order to include organisms producing these enzymes. Piperacillin/tazobactam seems to be effective options for the treatment of MBL-positive P. aeruginosa infections. Since novel therapeutics against Gram-negative rods is not a near prospect. The emergence of this resistance mechanism is a real threat for almost all available options and measures to control the spread of MBL mediated resistances are urgently required.

  References Top

1.Mehta M, Dutta P, Gupta V. Bacterial isolates from burn wound infections and their antibiograms: An eight-year study. Indian J Med Microbiol 2004;26:349-51.  Back to cited text no. 1
2.Rajput A, Singh KP, Vijay Kumar, Sexena R, Singh RK. Antibacterial resistance pattern of aerobic bacterial isolates from burn patients in tertiary care hospital. Biomed Res 2008;19:1998-2001.  Back to cited text no. 2
3.Sharma BR, Singh V, Bangar S, Gupta N. Septicemia-the principal killer of burns patients. Am J Infect Dis 2005;17:132-8.  Back to cited text no. 3
4.Bairy I, Shivananda PG. Aerobic bacterial flora of burn wound infection. Indian J Surg 1997;59:215-8.  Back to cited text no. 4
5.Rastegar Lari AR, Alaghehbandan R, Akhlaghi L. Burn wound infections and antimicrobial resistance in Tehran, Iran: An increasing problem. Ann Burns Fire Disasters 2005;18:68-73.  Back to cited text no. 5
6.Collee JG, Miles RS, Watt B. Tests for identification of bacteria in Mackie and McCartney Practical Medical Microbiology. 14 th ed. Edinburgh: Churchill; 1996. p. 131-51.  Back to cited text no. 6
7.Koneman EW, Alen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook of Diagnostic Microbiology. 5 th ed. Philadelphia: Lippincott; 1997  Back to cited text no. 7
8.Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk tests; Approved Standards. 9 th ed., vol. 26., no 1 CLSI Document M2-A9. Wayne; 2006.  Back to cited text no. 8
9.Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-beta-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002;40:3798-801.  Back to cited text no. 9
10.Vinitha CT, Tiwari P, Singh S, Rasania SK, Khokkar A, Talwar R. Pattern and extent of hospital acquired wound infections in burn patients in a Delhi tertiary care hospital. Indian J Prev Soc Med 2011;42:79-81.  Back to cited text no. 10
11.Horieh S, Zohreh K, Parviz O, Mohammad Ali B, Seyed Mohammad BA. Phenotypic Detection of metallo-beta-lactamase producing Pseudomonas aeruginosa strains isolated from burned patients. Iran J Pathol 2008;3:20-4.  Back to cited text no. 11
12.Mendiratta DK, Deotale V, Narang P. Metallo-beta-lactamase producing Pseudomonas aeruginosa in a hospital from rural area. Indian J Med Res 2005;121:701-3.  Back to cited text no. 12
13.Agarwal G, Lodhi RB, Kamalakar UP, Khadse RK, Jalgaonkar SV. Study of metallo-beta-lactamase production in clinical isolates of Pseudomonas aeruginosa. Indian J Med Microbiol 2008;26:349-51.  Back to cited text no. 13
14.Hemalatha V, Sekar U, Kamat V. Detection of metallo betalactamase producing Pseudomonas aeruginosa in hospitalized patients. Indian J Med Res 2005;122:148-52.  Back to cited text no. 14
15.Singh SP, Shariff M, Barua T, Thukral SS Comparitive evaluaton of phenotypic test for identifcaton of metallo beta-lactamase producing clinical isolates of Pseudomonas aeruginosa. Indian J Med Res 2009;129:713-5.  Back to cited text no. 15
16.Manoharan A, Chatterjee S, MathD. SARI Study Group. Detection and characterization of metallo beta lactamases producing Pseudomonas aeruginosa. Indian J Med Microbiol 2010;28:241-4.  Back to cited text no. 16
17.Navneeth 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;116:264-7.  Back to cited text no. 17
18.Bandekar N, Vinodkumar CS, Basavarajappa KG, Prabhakar PJ, Nagaraj P. Beta lactamases mediated resistance amongst gram negative bacilli in Burn infection. Int J Biol Med Res 2011;2:766-70.  Back to cited text no. 18
19.Arakawa Y, Shibata N, Shibayama K, Kurokawa H, Yagi T, Fujiwara H, et al. Convenient test for screening metallo-beta-lactamase-producing gram-negative bacteria by using thiol compounds. J Clin Microbiol 2000;38:40-3.  Back to cited text no. 19
20.Chacko B, Varaiya A, Dedhia B. Imipenem resistant metallo beta lactamse producing Pseudomonas aeruginosa. Indian J Med Microbiol 2008;26:398.  Back to cited text no. 20


  [Figure 1]

  [Table 1], [Table 2], [Table 3], [Table 4]


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