|Year : 2015 | Volume
| Issue : 1 | Page : 60-64
Prevalent resistance mechanisms in isolates from patients with burn wounds
Varsha Gupta1, Ritu Garg1, Manpreet Kaur1, Shivani Garg1, Ashok Kumar Attri2, Jagdish Chander1
1 Department of Microbiology, Government Medical College Hospital, Chandigarh, Punjab, India
2 Department of General Surgery, Government Medical College Hospital, Chandigarh, Punjab, India
|Date of Web Publication||11-Dec-2015|
Dr. Manpreet Kaur
Department of Microbiology, Government Medical College Hospital, Sector 32, Chandigarh, Punjab
Source of Support: None, Conflict of Interest: None
Context: Infections are the major cause of morbidity and mortality in burn patients. The Extended Spectrum Beta Lactamase (ESBL), AmpC Beta Lactamase (AmpC), and Metallo-Beta Lactamase (MBL) are the major mediators of antimicrobial resistance in Gram-negative organisms. Methicillin-resistant Staphylococcus aureus (MRSA) are also implicated in causing serious infections in burn patients. Aim: To find the prevalence of ESBL, AmpC, and MBL mediated resistance among Gram-negative organisms and MRSA in Staphylococcus aureus isolates in pus samples obtained from the burn unit. Materials and Methods: ESBL, Amp C, and MBL production was detected using ceftazidime and ceftazidime-clavulanic acid combination disc test, cefoxitin and cefoxitin/boronic acid disk test, and Imipenem-EDTA disk test, respectively. MRSA were screened using oxacillin disc by disc-diffusion technique. Results: High ESBL rate (37%) was seen among Escherichia coli isolates, whereas Acinetobacter cbc exhibited maximum (25%) MBL activity. Among Klebsiella pneumoniae isolates, 20% isolates were ESBL and AmpC producers, whereas no AmpC expression and no co-existence of these enzymes were seen in Escherichia coli. Co-existence of AmpC with MBLs was seen in 9.6% of Pseudomonas aeruginosa isolates while ESBL expression alone was seen in 16.1% of isolates. Conclusions: Drug resistance to antimicrobial agents is a serious threat in burn infection. Early detection of these β-lactamase producing isolates in a diagnostic laboratory could help to avoid treatment failure, as often the isolates producing this enzyme show a susceptible phenotype in routine susceptibility testing.
Keywords: Amp C, burns, ESBL, MBL, MRSA
|How to cite this article:|
Gupta V, Garg R, Kaur M, Garg S, Attri AK, Chander J. Prevalent resistance mechanisms in isolates from patients with burn wounds. Indian J Burns 2015;23:60-4
|How to cite this URL:|
Gupta V, Garg R, Kaur M, Garg S, Attri AK, Chander J. Prevalent resistance mechanisms in isolates from patients with burn wounds. Indian J Burns [serial online] 2015 [cited 2022 Aug 11];23:60-4. Available from: https://www.ijburns.com/text.asp?2015/23/1/60/171659
| Introduction|| |
In the current scenario of rising resistance to various antimicrobial agents, it becomes challenging for the microbiologists to detect various resistance mechanisms and formulate alternative antibiotic strategies for their judicious use. The Extended Spectrum Beta Lactamase (ESBL), AmpC Beta Lactamase (AmpC), and Metallo-Beta Lactamase (MBL) have gained importance as major mediators of antimicrobial resistance in Gram-negative organisms.  On the other hand, Methicillin resistant Staphylococcus aureus (MRSA) is prevailing as an important nosocomial pathogen. 
Burns are a significant public health problem and devastating form of trauma around the world, leading to high morbidity, long-term disability, and mortality especially in economically developing countries.  As thermal injury destroys the skin barriers that help prevent infection by microbes these patients become susceptible to infection due to loss of this protective barrier and decreased immunity.  Burnt areas are suitable sites which favor multiplication of organisms. The organisms which are usually associated with burn infections are aerobic organisms like Staphylococcus aureus (S aureus), Streptococcus pyogenes, Escherichia More Details coli (E coli), Klebsiella spp., Proteus, etc., anerobic organisms like Bacteroides fragilis, Peptostreptococcus, Propionibacterium spp., Fusobacterium spp., and fungi like Aspergillus spp., Candida spp., and mucormycetes.  Pseudomonas aeruginosa (P aeruginosa) and Acinetobacter calcoaceticus A. baumannii complex (Acinetobacter cbc) are also important nosocomial pathogens that are being increasingly isolated from burn patients. 
Nowadays, majority of the bacteria causing burn infections are multidrug resistant. The major hurdles in treating burn infections are ESBL, AmpC, and MBL producing organisms from the Gram-negative bacterial group. Among Gram-positive organisms, S aureus is a common colonizer resistant to several anti-microbial agents.  ESBLs are enzymes capable of hydrolyzing and inactivating a wide variety of β-lactams, including third-generation cephalosporins, penicillins, and aztreonam. These enzymes are the result of mutations of TEM-1 and TEM-2 and SHV-I. These enzymes are plasmid-mediated and confer resistance to other antimicrobials also.  AmpC β-lactamases are of major concern as they lead to resistance to a variety of β-lactams, β-lactams/β-lactamase inhibitor combinations, and monobactams. Two types of AmpC β-lactamases are plasmid-mediated and chromosomal or inducible AmpC. The transferable AmpC gene products are commonly called plasmid-mediated AmpC β-lactamases. Chromosomal AmpC enzymes are inducible by β-lactam antibiotics such as cefoxitin and imipenem but they are poorly induced by the third or fourth generation cephalosporins. These are seen in organisms such as Citrobacter freundii, Enterobacter cloacae, Morganella morganii, Hafnia alvei, and Serratia marcescens. MBLs are enzymes that lead to high level resistance to all β-lactams including carbapenem except aztreonam. MBL producers also show resistance to aminoglycosides and fluoroquinolones further limiting the therapeutic options.  MRSA is an antibiotic-resistant pathogen that causes serious infections and is one of the major cause of morbidity and mortality associated with hospitalized patients. 
The routine susceptibility tests fail to detect these resistance mechanisms; this, may mislead the treatment.  So the need of the hour is to know the prevalence of these resistant strains in a burn unit so as to formulate a policy of empirical therapy in high risk units where infections due to resistant organisms are much higher. It will help to avoid misuse of extended spectrum cephalosporins and carbapenems which are the mainstay of treatment in hospitalized patients.
The aim of the present study is to find the prevalence of ESBL-, AmpC-, and MBL-mediated resistance among Gram-negative organisms and MRSA in S aureus isolates in clinical samples obtained from the burn unit of our hospital.
| Materials and Methods|| |
This study was conducted in the Department of Microbiology, Government Medical College Hospital, Chandigarh on the pus specimens received from the burn unit over a period of 1 year. Ninety non-duplicate isolates of E coli, Klebsiella pneumonia (K pneumoniae), P aeruginosa, Acinetobacter cbc, Citrobacter, and S aureus were included in the study. The identification of the isolates was done by standard biochemical methods.  Antibiotic susceptibility testing was done using ceftazidime, cefotaxime, cefepime, cefoxitin, amikacin, gentamicin, ciprofloxacin, imipenem, amoxicillin-clavulanic acid, piperacillin-tazobactam, ampicillin-sulbactam, tobramycin, aztreonam for Gram-negative organisms. ESBL screening was done using ceftazidime, cefotaxime and cefipime. Cefoxitin helped in AmpC screening and imipenem was used for MBLs. Antibiotics used for S aureus were penicillin, oxacillin, cotrimoxazole, chloramphenicol, erythromycin, clindamycin, ciprofloxacin, doxycycline, gentamicin, linezolid, teicoplanin, and vancomycin. The sensitivity pattern of these isolates to various antibiotics was studied by Kirby-Bauer disk diffusion method according to Clinical Laboratory Standards Institute (CLSI) guidelines. 
Detection of ESBLs
ESBL production was detected by phenotypic confirmatory test given by CLSI using ceftazidime and ceftazidime-clavulanic acid combination disc.  Those strains which were found to be negative for ESBLs were further confirmed to be ESBL non-producers by modifying phenotypic confirmatory test for ESBLs detection using boronic acid (BA). , For preparation of BA solution 120 mg of 3-aminophenyl boronic acid (Sigma) was dissolved in 3 ml of dimethylsulfoxide (DMSO) and 3 ml of distilled water was added to it. Then 20 μl of this solution was dispensed onto each disk containing ceftazidime (CAZ; 30 μg) and ceftazidime/clavulanic acid (CAZ/CA; 30/10 μg) combination discs. The final amount of BA on the discs was 400 μg. The discs were allowed to dry for 60 minutes and used immediately. A lawn culture of the test strain was made on Mueller-Hinton Agar (MHA) and these discs, i.e., ceftazidime-boronic acid (CAZ/BA and ceftazidime-clavulanic acid-boronic acid (CAZ/CA/BA were placed on it like CLSI phenotypic confirmatory method for ESBL detection. The plate was incubated at 37°C overnight. A ≥3 mm increase in the zone diameter of CAZ/CA/BA disk versus CAZ/BA alone was considered positive for ESBL [Figure 1].
|Figure 1: An extended spectrum beta lactamase (ESBL) producing isolate showing increase in zone diameter of ceftazidime-clavulanic acid-boronic acid (CAZ/CA/BA) disk versus ceftazidime-boronic acid (CAZ/BA) alone|
Click here to view
Detection of AmpC
AmpC screening was done using cefoxitin disc. The strains which were found to be cefoxitin-resistant were confirmed by combination disk test using BA (cefoxitin and cefoxitin/boronic acid disc).  Twenty microliter of BA solution (prepared as above) was dispensed onto cefoxitin disks. A lawn culture of the test strain was made on MHA plate according to the CLSI guideline. Disks containing cefoxitin (FOX) and cefoxitin plus boronic acid (FOX/BA were placed on the MHA plate and incubated at 37°C overnight. An increase in the zone size of ≥5 mm for cefoxitin in the presence of BA compared with that of cefoxitin alone was considered as positive result [Figure 2].
|Figure 2: AmpC beta lactamase (AmpC) producer showing an increase in zone size of ≥5 mm for cefoxitin plus boronic acid (FOX/BA) disc as compared to cefoxitin (FOX) disc alone|
Click here to view
Detection of Metallo β-lactamase
MBL production was detected by Imipenem-ethylenediaminetetraacetic acid (EDTA) disk test. Two 10 μg imipenem disks were placed on the plate, and appropriate amounts of 10 μl of 0.5 M EDTA solution were added to one of them to obtain the described concentration (750 μg). The inhibition zones of imipenem and imipenem-EDTA disks were compared after 16-18 hours of incubation in air at 35°C. If the increase in inhibition zone with imipenem and EDTA disk was ≥7 mm than the imipenem disk alone, then it was considered to be an MBL producer  [Figure 3].
|Figure 3: Metallo-beta lactamase (MBL) detection using combination disk test. An MBL producer showing increase in zone diameter in the presence of dipicolinic acid (DPA)|
Click here to view
Combination disk test (CDT) with use of meropenem supplemented with dipicolinic acid (DPA)
CDT detects MBLs with the use of meropenem supplemented with 1000 μg of dipicolinic acid (DPA). MBL producers showed an increase in meropenem zone diameter, i.e., >5 mm in the presence of DPA. 
Screening for MRSA
MRSA were screened using oxacillin (1 mcg) disc by disc-diffusion technique. 
For ESBL: Klebsiella pneumonia ATCC 700603
For MRSA: Staphylococcus aureus ATCC 25923
| Results|| |
A total of 90 isolates were obtained after sample processing. Out of these 90 isolates, 31 (34.4%) were P aeruginosa, 20 (22.2%) were Acinetobacter cbc and K pneumonia, 8 (8.8%) were E coli and S aureus each, and 3 (3.3%) were Citrobacter spp. Twenty-nine (93.5%) out of 31 isolates of P aeruginosa showed cefoxitin resistance but AmpC production alone was seen in none of the isolates. None of the isolates showed co-existence of all the three β-lactamases. All the isolates were multidrug resistant. 
Out of 20 isolates of Acinetobacter cbc, co-existence of MBL with AmpC was seen in three (15%) isolates while two (10%) isolates were both MBL and ESBL producers. Co-expression of all the three enzymes was evident in five (25%) isolates.
Among K pneumoniae isolates, co-expression of ESBL and Amp C was seen in (5%) isolate.
S aureus was the only Gram-positive organism, which was isolated. Out of total eight isolates obtained, four (50%) were found to be MRSA. All the isolates were sensitive to vancomycin [Table 1].
| Discussion|| |
The most common pathogen isolated from burn wounds in our study was P aeruginosa (34.4%), followed by Acinetobacter cbc and K pneumonia (22.2%) each, E coli and S aureus (8.8%) each, and Citrobacter spp. (3.3%). However, in another study, P aeruginosa (30%) was found to be the most common pathogen, followed by S aureus (28%), Klebsiella spp. (16%), Proteus spp. (14%), E coli (6%), and Staphylococcus epidermidis (6%). 
Resistance to β-lactams in P aeruginosa and A baumannii is most commonly associated with production of high levels of naturally produced cephalosporinase (AmpC). , MDR strains may, however, arise due to unrelated mechanisms accumulating sequentially in an organism.  In our study, 93.5% of P aeruginosa showed cefoxitin resistance but AmpC production alone was seen in none of the isolates. MBL was shown by 22.5% of the isolates. In a study by Kumar et al., 32.04% of P aeruginosa isolates showed MBL production.  In a study on burns patient, prevalence of MBL producing P aeruginosa has been reported to be 16-19.5%.  We found that 16.1% of isolates showed ESBL expression and co-existence of ESBL and MBL was observed in 6.4% isolates. However, a very high incidence of ESBL was reported among P aeruginosa isolates (42.31%) in a study by Goel et al. 
We found that 25% isolates of Acinetobacter cbc were MBL producers while in another study, 48.72% isolates were MBL enzyme producing strains.  In this study, none of the isolates showed ESBL and AmpC production alone, whereas in another study, only 2% isolates showed ESBL production.  We found co-expression of all the three enzymes in 25% isolates. The emergence of these resistant strains is a serious threat and this reflect excessive use of carbapenems and at the same time pose a therapeutic challenge to clinicians as well as to the microbiologists.
Our study showed that 20% of isolates of K pneumonia were ESBL and AmpC each, whereas only 37.5% of isolates of E coli were ESBL producers. In a study by Singh et al., 7.6% and 15.3% of isolates of E coli and K pneumonia, respectively, were identified as ESBL producers.  As these micro-organisms are prevalent in the hospital environment, the patients are more at risk of acquiring these enzymes. Such isolates could be responsible for outbreaks of infections in hospitalized patients resulting in higher morbidity and mortality. As the third-generation cephalosporins are predominantly used in several hospitals, resistance to these antibiotics can lead to treatment failures. Hence, detection of isolates expressing ESBLs is very important so as to limit the spread of ESBL-producing isolates. We found that 5% of K pneumonia isolates showed co-expression of ESBL and AmpC, whereas there was no AmpC expression and no co-existence of these enzymes in E coli isolates.
We observed that S aureus was the only Gram-positive organism which was isolated and 50% were found to be MRSA, and all the isolates were sensitive to vancomycin. However, in a study at New Delhi, incidence of infection by MRSA isolates varies between 60-69% and is showing an upward trend in recent years. Also, all the isolates were sensitive to vancomycin.  The high prevalence of MRSA is a point of grave concern as this could be a consequence of the fact that a lot healthcare workers are the carriers of these resistant strains. Also, heavy usage of antibiotics could have built up a selective pressure to enrich such strains in the hospitals.
| Conclusion|| |
Drug resistance to antimicrobial agents is a serious threat in burn infection. Early detection of these β-lactamase-producing isolates in a diagnostic laboratory could help to avoid treatment failure, as often the isolates producing this enzyme show a susceptible phenotype in routine susceptibility testing. Aggressive infection control measures should be applied to limit the emergence and spread of these pathogens.
| References|| |
Bandekar N, Vinodkumar CS, Basavarajappa KG, Prabhakar PJ, Nagaraj P. The beta lactamases mediated resistance amongst the Gram-negative bacilli in burn infections. Int J Biol Res 2011;2:766-70.
Sikka A, Kasana D. Emergence of methicillin resistant Staphylococcus aureus
as an important cause of nosocomial infections in burns-ICU patients in a tertiary care centre. Int J Appl Microbiol Sci 2012;1:1-9.
Qader AR, Muhamad JA. Nosocomial infection in sulaimani burn hospital, Iraq. Ann Burns Fire Disasters 2010;23:177-81.
Wong TH, Tan BJ, Ling ML, Song C. Multi-resistant Acinetobacter baumannii
on a burns unit - clinical risk factors and prognosis. Burns 2002;28:349-57.
Revathi G, Puri J, Jain BK. Bacteriology of burns. Burns 1998;24:347-9.
Chaudhary U, Aggarwal R. Extended spectrum β-lactamases (ESBL) - an emerging threat to clinical therapeutics. Indian J Med Microbiol 2004;22:75-80.
Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type beta lactamases. Antimicrob Agents Chemother 2002;46:1-11.
Walsh TR. The emergence and implications of metallo-beta-lactamases in Gram-negative bacteria. Clin Microbiol Infect 2005;11:2-9.
Colle JG, Miles RS, Watt B. Tests for identification of bacteria. In: Collee JG, editors. Mackie and McCartney Practical Medical Microbiology. 14 th
ed. Edinburgh: Churchill Livingstone; 1996. p. 151-79.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 18 th
Informational Supplement. CLSI document M100-S18. Clinical and Laboratory Standards Institute, Wayne, PA; 2008.
Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; 14 th
Informational Supplement. CLSI document M100-S14. Clinical and Laboratory Standards Institute, Wayne, PA; 2004.
Glupczynski Y, Bogaerts P, Deplano A, Berhin C, Huang TD, Van Eldere J, et al
. Detection and characterization of class A extended-spectrum-beta-lactamase-producing Pseudomonas aeruginosa
isolates in Belgian hospitals. J Antimicrob Chemother 2010;65:866-71.
Song W, Bae IK, Lee YN, Lee CH, Lee SH, Jeong SH. Detection of extended-spectrum beta-lactamases by using boronic acid as an AmpC beta-lactamase inhibitor in clinical isolates of Klebsiella
spp. and Escherichia coli
. J Clin Microbiol 2007;45:1180-4.
Lee W, Jung B, Hong SG, Song W, Jeong SH, Lee K, et al
. Comparison of 3 phenotypic-detection methods for identifying plasmid-mediated AmpC beta-lactamase-producing Escherichia coli
, Klebsiella pneumonia
and Proteus mirabilis
strains. Korean J Lab Med 2009;29:448-54.
Lee K, Lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-beta-lactamase-producing isolates of Pseudomonas
spp. and Acinetobacter
spp. J Clin Microbiol 2003;41:4623-9.
Pasteran F, Veliz O, Faccone D, Guerriero L, Rapoport M, Mendez T, et al
. A simple test for the detection of KPC and metallo-β-lactamase carbapenemase-producing Pseudomonas aeruginosa
isolates with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin. Clin Microbiol Infect 2011;17:1438-41.
Baird D. Staphlococcus: Cluster-forming Gram-positive cocci. In: Collee JG, editor. Mackie and McCartney Practical Medical Microbiology. 14 th
ed. Edinburgh: Churchill Livingstone; 1996. p. 245-61.
Cervera C, Linares L, Bou G, Moreno A. Multidrug-resistant bacterial infection in solid organ transplant recipients. Enferm Infecc Microbiol Clin 2012;30:40-8.
Shaikh D, Zaidi SA, Shaikh K, Shaikh M, Naqvi BS, Shaikh MR, et al
. Incidence and resistance pattern of bacteria associated with burn wound sepsis. Pak J Pharmacol 2004;21:39-47.
Kumar SH, De AS, Baveja SM, Gore MA. Prevalence and risk factors of Metallo β-lactamase producing Pseudomonas aeruginosa
species in burns and surgical wards in a tertiary care hospital. J Lab Physicians 2012;4:39-42.
Khosravi AD, Mihahi F. Detection of metallo-beta-lactamase-producing Pseudomonas aeruginosa
strains isolated from burns patient in Ahwaz, Iran. Diagn Microbiol Infect Dis 2008;60:125-8.
Goel V, Hogade SA, Karadesai SG. Prevalence of Extended spectrum beta lactamases, AmpC beta lactamases and metallo beta lactamases producing Pseudomonas aeruginosa
and Acinetobacter baumannii
in an intensive care unit in a tertiary care hospital. J Sci Society 2013;40:28-31.
Jazani NH, Babazadeh H, Sohrabpour M, Zartoshti M, Rad MG. The prevalence of extended spectrum beta-lactamases in Acinetobacter baumannii
isolates from burn wounds in Iran. Int J Microbiol 2011;9:5580-7.
Singh RE, Veena M, Raghukumar KG, Vishwanath G, Rao PN, Murlimanju BV. ESBL production: Resistance pattern in Escherichia coli
and Klebsiella pneumoniae
, a study by DDST method. Int J Appl Biol Pharm Technol 2011;2:415-22.
[Figure 1], [Figure 2], [Figure 3]
|This article has been cited by|
||Microbiological Profile of Infections in a Tertiary Care Burns Unit
| ||Isabella Princess,Ebenezer R |
| ||Indian Journal of Critical Care Medicine. 2019; 23(9): 405 |
|[Pubmed] | [DOI]|
||Molecular characterisation of antimicrobial resistance in Pseudomonas aeruginosa and Acinetobacter baumannii during 2014 and 2015 collected across India
| ||AK Pragasam,S Vijayakumar,YD Bakthavatchalam,A Kapil,BK Das,P Ray,V Gautam,S Sistla,SC Parija,K Walia,VC Ohri,S Anandan,B Veeraraghavan |
| ||Indian Journal of Medical Microbiology. 2016; 34(4): 433 |
|[Pubmed] | [DOI]|