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ORIGINAL ARTICLE
Year : 2015  |  Volume : 23  |  Issue : 1  |  Page : 60-64

Prevalent resistance mechanisms in isolates from patients with burn wounds


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 Publication11-Dec-2015

Correspondence Address:
Dr. Manpreet Kaur
Department of Microbiology, Government Medical College Hospital, Sector 32, Chandigarh, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0971-653X.171659

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  Abstract 

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 Top


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. [1] On the other hand, Methicillin resistant Staphylococcus aureus (MRSA) is prevailing as an important nosocomial pathogen. [2]

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. [3] 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. [4] 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. [5] Pseudomonas aeruginosa (P aeruginosa) and  Acinetobacter calcoaceticus Scientific Name Search  A. baumannii complex (Acinetobacter cbc) are also important nosocomial pathogens that are being increasingly isolated from burn patients. [1]

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. [2] 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. [6] 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.[7] 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. [8] 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. [2]

The routine susceptibility tests fail to detect these resistance mechanisms; this, may mislead the treatment. [5] 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 Top


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. [9] 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. [10]

Detection of ESBLs

ESBL production was detected by phenotypic confirmatory test given by CLSI using ceftazidime and ceftazidime-clavulanic acid combination disc. [11] 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). [12],[13] 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


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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). [14] 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


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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 [15] [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)


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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. [16]

Screening for MRSA

MRSA were screened using oxacillin (1 mcg) disc by disc-diffusion technique. [17]

Controls

For ESBL: Klebsiella pneumonia ATCC 700603

For MRSA: Staphylococcus aureus ATCC 25923


  Results Top


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. [18]

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].
Table 1: Different b-lactamases among isolates obtained (n = 90)


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  Discussion Top


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%). [19]

Resistance to β-lactams in P aeruginosa and A baumannii is most commonly associated with production of high levels of naturally produced cephalosporinase (AmpC). [4],[12] MDR strains may, however, arise due to unrelated mechanisms accumulating sequentially in an organism. [6] 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. [20] In a study on burns patient, prevalence of MBL producing P aeruginosa has been reported to be 16-19.5%. [21] 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. [22]

We found that 25% isolates of Acinetobacter cbc were MBL producers while in another study, 48.72% isolates were MBL enzyme producing strains. [22] In this study, none of the isolates showed ESBL and AmpC production alone, whereas in another study, only 2% isolates showed ESBL production. [23] 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. [24] 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. [2] 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 Top


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 Top

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