burns 41 (2015) 812–819
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The analysis of distribution of multidrug resistant Pseudomonas and Bacillus species from burn patients and burn ward environment Manju Panghal a, Khushboo Singh a, Sangeeta Kadyan a, Uma Chaudary b, J.P. Yadav a,* a b
Department of Genetics, M. D. University, Rohtak, 124001, Haryana, India Department of Microbiology, Pt. B.D. Sharma University of Health Sciences, Rohtak 124001, Haryana, India
article info
abstract
Article history:
Introduction: Infections caused by multidrug resistant bacteria act as a risk factor for
Accepted 9 October 2014
mortality in burns patients. So keeping in view the crucial importance of reliable therapeutic decisions of multidrug resistance bacteria and role of hospital environment in bacteria
Keywords:
colonization, our study is based on the evaluation of distribution of Pseudomonas sp. and
Multidrug resistant bacteria
Bacillus sp. in burn patients and burn ward environment.
B. cereus
Methods: The present prospective analysis was conducted on the patients undergoing
P. aeruginosa
treatment in the Burn ward of Pt. B.D. Sharma University of Health Sciences, Rohtak,
Burn patients
Haryana, during the period of January 2012 to March 2013. The multidrug resistant bacteria
Burn ward environment
were characterized by following the CLSI guidelines. Molecular identification isolates were done by amplifying and sequencing 16S rDNA. Results: In our study out of 510 samples of 280 burn patients, 263 samples were observed sterile and bacterial isolates were obtained from 247 samples. In burn patients out of 247 samples 43 MDR strains, and in burn ward out of 60 samples 4 MDR strain were observed. After 16S rDNA amplification of MDR isolates the prevalent bacterium was belonged to the genus Bacillus (8 species; 26 isolates) followed by genus Pseudomonas (5 species; 17 isolates). The burn ward environment isolates were Pseudomonas aeruginosa, Pseudomonas stutzeri, Bacillus cereus and Acinetobacter baumanii. Conclusion: The major finding of our study is the predominance of B. cereus followed by P. aeruginosa in burn patients of Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana. While considering the role of hospital environment, no direct role of environmental isolates was observed in transfer of bacterial infection. # 2014 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Burn is one of the most common incidents that create different types of wound infections and act as an important * Corresponding author. Tel.: +91 9416474640. E-mail address: [email protected] (J.P. Yadav). http://dx.doi.org/10.1016/j.burns.2014.10.014 0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.
cause of death in patients with burns. In case of severe burns, wound infection becomes critical due to the destruction of skin. As skin act as the first barrier in front of foreign organisms and existence of necrosis, which provides a suitable environment for microbial growth and invasion [1].
burns 41 (2015) 812–819
In addition with that burn patients are relatively immunosuppressed caused by the impaired functioning of neutrophils, the cellular and humoral immune system and are at high risk of infections, in particular with nosocomial-acquired multidrug-resistant (MDR) organisms [2,3]. Multi-drug resistant organisms also increase death rates of patients with burnrelated sepsis from 42% to 86%. Hospitalized patients in burn care wards are specially at higher risk for hospital-associated infections due to the above mentioned reasons. Organisms associated with nosocomial infections in burn patients include organisms found in the patient’s own endogenous (normal) flora or from exogenous sources in the hospital environment [4]. The main exogenous sources that may be transferred to a patient’s skin surface via contact with contaminated external environmental surfaces, water, fomites, air, and the soiled hands of health care workers [5]. Numerous reports have demonstrated that hospital environment surfaces are a source of antibiotic-resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter sp., Klebsiella pneumonia, Clostridium difficile, and methicillin-resistant Staphylococcus aureus (MRSA). The contamination of wound surfaces by these agents occurs more frequently in developing countries [6,7]. Side by side these drug-resistant bacteria are easily transferred from one patient to another because overcrowding in burn units is another important factor for cross-infection [8]. The organisms that predominate as causative agents of burn wound infection in any burn treatment facility change over time. Gram-positive organisms (methicillin-resistant S. aureus), are initially prevalent, then gradually become predominate by Gram-negative opportunists (P. aeruginosa, A. baumannii, Enterobacter sp) that appear to have a greater propensity to invade [9,10]. Out of all MDR bacteria, P. aerugnosa (an opportunistic human pathogen) isolated from burn patients proved as most significant bacteria because this bacterium showed best growth on the moist surface of burn wounds and is highly pathogenic in immunocompromised patients [11]. Incidentally P. aeruginosa infection is awfully problematic since this organism is inherently resistant to many drug classes and is able to acquire resistance to all effective antimicrobial drugs that make infected burn wounds difficult to treat [12]. Parallel to Pseudomonas sp., our study also highlighted Bacillus sp. which has also emerged as one of the new Gram-positive pathogens to cause serious infection in immunocompromised patients. Members of the genus Bacillus are aerobic or facultative anaerobic Gram-positive or Gramvariable, spore-forming rods that are ubiquitous in the hospital environment; they may be part of the normal flora, particularly in patients hospitalized for prolonged periods [13]. Bacillus causes number of systemic and local infections, including fulminant bacteraemia, central nervous system involvement, catheter related blood stream infection and pneumonia and severe burn wounds [14,15]. The accurate bacterial identification is important in analyzing the epidemiology, antibiotic resistance patterns and outcomes of infections. Traditionally, identification of bacteria in clinical microbiology laboratories was performed using phenotypic tests, including Gram smear and biochemical tests. However, these methods of bacterial identification have major limitations because the organisms with
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biochemical characteristics that do not fit into the patterns of any known genus and species are difficult to be identified. Second, they cannot be used for uncultivable organisms [16]. Using 16S rDNA sequencing, these problems can be overcome by a single technology, which also facilitates the discovery of novel genera and species. Among the three domains of life, the largest amount of rDNA gene sequencing work concerns bacteria. Using 16S rDNA sequences, numerous bacterial genera and species have been reclassified and renamed; classification of uncultivable bacteria has been made possible [17]. Because of above mentioned reasons for characterization of Pseudomonas sp. and Bacillus sp. we took advantage of 16S ribosomal DNA (rDNA) sequence data to identify genus- and species- specific 16S rDNA signature sequences of both bacteria’s. So in the nutshell, keeping in view the role of Pseudomonas sp. and Bacillus sp. (MDR) in immunocompromised burn patients and hospital environment the aim of our study is to evaluate the distribution of multidrug resistant Pseudomonas sp. and Bacillus sp. in burn patients and burn ward by using 16S rRNA- PCR sequencing tools. In this study an attempt was made to track the path of infection in burn patients i.e. whether the bacterial infection in burn cases come through hospital environment or through cross infection. Because by understanding the routes of colonization one can develop the effective preventive measures against infection.
2.
Material and methods
2.1.
Study design
The present prospective analysis was conducted on the patients undergoing treatment in the Burn ward of Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana, during the period of January 2012–March 2013. Bacterial isolates were obtained from the various samples received in Microbiology department of Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana. The sources of the bacterial isolates were urine, pus, wounds, blood, and body fluids of burn cases. The purity of isolates was determined by macroscopic examination of colonies and microscopic examination of bacteria after Gram staining, and identity of each isolate was confirmed in laboratory by using standard microbiological and other biochemical methods. Bacterial isolates were also grown on selective media like cetrimide agar, Bacillus cereus agar base etc. [18,19]. Different isolates were kept in 10% glycerol and frozen at 40 8C for further study. For burn ward environmental sampling various environmental samples (tap water/sink swab/detergents/soap swab/floor/tables/air/hands of nurses and other damp surfaces) were taken once in a week from the various places of burn ward. Environmental samples were taken by using standard protocols like for air sampling we have used sedimentation or depositional method by placing Nutrient agar plates (Simple and inexpensive; best suited for qualitative sampling). Nutrient agar plates were placed in different regions of burn ward keeping in view the temperature, place and humidity [20]. The Inclusion criterion of our study was about 108 CFU/ml of bacteria cells were considered as
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burns 41 (2015) 812–819
version 5.1 (MEGA 5.1) for construction of Phylogenetic tree by using Neighbor–Joining method and with boot strap values up to 500 [25].
pathogenic. The patients were excluded from the study if they had clinical or microbiological evidence of infection of unknown origin. Common skin isolates, including Coryneforms excluded from analysis.
2.2.
3.
Antimicrobial susceptibility testing
The antimicrobial susceptibility of all the isolates was performed by estimating minimum inhibitory concentration (MIC50 and MIC90) and disc diffusion method. Values for MICs at which 50% of isolates were inhibited MIC50 and MIC90 were determined by the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [21]. The definition of MDR strains in our study was taken as non-susceptibility to at least one agent in three or more antimicrobial categories [22]. The antimicrobial sensitivity was checked with nine antibiotics Netilimicin, NET; Amikacin, AMK; Gentamicin, GEN, Pipiercilline, PIP; Azlocillin, AZL, Ceftizoxime, ZOX, Cefepime, FEP, Ofloxine, OFX and Impienem, IPM.
2.3.
Results
Out of 280 cases of burn patients, MDR strains were isolated from 43 patients (30 females with age of 19–47; 26 males with age of 18–65). The hospital stays of these burn patients vary from 10 to 25 days. The main cause of burn in the hospitalized patients was accidental fire due to kerosene oil/gas stoves/gas cylinder. Out of total 510 samples of burn patients, 263 samples were sterile and bacterial isolates were obtained from 247 samples. Out of 247 isolates, 37 isolates were isolated twice from same patients during patients hospital stay. The main source of bacteria in burn patients was the pus collected from burn wounds. In case of environmental samples out of 680 samples, bacteria were isolated from 60 and rest of the samples were sterile. The main source of bacterial contamination in burn ward environment was sink and air. To get more environmental samples we have further taken environmental samples from March 2013 to January 2014 but all samples were sterile. The reason may be that hospital authority has taken preventive measures to control environmental contamination in burn ward after our study results.
16S rRNA characterization of MDR isolates
DNA of all MDR strains was prepared by using the Bangalore Genei DNA isolation kit according to the instructions of the manufacturer. Eluted DNA was stored at 20 8C until further use. For molecular identification 16S rDNA sequencing was used. Gene coding for 16S rDNA was amplified by using universal primers, most common primer pair referred as 27F and 1492R [23]. PCR amplified rDNA genes were then purified with Quick PCR purification kit (Bangalore GENEI, India). The purified products were sequenced by using the facility of Eurofins Genomics India Pvt. Ltd., India. After gene sequencing matching of sequences was done by using the basic local alignment search tool (BLAST) to characterize the MDR bacterial isolates. For molecular phylogenetic analyses, the required reference sequences for comparison were downloaded from the NCBI gene bank database using the website http://www.ncbi.nlm.nih.gov/Genbank. All of the sequences of 16S rDNA genes were aligned with reference sequences using the multiple sequence alignment programs CLUSTAL X 2.0 [24]. Later on alignment file was used in Molecular Evolutionary Genetic Analysis software
3.1.
Antibacterial assay for MDR characterization
About 43 isolates of burn cases and four from burn ward enviornmet isolates were characterized as MDR on the basis of resistance against various antibiotics. The detail of antibacterial activity of antibiotics against isolates, depicting zone of inhibition, MIC50 and MIC90 values have been shown in Table 1. The %resistance of different drugs against 247 isolates has been observed ranged from 14 to 65% and %sensitivity ranged from 34 to 85%. But the %resistance in burn ward isolates was found ranged from 18 to 45% and %sensitivity ranged from 55 to 78%. Both %resistance and %sensitivity of all antibiotics have been shown in Fig. 1. The highest resistance was shown by ZOX (65%) followed by GEN (62%) against burn patients isolates but in burn ward isolates the most resistant drug observed was ZOX (45%) followed by FEM (31%) respectively.
Table 1 – The detail antibacterial activity of antibiotics against multidrug resistant isolates depicting zone of inhibition, MIC50 and MIC90 values. Antimicrobial agents
Disk content
AMK AZL FEM GEN IPM OFX PIP NET ZOX
30 mg 30 mg 30 mg 10 mg 10 mg 5 mg 10 mg 30 mg 30 mg
MDR isolates
Zone of diameter (mm) of bacterial isolates R
I
S
14 15 14 12 13 12 17 12 14
15–16 16–21 15–17 13–14 14–15 13–14 14–16 13–14 15–19
17 22 18 17 16 16 18 15 20
Range (mg/ml) 32–256 16 512 8 32 0.25–32 32 512 32 128 128 512 32 128 8 32
MIC50 (mg/ml)
MIC90 (mg/ml)
128 >128 >32 16 256 >128 >512 >128 >32
256 >128 >32 16 512 >128 >512 >128 >32
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burns 41 (2015) 812–819
Fig. 1 – Antibacterial activity i.e. sensitivity and resistance (%) of antibiotics against bacterial isolates of Burn patients and Burn Ward environment.
Table 2 – Taxonomic details of Burn patients and Burn ward isolates along with the accession numbers as identified on the basis of 16S rDNA gene partial sequence. Isolate code B. Env. 2; B. Pat. 1; B. Pat. 4; B. Pat. 7; B. Pat. 14; B. Pat. 8; B. Pat. 18; B. Pat. 12 B. Env. 1; B. Pat. 2; B. Pat. 3; B. Pat. 6 B. Pat. 9 B. Pat. 13 B. Pat. 15; B. Pat. 16 B. Pat. 17 B. Pat. 10 B. Pat. 11 B. Pat. 25 B. Pat. 39; B. Pat. 40; B. Pat. 49; B. Pat. 46 B. Pat. 47 B. Pat. 48 B. Pat. 52 B. Pat. 30 B. Pat. 33 B. Env. 3 B. Pat. 18 B. Pat. 20 B. Pat. 23; B. Pat. 26 B. Pat. 56 B. Pat. 21; B. Pat. 29 B. Pat. 22; B. Pat. 38; B. Pat. 24 B. Pat. 28 B. Pat. 55 B. Pat. 54 B. Pat. 41 B. Pat. 45 B. Pat. 51 B. Env. 4
Accession no. of isolates KF862889 KF862888 KF951532 KF862896; KF862897; KF862901 KF862890; KF862892; KF862893 KF862898 KF862902; KF862906 KF862899 KF862900 KF862914 KF862925; KF862935 KF862932 KF862933 KF862934 KF862938 KF862919 KF862921 KF862894 KF862908 KF862909 KF862912; KF862942 KF862910; KF862911; KF862913 KF862917 KF862941 KF862940 KF862927 KF862931 KF862937 KF862895
KF862903 KF862907 KF862889; KF862893
KF862905
KF862926;
KF862915 KF862918 KF862924;
Bacterial strain names showing % similarity with isolates P. P. P. P. P. P. P.
aeruginosa strain FN826303 (100%) aeruginosa strain JQ6599661 (99%) aeruginosa strain HO8090971 (100%) aeruginosa strain GU451298 (100%) aeruginosa strain CP006832 (99%) aeruginosa strain KF583972 (100%) stutzeri strain KF607058 (100%)
No. of isolates 1 1 1 2 2 1 4
P. stutzeri strain HM209781 (99%) P. stutzeri strain KF4539531 (100%) P. stutzeri strain KF5766511 (100%) P. mendocina strain KC895888 (100%) Pseudomonas bacterium strain HE583117 (99%) Pseudomaceae strain KF560362 (99%) B. cereus strain KC876035 (100%) B. cereus strain KF054992 (99%) B. cereus strain JX050218 (99%) B. cereus strain JX088534 (99%) B. cereus strain JX088538 (99%) B. cereus strain JN411376 (100%) B. cereus strain JQ284035 (99%) B. cereus strain KF612021 (99%) B. cereus strain JQ818396 (96%)
1 1 2 1 1 1 1 3 1 1 1 1 1 1 1
B. B. B. B. B. B.
1 1 2 1 2 3
subtilis strain JN641293 (99%) subtilis strain JQ653049 (99%) subtilis strain KF582786 (99%) subtilis strain JX286682 (99%) anthracis strain KF017366 (99%) anthracis strain GU939625 (99%)
B. tequilensia KF436736 (99%) Bacillus safensis HQ22459 (99%) Bacillus pumilius EU780103(99%) Bacillus thuringensis JX860329 (99%) Bacillus stratosphercium HM0327861 (99%) Bacterium NLAE JX006771 (99%) A. baumanii KF060283 (99%)
1 1 1 1 1 1 1
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burns 41 (2015) 812–819
Fig. 2 – Phylogenetic tree (based on neighboring–joining method with boot strap values on node) of the bacterial isolates from Burn cases and Burn Ward Environment. Number in parentheses are Gene Bank accession Numbers for 16S rDNA nucleotide sequences of isolates.
burns 41 (2015) 812–819
In case of environmental samples only four multidrug resistance isolates were observed out of 60 isolates. So, 22% MDR bacterial isolates were isolated from burn cases and 6% from burn ward. No repeatability was observed in MDR strains of burn patients and burn ward environment.
3.2. 16S rDNA gene partial sequencing of characterized MDR isolates The details of bacterial isolate code, their accession number provided by NCBI and bacterial name showing % similarity with our isolates has been shown in Table 2. The taxonomic information obtained by molecular characterization on the basis of 16S rDNA gene partial sequence has shown that all of the isolates have shown 97–100% of similarity with the NCBI strains. Out of all MDR isolates maximum number were belonged to the genus Bacillus (eight species and 26 isolates) followed by genus Pseudomonas (five species and 17 isolates). Some bacterial strains isolated from burn patient (B. Pat. 27, B. Pat. 31) and one burn ward isolates i.e. B. Env. 2 have shown 16S rDNA similarities up to 96–97% probably may be new bacterial species. So we found that in case of burn patients, B. cereus species has showed predominant diversity having 11 numbers of isolates followed by P. aeruginosa and P. stutzeri having seven isolates. Another important finding in our study was the presence of five strains (B. Pat. 21; B. Pat. 29; B. Pat. 22; B. Pat. 38; B. Pat. 24) of Bacillus anthracis. In case of burn ward MDR isolates were belong to P. aeruginosa (B. Env. 1), P. stutzeri (B. Env. 2), B. cereus (B. Env. 3) and Acinetobacter baumanii (B. Env. 4). Phylogenetic analysis based on 16S rDNA gene sequence was determined. Phylogenetic relationship of the burn patients isolates and burn ward isolates was calculated by the multiple alignments of the 16S rDNA gene sequences has been diagrammed in Fig. 2.
4.
Discussion
In recent decades, bacteria isolated from burn patients become resistant to antibiotics. In high-infection rate units, periodic monitoring of the microbial species and their respective susceptibility to antibiotics is important in selection of effective empiric antimicrobial therapy against bacteria isolated from the burn patients [26]. In the present study, we have carried out a prospective study of the antibiotic resistance profiling and molecular characterization by 16S rDNA sequencing of MDR isolates of burn cases and burns unit environment. In our study we have isolated 43 MDR bacteria’s from burn patients and four from the burn ward. While checking antibiotic sensitivity of isolates we found that most resistance antimicrobial class was aminoglycosides (Gentamicin, Amikacin) in both burn patients and burn ward disparately, imipenem was effective drug. Similar reports of aminoglycosides resistant were also elucidated by other studies done on burn patients in different parts of India [10,27]. This could be due to the reason that imipenem is reserve drug and has been used as last options drug against resistant bacteria for burn patients. While inspecting distribution of P. aeruginosa and Bacillus sp., the noteworthy finding of our study was predominance of
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B. cereus with 11 isolates followed by P. aeruginosa with seven isolates in burn patients. It is a well known fact that members of the genus Bacillus are spore forming that are ubiquitous in the hospital environment. This bacterial genus is part of the normal flora and nowadays this genus has been getting appreciation for its potential as an opportunistic pathogen in immunocompromised hospitalized patients [13]. The hypothesis that B. cereus spores present in air also been justified by our study because we have isolated one MDR B. cereus in our burn ward air sample. But due to presence of only one air MDR isolate we cannot predict air as sole causative agent of B. cereus contamination in burn patients. The cross contamination between the patients may be the main factor of B. cereus infection. The outbreak caused by B. cereus in immunosuppressed hospitalized patients has been also studied by numerous studies conducted on cancer patients, acute leukemia cases, infants, newborn babies, and ICU units [28,29]. The presence of four strains of Bacillus thuringiensis was observed in causing infection in burn wound and also from water used in the treatment of burn wounds [15]. The role of non sterile gloves as contaminate in B. cereus infection in burn unit was also observed in one study [30]. To the best of our knowledge our study is the first study which is evaluating distribution of Bacillus sp. in burn patients and burn ward environment. In the present our study when we discuss the distribution of P. aeruginosa than it was observed as second prevalent MDR isolates. In hospital ward this bacterium was isolated from sink. The presence of P. aeruginosa in burn patients is a well known fact and indeed also proved by other studies [31,32]. The main reason behind remarkably high prevalence of this bacterium in burn cases and burn unit may be that the devitalised tissue and moist environment of burns provide an ideal environment for colonization and infection with P. aeruginosa [33]. In our study both MDR species of Pseudomonas (P. aeruginosa and P. stutzeri) were isolated from sink, on this basis we may say that sink may be the main source of Pseudomonas infection in burn patients. The presence of P. aeruginosa in sink may be due to nutritional requirements and its capability to thrives in moist environments (such as drains) that allow it to grow in macrocolonies within plumbing systems [34]. But owing to the fact that there were only two MDR Pseudomonas sp. in burn ward we cannot predict the sole role of burn ward environment in contamination of Pseudomonas sp. in burn patients again like Bacillus sp. The other reasons of Pseudomonas infection in burn patients may be there due to cross contamination, due to other exogenous or endogenous sources. The contamination of P. aeruginosa in burn cases via cross infection has been also proved by numerous studies conducted in various parts of world [8,35].
5.
Conclusion:
To the best of our knowledge our study is the first study which is based on evaluation of distribution of Bacillus sp. in burn patients and burn ward environment. On the basis of 16S rDNA amplification we conclude that B. cereus was major source of infection in burn patients followed by P. aeruginosa. The prevalence of B. cereus depicts importance for our burn institution to determine its specific pattern of burn microbial
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burns 41 (2015) 812–819
colonization, antimicrobial resistance profiles and timerelated changes in predominant flora. This would allow early management of septic episodes with proper empirical systemic antibiotics before the results of microbiologic cultures become available, thus improving the overall infection-related morbidity and mortality. In conclusion, our results may be useful guidelines for choosing effective antimicrobial therapy in our burn ward against multidrug resistant isolates throughout the patients’ hospital stay.
Conflict of interest We as authors do not have a conflict of interest.
Acknowledgments Dr Manju Panghal is thankful to Department of Science and Technology, New Delhi for providing her financial support under scheme of Women Scientist programme (WOS–A) (Grant no - SR/WOS- A/LS-259/2011(G) & Date- 19/12/2011) to carry out her research work. Authors are also thankful to Department of Science and Technology, New Delhi for the award of DST-FIST grant (SR/FST/LS1-038/2011) to the Department.
references
[1] Mayhall CG. The epidemiology of burn wound infections: then and now. Clin Infect Dis 2003;37:543–50. [2] Macedo JLS, Rosa SC, Castro C. Sepsis in burned patients. Rev Bras Med Trop 2003;36:647–52. [3] Jessie S, Glasser JS, Guymon GH, Mende K, Wolf SE, Hospenthal DR, et al. Activity of topical antimicrobial agents against multidrug-resistant bacteria recovered from burn patients. Burns 2010;36:1172–84. [4] Magnotti LJ, Deitch EA. Burns, bacterial translocation, gut barrier function, and failure. J Burn Care Rehabil 2005;26(5):383–91. [5] Gales AC, Jones RN, Turnidge J, Rennie R, Ramphal R. Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis 2001;32(Suppl. 2):S146–55. [6] Macedo JLS, Santos JB. Nosocomial infections in a Brazilian Burn Unit. Burns 2006;32:477–81. [7] Hota B, Blom DW, Lyle EA, Weinstein RA, Hayden MK. Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product, or procedure. J Hosp Infect 2009;71:123–31. [8] Douglas MW, Mulholland K, Denyer V, Gottlieb T. Multidrug resistant Pseudomonas aeruginosa outbreak in a burns unit-an infection control study. Burns 2001;27:131–5. [9] Altoparlak U, Erol S, Akcay MN, Celebi F, Kadanali A. The time-related changes of antimicrobial resistance patterns and predominant bacterial profiles of burn wounds and body flora of burned patients. Burns 2004;30:660–4. [10] Agnihotri N, Gupta V, Joshi RM. Aerobic bacterial isolates from burn wound infections and their antibiograms: a five year study. Burns 2004;30:241–3.
[11] Williams FN, Herndon DN, Hawkins HK, Lee JO, Cox RA, Kulp GA. The leading causes of death after burn injury in a single pediatric burn center. Crit Care 2009;13:R183. [12] Hsueh PR, Teng LJ, Yang PC, Chen YC, Ho SW, Luh KT. Persistence of a multidrug-resistant Pseudomonas aeruginosa clone in an intensive care burn unit. J Clin Microbiol 1998;36:1347–51. [13] Drobniewsky FA. Bacillus cereus and related species. Clin Microbiol Rev 1993;6:324. [14] Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 2010;23(2):382–98. [15] Damgaard PH1, Granum PE, Bresciani J, Torregrossa MV, Eilenberg J, Valentino L. Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunol Med Microbiol 1997;18(1):47–53. [16] Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen KY. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 2008;14:908–34. [17] Snel B, Bork P, Huynen MA. Genome phylogeny based on gene content. Nat Genet 1999;21:108–10. [18] Hawkey P, Law D. Medical bacteriology. NewYork: Publication Co, Oxford University Press; 2004. [19] Pawar VB, Dutta D. A procedure manual for routine diagnostic test. In: Mukherjee KL, editor. Diagnostic bacteriology. 2nd ed. New Delhi: Publication Co. Tata MacGrew -Hill Publishing Company Limited; 2006. [20] Sehulster LM, Chinn RYW, Arduino MJ, Carpenter J, Donlan R, Ashford D, et al. Guidelines for environmental infection control in health-care facilities. Recommendations from CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). Chicago, IL: American Society for Healthcare Engineering/American Hospital Association; 2004. [21] Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard ninth edition approved standard M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. [22] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drugresistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268–81. [23] Jiang H, Dong H, Zhang G, Yu B, Chapman LR, Fields MW. Microbiology diversity in water and sediments of lake chaka, an Athalassonhaline Lake in Norhwestern China. Appl Environ Microbiol 2006;72(6):3832–45. [24] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam HV, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947–8. [25] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony method. Mol Biol Evol 2011;28:2731–9. [26] Ozumba UC, Jiburum BC. Bacteriology of burn wounds in Enugu, Nigeria. Burns 2000;26:178–80. [27] Singh NP, Goyal R, Manchanda V, Das S, Kaur I, Talwar V. Changing trends in bacteriology of burns in the burns unit: Delhi, India. Burns 2003;29(2):129–32. [28] Hilliard NJ, Schelonka RLRL, Waites KB. Bacillus cereus: bacteremia in a Preterm Neonate. J Clin Microbiol 2003;41(7):3441–4. [29] Ginsburg AS, Salazar LG, True LD, Disis ML. Fatal Bacillus cereus sepsis following resolving neutropenic enterocolitis during the treatment of acute leukemia. Am J Hematol 2003;72:204–8.
burns 41 (2015) 812–819
[30] Jeurissen A, Weyers L, Cossey V, Muller J, Schuermans AJ. Dissemination of Bacillus cereus in the burn unit due to contaminated non-sterile gloves. Hosp Infect 2010;76(1): 92–3. [31] Japoni A, Alborzi A, Kalani M, Nasiri J, Hayati M, Farshad S. Susceptibility patterns and cross-resistance of antibiotics against Pseudomonas aeruginosa isolated from burn patients in the South of Iran. Burns 2006;32:343–7. [32] Zhang JP1, Yang XS, Chen J, Peng YZ, Huang YS. Clinical assessment of colistin in treating infections caused by multidrug-resistant gram-negative bacillus in patients
819
with severe burn. Zhonghua Shao Shang Za Zhi 2009;25(5):372–6. [33] Atoyebi OA, Sowemimo GOA, Odugbemi T. Bacterial flora of burn wounds in Lagos, Nigeria: a prospective study. Burns 1992;18:448–51. [34] Labarca JA, Pegues DA, Wagar EA, Hindler JA, Bruckner DA. Something’s rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa. Clin Infect Dis 1998;26:1440–6. [35] Komolafe OO, James J, Kalongolera L, Makoka M. Bacteriology of burns at the Queen Elizabeth Central Hospital, Blantyre, Malawi. Burns 2003;29:235–8.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/burns
The analysis of distribution of multidrug resistant Pseudomonas and Bacillus species from burn patients and burn ward environment Manju Panghal a, Khushboo Singh a, Sangeeta Kadyan a, Uma Chaudary b, J.P. Yadav a,* a b
Department of Genetics, M. D. University, Rohtak, 124001, Haryana, India Department of Microbiology, Pt. B.D. Sharma University of Health Sciences, Rohtak 124001, Haryana, India
article info
abstract
Article history:
Introduction: Infections caused by multidrug resistant bacteria act as a risk factor for
Accepted 9 October 2014
mortality in burns patients. So keeping in view the crucial importance of reliable therapeutic decisions of multidrug resistance bacteria and role of hospital environment in bacteria
Keywords:
colonization, our study is based on the evaluation of distribution of Pseudomonas sp. and
Multidrug resistant bacteria
Bacillus sp. in burn patients and burn ward environment.
B. cereus
Methods: The present prospective analysis was conducted on the patients undergoing
P. aeruginosa
treatment in the Burn ward of Pt. B.D. Sharma University of Health Sciences, Rohtak,
Burn patients
Haryana, during the period of January 2012 to March 2013. The multidrug resistant bacteria
Burn ward environment
were characterized by following the CLSI guidelines. Molecular identification isolates were done by amplifying and sequencing 16S rDNA. Results: In our study out of 510 samples of 280 burn patients, 263 samples were observed sterile and bacterial isolates were obtained from 247 samples. In burn patients out of 247 samples 43 MDR strains, and in burn ward out of 60 samples 4 MDR strain were observed. After 16S rDNA amplification of MDR isolates the prevalent bacterium was belonged to the genus Bacillus (8 species; 26 isolates) followed by genus Pseudomonas (5 species; 17 isolates). The burn ward environment isolates were Pseudomonas aeruginosa, Pseudomonas stutzeri, Bacillus cereus and Acinetobacter baumanii. Conclusion: The major finding of our study is the predominance of B. cereus followed by P. aeruginosa in burn patients of Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana. While considering the role of hospital environment, no direct role of environmental isolates was observed in transfer of bacterial infection. # 2014 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
Burn is one of the most common incidents that create different types of wound infections and act as an important * Corresponding author. Tel.: +91 9416474640. E-mail address: [email protected] (J.P. Yadav). http://dx.doi.org/10.1016/j.burns.2014.10.014 0305-4179/# 2014 Elsevier Ltd and ISBI. All rights reserved.
cause of death in patients with burns. In case of severe burns, wound infection becomes critical due to the destruction of skin. As skin act as the first barrier in front of foreign organisms and existence of necrosis, which provides a suitable environment for microbial growth and invasion [1].
burns 41 (2015) 812–819
In addition with that burn patients are relatively immunosuppressed caused by the impaired functioning of neutrophils, the cellular and humoral immune system and are at high risk of infections, in particular with nosocomial-acquired multidrug-resistant (MDR) organisms [2,3]. Multi-drug resistant organisms also increase death rates of patients with burnrelated sepsis from 42% to 86%. Hospitalized patients in burn care wards are specially at higher risk for hospital-associated infections due to the above mentioned reasons. Organisms associated with nosocomial infections in burn patients include organisms found in the patient’s own endogenous (normal) flora or from exogenous sources in the hospital environment [4]. The main exogenous sources that may be transferred to a patient’s skin surface via contact with contaminated external environmental surfaces, water, fomites, air, and the soiled hands of health care workers [5]. Numerous reports have demonstrated that hospital environment surfaces are a source of antibiotic-resistant bacteria such as Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter sp., Klebsiella pneumonia, Clostridium difficile, and methicillin-resistant Staphylococcus aureus (MRSA). The contamination of wound surfaces by these agents occurs more frequently in developing countries [6,7]. Side by side these drug-resistant bacteria are easily transferred from one patient to another because overcrowding in burn units is another important factor for cross-infection [8]. The organisms that predominate as causative agents of burn wound infection in any burn treatment facility change over time. Gram-positive organisms (methicillin-resistant S. aureus), are initially prevalent, then gradually become predominate by Gram-negative opportunists (P. aeruginosa, A. baumannii, Enterobacter sp) that appear to have a greater propensity to invade [9,10]. Out of all MDR bacteria, P. aerugnosa (an opportunistic human pathogen) isolated from burn patients proved as most significant bacteria because this bacterium showed best growth on the moist surface of burn wounds and is highly pathogenic in immunocompromised patients [11]. Incidentally P. aeruginosa infection is awfully problematic since this organism is inherently resistant to many drug classes and is able to acquire resistance to all effective antimicrobial drugs that make infected burn wounds difficult to treat [12]. Parallel to Pseudomonas sp., our study also highlighted Bacillus sp. which has also emerged as one of the new Gram-positive pathogens to cause serious infection in immunocompromised patients. Members of the genus Bacillus are aerobic or facultative anaerobic Gram-positive or Gramvariable, spore-forming rods that are ubiquitous in the hospital environment; they may be part of the normal flora, particularly in patients hospitalized for prolonged periods [13]. Bacillus causes number of systemic and local infections, including fulminant bacteraemia, central nervous system involvement, catheter related blood stream infection and pneumonia and severe burn wounds [14,15]. The accurate bacterial identification is important in analyzing the epidemiology, antibiotic resistance patterns and outcomes of infections. Traditionally, identification of bacteria in clinical microbiology laboratories was performed using phenotypic tests, including Gram smear and biochemical tests. However, these methods of bacterial identification have major limitations because the organisms with
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biochemical characteristics that do not fit into the patterns of any known genus and species are difficult to be identified. Second, they cannot be used for uncultivable organisms [16]. Using 16S rDNA sequencing, these problems can be overcome by a single technology, which also facilitates the discovery of novel genera and species. Among the three domains of life, the largest amount of rDNA gene sequencing work concerns bacteria. Using 16S rDNA sequences, numerous bacterial genera and species have been reclassified and renamed; classification of uncultivable bacteria has been made possible [17]. Because of above mentioned reasons for characterization of Pseudomonas sp. and Bacillus sp. we took advantage of 16S ribosomal DNA (rDNA) sequence data to identify genus- and species- specific 16S rDNA signature sequences of both bacteria’s. So in the nutshell, keeping in view the role of Pseudomonas sp. and Bacillus sp. (MDR) in immunocompromised burn patients and hospital environment the aim of our study is to evaluate the distribution of multidrug resistant Pseudomonas sp. and Bacillus sp. in burn patients and burn ward by using 16S rRNA- PCR sequencing tools. In this study an attempt was made to track the path of infection in burn patients i.e. whether the bacterial infection in burn cases come through hospital environment or through cross infection. Because by understanding the routes of colonization one can develop the effective preventive measures against infection.
2.
Material and methods
2.1.
Study design
The present prospective analysis was conducted on the patients undergoing treatment in the Burn ward of Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana, during the period of January 2012–March 2013. Bacterial isolates were obtained from the various samples received in Microbiology department of Pt. B.D. Sharma University of Health Sciences, Rohtak, Haryana. The sources of the bacterial isolates were urine, pus, wounds, blood, and body fluids of burn cases. The purity of isolates was determined by macroscopic examination of colonies and microscopic examination of bacteria after Gram staining, and identity of each isolate was confirmed in laboratory by using standard microbiological and other biochemical methods. Bacterial isolates were also grown on selective media like cetrimide agar, Bacillus cereus agar base etc. [18,19]. Different isolates were kept in 10% glycerol and frozen at 40 8C for further study. For burn ward environmental sampling various environmental samples (tap water/sink swab/detergents/soap swab/floor/tables/air/hands of nurses and other damp surfaces) were taken once in a week from the various places of burn ward. Environmental samples were taken by using standard protocols like for air sampling we have used sedimentation or depositional method by placing Nutrient agar plates (Simple and inexpensive; best suited for qualitative sampling). Nutrient agar plates were placed in different regions of burn ward keeping in view the temperature, place and humidity [20]. The Inclusion criterion of our study was about 108 CFU/ml of bacteria cells were considered as
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version 5.1 (MEGA 5.1) for construction of Phylogenetic tree by using Neighbor–Joining method and with boot strap values up to 500 [25].
pathogenic. The patients were excluded from the study if they had clinical or microbiological evidence of infection of unknown origin. Common skin isolates, including Coryneforms excluded from analysis.
2.2.
3.
Antimicrobial susceptibility testing
The antimicrobial susceptibility of all the isolates was performed by estimating minimum inhibitory concentration (MIC50 and MIC90) and disc diffusion method. Values for MICs at which 50% of isolates were inhibited MIC50 and MIC90 were determined by the microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [21]. The definition of MDR strains in our study was taken as non-susceptibility to at least one agent in three or more antimicrobial categories [22]. The antimicrobial sensitivity was checked with nine antibiotics Netilimicin, NET; Amikacin, AMK; Gentamicin, GEN, Pipiercilline, PIP; Azlocillin, AZL, Ceftizoxime, ZOX, Cefepime, FEP, Ofloxine, OFX and Impienem, IPM.
2.3.
Results
Out of 280 cases of burn patients, MDR strains were isolated from 43 patients (30 females with age of 19–47; 26 males with age of 18–65). The hospital stays of these burn patients vary from 10 to 25 days. The main cause of burn in the hospitalized patients was accidental fire due to kerosene oil/gas stoves/gas cylinder. Out of total 510 samples of burn patients, 263 samples were sterile and bacterial isolates were obtained from 247 samples. Out of 247 isolates, 37 isolates were isolated twice from same patients during patients hospital stay. The main source of bacteria in burn patients was the pus collected from burn wounds. In case of environmental samples out of 680 samples, bacteria were isolated from 60 and rest of the samples were sterile. The main source of bacterial contamination in burn ward environment was sink and air. To get more environmental samples we have further taken environmental samples from March 2013 to January 2014 but all samples were sterile. The reason may be that hospital authority has taken preventive measures to control environmental contamination in burn ward after our study results.
16S rRNA characterization of MDR isolates
DNA of all MDR strains was prepared by using the Bangalore Genei DNA isolation kit according to the instructions of the manufacturer. Eluted DNA was stored at 20 8C until further use. For molecular identification 16S rDNA sequencing was used. Gene coding for 16S rDNA was amplified by using universal primers, most common primer pair referred as 27F and 1492R [23]. PCR amplified rDNA genes were then purified with Quick PCR purification kit (Bangalore GENEI, India). The purified products were sequenced by using the facility of Eurofins Genomics India Pvt. Ltd., India. After gene sequencing matching of sequences was done by using the basic local alignment search tool (BLAST) to characterize the MDR bacterial isolates. For molecular phylogenetic analyses, the required reference sequences for comparison were downloaded from the NCBI gene bank database using the website http://www.ncbi.nlm.nih.gov/Genbank. All of the sequences of 16S rDNA genes were aligned with reference sequences using the multiple sequence alignment programs CLUSTAL X 2.0 [24]. Later on alignment file was used in Molecular Evolutionary Genetic Analysis software
3.1.
Antibacterial assay for MDR characterization
About 43 isolates of burn cases and four from burn ward enviornmet isolates were characterized as MDR on the basis of resistance against various antibiotics. The detail of antibacterial activity of antibiotics against isolates, depicting zone of inhibition, MIC50 and MIC90 values have been shown in Table 1. The %resistance of different drugs against 247 isolates has been observed ranged from 14 to 65% and %sensitivity ranged from 34 to 85%. But the %resistance in burn ward isolates was found ranged from 18 to 45% and %sensitivity ranged from 55 to 78%. Both %resistance and %sensitivity of all antibiotics have been shown in Fig. 1. The highest resistance was shown by ZOX (65%) followed by GEN (62%) against burn patients isolates but in burn ward isolates the most resistant drug observed was ZOX (45%) followed by FEM (31%) respectively.
Table 1 – The detail antibacterial activity of antibiotics against multidrug resistant isolates depicting zone of inhibition, MIC50 and MIC90 values. Antimicrobial agents
Disk content
AMK AZL FEM GEN IPM OFX PIP NET ZOX
30 mg 30 mg 30 mg 10 mg 10 mg 5 mg 10 mg 30 mg 30 mg
MDR isolates
Zone of diameter (mm) of bacterial isolates R
I
S
14 15 14 12 13 12 17 12 14
15–16 16–21 15–17 13–14 14–15 13–14 14–16 13–14 15–19
17 22 18 17 16 16 18 15 20
Range (mg/ml) 32–256 16 512 8 32 0.25–32 32 512 32 128 128 512 32 128 8 32
MIC50 (mg/ml)
MIC90 (mg/ml)
128 >128 >32 16 256 >128 >512 >128 >32
256 >128 >32 16 512 >128 >512 >128 >32
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Fig. 1 – Antibacterial activity i.e. sensitivity and resistance (%) of antibiotics against bacterial isolates of Burn patients and Burn Ward environment.
Table 2 – Taxonomic details of Burn patients and Burn ward isolates along with the accession numbers as identified on the basis of 16S rDNA gene partial sequence. Isolate code B. Env. 2; B. Pat. 1; B. Pat. 4; B. Pat. 7; B. Pat. 14; B. Pat. 8; B. Pat. 18; B. Pat. 12 B. Env. 1; B. Pat. 2; B. Pat. 3; B. Pat. 6 B. Pat. 9 B. Pat. 13 B. Pat. 15; B. Pat. 16 B. Pat. 17 B. Pat. 10 B. Pat. 11 B. Pat. 25 B. Pat. 39; B. Pat. 40; B. Pat. 49; B. Pat. 46 B. Pat. 47 B. Pat. 48 B. Pat. 52 B. Pat. 30 B. Pat. 33 B. Env. 3 B. Pat. 18 B. Pat. 20 B. Pat. 23; B. Pat. 26 B. Pat. 56 B. Pat. 21; B. Pat. 29 B. Pat. 22; B. Pat. 38; B. Pat. 24 B. Pat. 28 B. Pat. 55 B. Pat. 54 B. Pat. 41 B. Pat. 45 B. Pat. 51 B. Env. 4
Accession no. of isolates KF862889 KF862888 KF951532 KF862896; KF862897; KF862901 KF862890; KF862892; KF862893 KF862898 KF862902; KF862906 KF862899 KF862900 KF862914 KF862925; KF862935 KF862932 KF862933 KF862934 KF862938 KF862919 KF862921 KF862894 KF862908 KF862909 KF862912; KF862942 KF862910; KF862911; KF862913 KF862917 KF862941 KF862940 KF862927 KF862931 KF862937 KF862895
KF862903 KF862907 KF862889; KF862893
KF862905
KF862926;
KF862915 KF862918 KF862924;
Bacterial strain names showing % similarity with isolates P. P. P. P. P. P. P.
aeruginosa strain FN826303 (100%) aeruginosa strain JQ6599661 (99%) aeruginosa strain HO8090971 (100%) aeruginosa strain GU451298 (100%) aeruginosa strain CP006832 (99%) aeruginosa strain KF583972 (100%) stutzeri strain KF607058 (100%)
No. of isolates 1 1 1 2 2 1 4
P. stutzeri strain HM209781 (99%) P. stutzeri strain KF4539531 (100%) P. stutzeri strain KF5766511 (100%) P. mendocina strain KC895888 (100%) Pseudomonas bacterium strain HE583117 (99%) Pseudomaceae strain KF560362 (99%) B. cereus strain KC876035 (100%) B. cereus strain KF054992 (99%) B. cereus strain JX050218 (99%) B. cereus strain JX088534 (99%) B. cereus strain JX088538 (99%) B. cereus strain JN411376 (100%) B. cereus strain JQ284035 (99%) B. cereus strain KF612021 (99%) B. cereus strain JQ818396 (96%)
1 1 2 1 1 1 1 3 1 1 1 1 1 1 1
B. B. B. B. B. B.
1 1 2 1 2 3
subtilis strain JN641293 (99%) subtilis strain JQ653049 (99%) subtilis strain KF582786 (99%) subtilis strain JX286682 (99%) anthracis strain KF017366 (99%) anthracis strain GU939625 (99%)
B. tequilensia KF436736 (99%) Bacillus safensis HQ22459 (99%) Bacillus pumilius EU780103(99%) Bacillus thuringensis JX860329 (99%) Bacillus stratosphercium HM0327861 (99%) Bacterium NLAE JX006771 (99%) A. baumanii KF060283 (99%)
1 1 1 1 1 1 1
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Fig. 2 – Phylogenetic tree (based on neighboring–joining method with boot strap values on node) of the bacterial isolates from Burn cases and Burn Ward Environment. Number in parentheses are Gene Bank accession Numbers for 16S rDNA nucleotide sequences of isolates.
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In case of environmental samples only four multidrug resistance isolates were observed out of 60 isolates. So, 22% MDR bacterial isolates were isolated from burn cases and 6% from burn ward. No repeatability was observed in MDR strains of burn patients and burn ward environment.
3.2. 16S rDNA gene partial sequencing of characterized MDR isolates The details of bacterial isolate code, their accession number provided by NCBI and bacterial name showing % similarity with our isolates has been shown in Table 2. The taxonomic information obtained by molecular characterization on the basis of 16S rDNA gene partial sequence has shown that all of the isolates have shown 97–100% of similarity with the NCBI strains. Out of all MDR isolates maximum number were belonged to the genus Bacillus (eight species and 26 isolates) followed by genus Pseudomonas (five species and 17 isolates). Some bacterial strains isolated from burn patient (B. Pat. 27, B. Pat. 31) and one burn ward isolates i.e. B. Env. 2 have shown 16S rDNA similarities up to 96–97% probably may be new bacterial species. So we found that in case of burn patients, B. cereus species has showed predominant diversity having 11 numbers of isolates followed by P. aeruginosa and P. stutzeri having seven isolates. Another important finding in our study was the presence of five strains (B. Pat. 21; B. Pat. 29; B. Pat. 22; B. Pat. 38; B. Pat. 24) of Bacillus anthracis. In case of burn ward MDR isolates were belong to P. aeruginosa (B. Env. 1), P. stutzeri (B. Env. 2), B. cereus (B. Env. 3) and Acinetobacter baumanii (B. Env. 4). Phylogenetic analysis based on 16S rDNA gene sequence was determined. Phylogenetic relationship of the burn patients isolates and burn ward isolates was calculated by the multiple alignments of the 16S rDNA gene sequences has been diagrammed in Fig. 2.
4.
Discussion
In recent decades, bacteria isolated from burn patients become resistant to antibiotics. In high-infection rate units, periodic monitoring of the microbial species and their respective susceptibility to antibiotics is important in selection of effective empiric antimicrobial therapy against bacteria isolated from the burn patients [26]. In the present study, we have carried out a prospective study of the antibiotic resistance profiling and molecular characterization by 16S rDNA sequencing of MDR isolates of burn cases and burns unit environment. In our study we have isolated 43 MDR bacteria’s from burn patients and four from the burn ward. While checking antibiotic sensitivity of isolates we found that most resistance antimicrobial class was aminoglycosides (Gentamicin, Amikacin) in both burn patients and burn ward disparately, imipenem was effective drug. Similar reports of aminoglycosides resistant were also elucidated by other studies done on burn patients in different parts of India [10,27]. This could be due to the reason that imipenem is reserve drug and has been used as last options drug against resistant bacteria for burn patients. While inspecting distribution of P. aeruginosa and Bacillus sp., the noteworthy finding of our study was predominance of
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B. cereus with 11 isolates followed by P. aeruginosa with seven isolates in burn patients. It is a well known fact that members of the genus Bacillus are spore forming that are ubiquitous in the hospital environment. This bacterial genus is part of the normal flora and nowadays this genus has been getting appreciation for its potential as an opportunistic pathogen in immunocompromised hospitalized patients [13]. The hypothesis that B. cereus spores present in air also been justified by our study because we have isolated one MDR B. cereus in our burn ward air sample. But due to presence of only one air MDR isolate we cannot predict air as sole causative agent of B. cereus contamination in burn patients. The cross contamination between the patients may be the main factor of B. cereus infection. The outbreak caused by B. cereus in immunosuppressed hospitalized patients has been also studied by numerous studies conducted on cancer patients, acute leukemia cases, infants, newborn babies, and ICU units [28,29]. The presence of four strains of Bacillus thuringiensis was observed in causing infection in burn wound and also from water used in the treatment of burn wounds [15]. The role of non sterile gloves as contaminate in B. cereus infection in burn unit was also observed in one study [30]. To the best of our knowledge our study is the first study which is evaluating distribution of Bacillus sp. in burn patients and burn ward environment. In the present our study when we discuss the distribution of P. aeruginosa than it was observed as second prevalent MDR isolates. In hospital ward this bacterium was isolated from sink. The presence of P. aeruginosa in burn patients is a well known fact and indeed also proved by other studies [31,32]. The main reason behind remarkably high prevalence of this bacterium in burn cases and burn unit may be that the devitalised tissue and moist environment of burns provide an ideal environment for colonization and infection with P. aeruginosa [33]. In our study both MDR species of Pseudomonas (P. aeruginosa and P. stutzeri) were isolated from sink, on this basis we may say that sink may be the main source of Pseudomonas infection in burn patients. The presence of P. aeruginosa in sink may be due to nutritional requirements and its capability to thrives in moist environments (such as drains) that allow it to grow in macrocolonies within plumbing systems [34]. But owing to the fact that there were only two MDR Pseudomonas sp. in burn ward we cannot predict the sole role of burn ward environment in contamination of Pseudomonas sp. in burn patients again like Bacillus sp. The other reasons of Pseudomonas infection in burn patients may be there due to cross contamination, due to other exogenous or endogenous sources. The contamination of P. aeruginosa in burn cases via cross infection has been also proved by numerous studies conducted in various parts of world [8,35].
5.
Conclusion:
To the best of our knowledge our study is the first study which is based on evaluation of distribution of Bacillus sp. in burn patients and burn ward environment. On the basis of 16S rDNA amplification we conclude that B. cereus was major source of infection in burn patients followed by P. aeruginosa. The prevalence of B. cereus depicts importance for our burn institution to determine its specific pattern of burn microbial
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colonization, antimicrobial resistance profiles and timerelated changes in predominant flora. This would allow early management of septic episodes with proper empirical systemic antibiotics before the results of microbiologic cultures become available, thus improving the overall infection-related morbidity and mortality. In conclusion, our results may be useful guidelines for choosing effective antimicrobial therapy in our burn ward against multidrug resistant isolates throughout the patients’ hospital stay.
Conflict of interest We as authors do not have a conflict of interest.
Acknowledgments Dr Manju Panghal is thankful to Department of Science and Technology, New Delhi for providing her financial support under scheme of Women Scientist programme (WOS–A) (Grant no - SR/WOS- A/LS-259/2011(G) & Date- 19/12/2011) to carry out her research work. Authors are also thankful to Department of Science and Technology, New Delhi for the award of DST-FIST grant (SR/FST/LS1-038/2011) to the Department.
references
[1] Mayhall CG. The epidemiology of burn wound infections: then and now. Clin Infect Dis 2003;37:543–50. [2] Macedo JLS, Rosa SC, Castro C. Sepsis in burned patients. Rev Bras Med Trop 2003;36:647–52. [3] Jessie S, Glasser JS, Guymon GH, Mende K, Wolf SE, Hospenthal DR, et al. Activity of topical antimicrobial agents against multidrug-resistant bacteria recovered from burn patients. Burns 2010;36:1172–84. [4] Magnotti LJ, Deitch EA. Burns, bacterial translocation, gut barrier function, and failure. J Burn Care Rehabil 2005;26(5):383–91. [5] Gales AC, Jones RN, Turnidge J, Rennie R, Ramphal R. Characterization of Pseudomonas aeruginosa isolates: occurrence rates, antimicrobial susceptibility patterns, and molecular typing in the global SENTRY Antimicrobial Surveillance Program, 1997–1999. Clin Infect Dis 2001;32(Suppl. 2):S146–55. [6] Macedo JLS, Santos JB. Nosocomial infections in a Brazilian Burn Unit. Burns 2006;32:477–81. [7] Hota B, Blom DW, Lyle EA, Weinstein RA, Hayden MK. Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product, or procedure. J Hosp Infect 2009;71:123–31. [8] Douglas MW, Mulholland K, Denyer V, Gottlieb T. Multidrug resistant Pseudomonas aeruginosa outbreak in a burns unit-an infection control study. Burns 2001;27:131–5. [9] Altoparlak U, Erol S, Akcay MN, Celebi F, Kadanali A. The time-related changes of antimicrobial resistance patterns and predominant bacterial profiles of burn wounds and body flora of burned patients. Burns 2004;30:660–4. [10] Agnihotri N, Gupta V, Joshi RM. Aerobic bacterial isolates from burn wound infections and their antibiograms: a five year study. Burns 2004;30:241–3.
[11] Williams FN, Herndon DN, Hawkins HK, Lee JO, Cox RA, Kulp GA. The leading causes of death after burn injury in a single pediatric burn center. Crit Care 2009;13:R183. [12] Hsueh PR, Teng LJ, Yang PC, Chen YC, Ho SW, Luh KT. Persistence of a multidrug-resistant Pseudomonas aeruginosa clone in an intensive care burn unit. J Clin Microbiol 1998;36:1347–51. [13] Drobniewsky FA. Bacillus cereus and related species. Clin Microbiol Rev 1993;6:324. [14] Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 2010;23(2):382–98. [15] Damgaard PH1, Granum PE, Bresciani J, Torregrossa MV, Eilenberg J, Valentino L. Characterization of Bacillus thuringiensis isolated from infections in burn wounds. FEMS Immunol Med Microbiol 1997;18(1):47–53. [16] Woo PCY, Lau SKP, Teng JLL, Tse H, Yuen KY. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 2008;14:908–34. [17] Snel B, Bork P, Huynen MA. Genome phylogeny based on gene content. Nat Genet 1999;21:108–10. [18] Hawkey P, Law D. Medical bacteriology. NewYork: Publication Co, Oxford University Press; 2004. [19] Pawar VB, Dutta D. A procedure manual for routine diagnostic test. In: Mukherjee KL, editor. Diagnostic bacteriology. 2nd ed. New Delhi: Publication Co. Tata MacGrew -Hill Publishing Company Limited; 2006. [20] Sehulster LM, Chinn RYW, Arduino MJ, Carpenter J, Donlan R, Ashford D, et al. Guidelines for environmental infection control in health-care facilities. Recommendations from CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). Chicago, IL: American Society for Healthcare Engineering/American Hospital Association; 2004. [21] Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard ninth edition approved standard M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. [22] Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drugresistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268–81. [23] Jiang H, Dong H, Zhang G, Yu B, Chapman LR, Fields MW. Microbiology diversity in water and sediments of lake chaka, an Athalassonhaline Lake in Norhwestern China. Appl Environ Microbiol 2006;72(6):3832–45. [24] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam HV, et al. Clustal W and Clustal X version 2.0. Bioinformatics 2007;23:2947–8. [25] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony method. Mol Biol Evol 2011;28:2731–9. [26] Ozumba UC, Jiburum BC. Bacteriology of burn wounds in Enugu, Nigeria. Burns 2000;26:178–80. [27] Singh NP, Goyal R, Manchanda V, Das S, Kaur I, Talwar V. Changing trends in bacteriology of burns in the burns unit: Delhi, India. Burns 2003;29(2):129–32. [28] Hilliard NJ, Schelonka RLRL, Waites KB. Bacillus cereus: bacteremia in a Preterm Neonate. J Clin Microbiol 2003;41(7):3441–4. [29] Ginsburg AS, Salazar LG, True LD, Disis ML. Fatal Bacillus cereus sepsis following resolving neutropenic enterocolitis during the treatment of acute leukemia. Am J Hematol 2003;72:204–8.
burns 41 (2015) 812–819
[30] Jeurissen A, Weyers L, Cossey V, Muller J, Schuermans AJ. Dissemination of Bacillus cereus in the burn unit due to contaminated non-sterile gloves. Hosp Infect 2010;76(1): 92–3. [31] Japoni A, Alborzi A, Kalani M, Nasiri J, Hayati M, Farshad S. Susceptibility patterns and cross-resistance of antibiotics against Pseudomonas aeruginosa isolated from burn patients in the South of Iran. Burns 2006;32:343–7. [32] Zhang JP1, Yang XS, Chen J, Peng YZ, Huang YS. Clinical assessment of colistin in treating infections caused by multidrug-resistant gram-negative bacillus in patients
819
with severe burn. Zhonghua Shao Shang Za Zhi 2009;25(5):372–6. [33] Atoyebi OA, Sowemimo GOA, Odugbemi T. Bacterial flora of burn wounds in Lagos, Nigeria: a prospective study. Burns 1992;18:448–51. [34] Labarca JA, Pegues DA, Wagar EA, Hindler JA, Bruckner DA. Something’s rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa. Clin Infect Dis 1998;26:1440–6. [35] Komolafe OO, James J, Kalongolera L, Makoka M. Bacteriology of burns at the Queen Elizabeth Central Hospital, Blantyre, Malawi. Burns 2003;29:235–8.
