International Journal of Biological & Medical Research

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1 Int J Biol Med es. ; (): -0 Int J Biol Med es Volume, Issue, July BioMedSiDiret Publiations Contents lists available at BioMedSiDiret Publiations International Journal of Biologial & Medial esearh Journal homepage: International Journal of BIOLOGICAL AND MEDICAL ESEACH Original Artile Beta latamases mediated resistane amongst gram negative bailli in Burn infetion b* a d e Nitin Bandekar, Vinodkumar C.S, Basavarajappa K.G, Prabhakar P.J, Nagaraj P a Assoiate Professor, Department of Surgery, S. S. Institute of Medial Sienes and esearh Centre, Davangere-00, Karnataka, INDIA b Assistant Professor Department of Mirobiology, S. S. Institute of Medial Sienes and esearh Centre, Davangere-00, Karnataka, INDIA Professor & Head, Department of Mirobiology, S. S. Institute of Medial Sienes and esearh Centre, Davangere-00, Karnataka, INDIA d Professor & Head, Department of Surgery, S. S. Institute of Medial Sienes and esearh Centre, Davangere-00, Karnataka, INDIA. e Prinipal, S. S. Institute of Medial Sienes and esearh Centre, Davangere-00, Karnataka, INDIA A T I C L E I N F O A B S T A C T Keywords: β-latamases Burn infetion Drug resistane Introdution: Burn is one of the most ommon and devastating forms of trauma. Patients with serious thermal injury require immediate speialized are in order to minimize morbidity and mortality. Signifiant thermal injuries indue a state of Immuno- suppression, whih predisposes infetious ompliations in burn patients. In burn patient infetions arise from multiple soures and infet burn wounds by a variety of miro-organisms. Gram negative baterial infetion results from transloation from olon, further more burn patients are infeted by Hospital aquired bateria by various invasive and non invasive proedures Early diagnosis of mirobial infetions and sreening for mehanism of drug resistane is aimed to institute the appropriate antibaterial therapy and to avoid further ompliations. Objetives: Beta latamases are enzymes responsible for the resistane to beta latam antibiotis. This study is aimed at the detetion of various types of beta latamases present among the gram negative bailli isolated from burn infetion. Methods: patients admitted to burn intensive are unit were inluded in the study. gram negative bailli were isolated and sreened for the presene of extended spetrum beta latamase, AmpC latamase, Metallo beta latamase and onfirmed by the respetive onfirmatory tests. esults:. produed extended spetrum beta latamases,. AmpC beta latamase and. strains produed metallo beta latamases. Pseudomonas aeruginosa was the predominant bateria produing ESBL and AmpC mediated resistane, whereas Ainetobater baumannii was the predominant MBL produer. Conlusion: It is important to monitor the bateriology in burn patients at all time, and understand the hanging pattern of baterial flora, antibioti suseptibility and baterial strains spreading in burn ward. Extended-spetrum beta-latamases is the ause of resistane. After sulbatam added to the third generation of ephalosporins, the beta-latamases were inhibited, but it lead to inrease use of arbapenems leading to emergene of metallo beta latamase mediated resistane. Hene, sreening tehniques should be performed routinely to detet these β-latamase produers so that suitable antimirobial therapy an be instituted.. Introdution Copyright 0 BioMedSiDiret Publiations IJBM - ISSN: 0:. All rights reserved. Burns provide a suitable site for baterial multipliation and are more persistent riher soures of infetion than surgial wounds, mainly beause of the larger area involved and longer duration of * Corresponding Author : Dr. VinodKumar C. S, PhD, FAGE patient stay in the hospital []. Infetion is a major ause of Assistant Professor Department of Mirobiology, morbidity and mortality in hospitalized burn patients[]. It is now S. S. Institute of Medial Sienes and esearh Centre, estimated that about of the mortality following burn injuries Vidhyanagar post box- is related to infetions rather than osmoti shok and hypovolemia NH-, Bypass, Davanager-00, Karnataka Mobile: 00 []. The pattern of infetion differs from hospital to hospital; the vinodmiro@yahoo.om, vinodmiro@gmail.om Copyright 0 BioMedSiDiret Publiations. All rights reserved. varied baterial flora of infeted wound may hange onsiderably during the healing periodin the past years, antibiotis have been ritial in the fight against infetious disease aused by the

2 VinodKumar et.al / Int J Biol Med es. ; (): -0 bateria and other mirobes. Antimirobial hemotherapy has been a leading ause for the dramati rise of average life expetany in the Twentieth Century. Despite the advanes in patient are and the use of a large number of antimirobial agents, infetions whih ompliate the linial ourse of patients who had sustained severe thermal injures ontinue to be a major unsolved problem. Nowadays, majority of the bateria that ause burn infetion in hospitals are resistant to at least one of the drugs most ommonly used for treatment []. Extended Spetrum β-latamases (ESBL), AmpC β-latamase and Metallo β-latamase produing organisms pose a major problem for treating burn vitims [,]. The routine suseptibility tests done by linial laboratories fail to detet β- latamases positive strains and an erroneously detet isolates sometimes to be sensitive to any of the broad-spetrum ephalosporin like efotaxime, eftazidime, eftriaxone and for imipenem or meropenem [-]. Hene, it is neessary to know the prevalene of β-latamase positive strains in a burn unit so as to formulate a poliy of empirial therapy in high risk units where infetions due to resistant organisms are muh higher. Equally, important is the information on an isolate from a patient to avoid misuse of extended spetrum ephalosporins, and arbapenems whih still remain an important omponent of antimirobial therapy in burn wards [0,]. There is not enough information from the Indian subontinent regarding the prevalene of β- latamases mediated resistane among gram negative bateria in burn infetion. The aim of the present study is to find the prevalene of β-latamases mediated resistane among gram negative bateria in burn infetion.. Materials and methods A prospetive study was arried out on 0 burn patients over a period of two years at S.S. Institute of Medial College and esearh Centre, Davangere gram negative baterial were isolated and identified by standard laboratory tehniques []. Antimirobial sensitivity testing was performed on Mueller- Hinton agar plates with ommerially available diss (Hi-Media, Mumbai) by the Kirby-Bauer dis diffusion method []. The results were reorded and interpreted as per CLSI reommendations []...Tests for ESBL prodution...double disk approximation test for sreening: The test organisms were applied on to a Mueller Hinton agar plate by adjusting turbidity to MFarland no 0. tube. Antibioti diss of Amoxiillin / Clavulani aid (0/0 µg) and efotaxime (0 µg) were plaed at a distane of mm apart and inubated. Organisms that showed a lear extension of efotaxime inhibition zone towards the dis ontaining Clavulanate were onsidered as [] ESBL produer. The organisms whih were sreened and found positive for ESBL prodution were subjeted to onfirmatory test....nccls phenotypi onfirmatory test Ceftazidime (0 µg) and eftazidime plus Clavulani aid (0/0 µg) were plaed on Mueller Hinton agar and inubated. Organism was onsidered as ESBL produer if there was a mm inrease in diameter of Ceftazidime plus Clavulani dis and that of eftazidime dis alone [,]....Amp C Disk Test A lawn ulture of E.oli ATCC was prepared on MHA plate. Sterile disks (mm) was moistened with sterile saline (0 µl) and inoulated with several olonies of test organisms. The inoulated disk was then plaed mm beside a efoxitin dis. Plates were inubated overnight at C. A positive test was appeared as a flattening or indentation of the efoxitin inhibition zone in the viinity of the test dis [-0]. A negative test had an undisturbed zone...metallo β-latamase (MBL) prodution Gram negative organisms that showed resistane to Imipenem were seleted for MBL prodution... Imipenem-EDTA ombined dis test This test was performed aording to Yong et al. test organisms were inoulated onto Mueller Hinton agar plates as per the CLSI reommendations. Two 0µg imipenem disks were plaed on the plate and 0 µl of sterile 0. M EDTA solution was added to one of the imipenem disk. The inhibition zones of the imipenem and imipenem plus EDTA disks were ompared after inoulation []. If the inrease in inhibition zone with the imipenem plus EDTA dis was mm than the imipenem dis alone, it was onsidered as MBL positive.. esults Perentage of resistane exhibited by gram negative bailli isolated from burn infetion to various antimirobial agents is shown in table-. All the strains were resistant to more than or more drugs hene all the bateria were designated as multidrug resistane gram negative bailli (MDGNB) All gram negative bailli were sreened for ESβL prodution, Amp C β latamase prodution.. of gram negative bailli were ESβL produers. Pseudomonas aeruginosa (0.) was the predominant ESβL produer followed by Klebsiella pneumonia (.), Ainetobater baumanni (.), Proteus mirabilis (.) and Enterobater loaae (.) (Table-). of gram negative bailli were Amp C produers and Pseudomonas aeruginosa (.) was the predominant AmpC produer followed by Klebsiella pneumonia (.) and Proteus mirabilis (.) (Table )... MBL produers Not all gram negative bateria were tested for MBL prodution. Only those gram negative bailli resistant to imipenem (Table-) were sreened for MBL prodution. out of Pseudomonas aeruginosa were resistant to imipenem. Similarly 0 Klebsiella pneumoniae out of were resistant. Among Ainetobater baumanni 0 were resistant. (Table-) Ainetobater baumanni (.0) was the predominant MBL produer followed by Pseudomonas aeruginosa (.), Klebsiella pneumonia (.) and Proteus mirabilis (.)

3 VinodKumar et.al / Int J Biol Med es. ; (): -0 Table : Antibioti esistane pattern of Gram negative baterial isolates from burn infetion Baterial isolates Ps. aeruginosa Klebsiella pneumoniae Ainetobater baumannii Proteus mirabilis Enterobater loaae Citrobater freundii Drugs Amikain.... Ampiillin. Aztreonam.. Gentamiin.. Cephotaxime.... Ceftriaxone... Ceftazadime.. Cefoperozone... Ciprofloxain.... Imipenem... Piperaillin-tazobatam Piperaillin... Amoxyillin... Gatifloxain Table. Prevalene of ESBL and Amp C produers in Burn infetion Organisms Pseudomonas aeruginosa Klebsiella pneumonia Ainetobater baumanni Proteus mirabilis Enterobater loaae Citrobater freundii Total Table. Prevalene of Metallo beta latamase produers in burn infetion Organisms Pseudomonas aeruginosa Klebsiella pneumonia Ainetobater baumanni Proteus mirabilis Enterobater loaae Citrobater freundii Total No. Tested No. of GNB isolated ESBL produers No Imipenem resistant (.0) No No. Amp C produers MBL produers Disussion The ability to produe β-latamases enzymes is the major ause of resistane of bateria to β-latam antibiotis. Numerous β- latamases are enoded either by hromosomal genes or transferable genes loated on plasmids or transposons []. Based on amino aid and nuleotide sequene studies, four distint lasses of β-latamases have been defined. Class A (Extended spetrum β-latamases) lass B (Metallo β- latamases), lass C (AmpC β-latamases) and Class D (Cloxaillin hydrolysing β-latamases) [,]. Extended spetrum β-latamases are plasmid mediated TEM and SHV derived enzymes isolated for first time in Western Europe in mid 0s []. Initially theses enzymes were ommonly found in Klebsiella speies and E.oli, [] but now these enzymes are produed by all the members of Enterobateriaeae and few other gram negative bailli [,]. These enzymes are apable of hydrolysing broad spetrum ephalosporins and monobatams and inative against ephamyins and imipenem. In the present study. of gram negative bateria were ESBL produers. No studies are available on extended spetrum beta latamases resistane in burn infetion. But few studies in India on different linial ondition have reported the prevalene of ESBL in the range of to. [,,,] Our prevalene rate is lesser than other reports ompared with different linial onditions from India and abroad, sine the isolates were obtained from infetion in burn infetions whih is not a hroni infetion they might be wide disparity in the prevalene rate of ESL produing gram-negative bateria when ompared to other

4 VinodKumar et.al / Int J Biol Med es. ; (): -0 reports. Pseudomonas aeruginosa was the predominant ESL produer followed by Klebsiella pneumoniae, Ainetobater baumannii and Proteus mirabilis. In addition to the intrinsi resistane to ephalosporins and aztreonam, ESBL produing organism's exhibit o-resistane to many other lasses of antibiotis like quinolones and aminoglyosides resulting in limitation of therapeuti options. In the present study we found suh assoiated resistane with Ciprofloxain (.). As quinolones are strong seletors of ESBL produers, their use should be restrited as far as possible. Major risk fators for olonization or infetion with ESBL produing organisms are long term antibioti exposure, prolonged hospital stay, high rates of the third generation ephalosporin use and invasive proedures. However in the present study we ould not retrieve the nosoomial pathogens and their sensitivity to reinfore the above arguments. Treatment of ESBL produing strains of Enterobateriaeae has emerged as a major hallenge in hospitalized as well as ommunity patients. There are many fators whih determine the hoie of antibiotis and the management of burn infetions. Although β- latamases inhibitors have signifiant ativity against ESBL invitro, their linial effetiveness against serious infetions due to ESBL produing organisms is ontroversial. ESBL produing strains might show a false sensitive zone of inhibition in the Kirby Bauer's dis diffusion method. The antibiotis for the treatment inlude arbapenems, aminoglyoside and β-latamases inhibitor ombinations. AmpC β-latamases are linially important ephalosporinases enoded on hromosomes of many of the Enterobateriaeae and a few other organisms [], where they mediate resistane to ephalothin efazolin, efoxitin, most of the peniillins and β- latamase inhibitor [,]. In many bateria Amp C enzymes are induible and an be expressed at high levels by mutation [,]. Over expression onfers resistane to broad spetrum ephalosporins. In the present study. were AmpC produers and Pseudomonas aeruginosa was the predominant Amp C produer followed by Klebsiella pneumonia, Ainetobater baumannii and Proteus mirabilis. There are no reports to ompare the inidene of Amp C mediated resistane among burn infetion in India and aboard. Metallo β-latamase (MBL) is a group of arbapenem hydrolysing β-latamase [0]. They have been reported from many ountries, as well as from different parts of Indian subontinent, partiularly in multidrug resistane pathogens like Pseudomonas aeruginosa and Ainetobater speies. The MBLs are inhibited invitro by CuCl, FeCl, EDTA and thiol ompounds like meraptopropioni aid, sodium meraptoaetoi aid and meraptoethanal, but not by β-latamase inhibitors like Clavulani aid, sulbatum or tazobatam []. Detetion of MBL prodution in MD organisms from burn infetion has tremendous therapeuti onsequenes, as the treatment option for suh isolates are aztreonam or potentially toxi polymyxin B and olistin. In the present study not all gram negative bateria were tested for MBL prodution. Only those gram negative bailli resistant to imipenem were sreened for MBL prodution and. of them were metallo β-latamase produers. Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis were the predominant MBL produers.. Conlusion In onlusion, the present study highlights the high prevalene of β-latamases among the multi drug resistant gram negative isolates in burn infetion. It also reflets grim future of the treatment options available for these notorious pathogens. The high inidene of β-latamases prodution due to multiple mehanisms in burn infetion is alarming and urgent ation needs to be taken from both the therapeuti and infetion ontrol perspetive. Clinial mirobiology laboratories should perform the sreening tehniques to detet these β-latamases routinely so that the suitable antimirobial therapy an be instituted and the dissemination of these isolates may be prevented by employing appropriate ontrol measures.. eferenes [] Agnihotri N,Gupta V, Joshi M., Aerobi baterial isolate from burn wound infetions and their antibiograms- a five- year study. Burns. 00; 0: -. [] M Manus A.T., Mason A.D..jr, MManus W.F., Pruitt B.A,Jr: A deade of redued Gram -negative infetions and mortality improved isolation of burned patients. Arh Surg. ; : 0-0. [] Donati I, Sammazo F, Gervasoni M, Magliano A, Stankov B, Frashini F. Infetion and antibioti therapy in 000 burned patients treated in Milan, Italy, between and. Burns. ; : -. [] Fridkin SK, Steward CD, Edwards J, Pryor E, MGowan JE Jr, Arhibald LK, et al. Surveillane of antimirobial use and antimirobial resistane in United States hospitals: projet ICAE phase. Clin Infet Dis. ; ():. [] Pfaller MA, Jones N. MYSTIC (Meropenem Yearly Suseptibility Test Information Colletion) results from the Amerias: resistane impliations in the treatment of serious infetions. J Antimirob Chemother. 000; :. [] Goossens H. MYSTIC (Meropenem Yearly Suseptibility Test Information Colletion) results from Europe: omparison of antibioti suseptibilities between ountries and entre types. J Antimirob Chemother. 000;:. [] Jerestin BH, Vandana Agarwal Pathat M. Extended spetrum -latamases mediated resistane to third generation ephalosporins in Klebsiella pneumoniae in Nagpur, entral India. Indian Journal Med es. ;0:-. [] Mathur P, Kapil A, Das B, Dhawan B. Prevalene of extended spetrum beta latamase produing gram negative bateria in a tertiary are hospital. Indian J Med es. 00; :- [] Chaudhary U, Aggarwal. Extended spetrum β-latamases and emerging threat to linial therapeutis. Indian J Med Mirbiol. 00;():-0 [0] Abigali S, Mathur, Jesudasan MV. Ceftazidime resistane among Klebsiella pneumoniae in South India. Indian Journal Med esearh. ;0:- [] Cunha BA. Antibioti seletion for diabeti foot infetions: a review. J Foot Ankle Surg. 000;:. [] Baird D. Staphyloous: Cluster-forming gram-positive oi. In : Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Makie and MCartney Pratial Medial Mirobiology. th ed. New York: Churhill Livingstone;. p. -. [] Bauer A W, Kirby W M M, Sherris JC, Jurek M Antibioti suseptibility testing by a standardized dis method. Amerian Journal Clinial Pathology. ;:-.

5 0 VinodKumar et.al / Int J Biol Med es. ; (): -0 [] Clinial laboratory Standards Institute (CLSI) performane standards for antimirobial disk suseptibility testing th informational supplement. CLSI doument M-A. Wayne (PA), 00.. [] Brun-Buisson C, Legrand P, Philippon A, Montravers F, Ansquer M, Duval J et al. Transferable enzymati resistane to third-generation ephalosporins during a nosoomial outbreak of multiresistant Klebsiella pneumoniae. Lanet. ; (): 0-0. [] Coudron PE, Moland ES, Sanders CC. Ourrene and Detetion of Extended-Spetrum ß-latamases in members of the family Enterobateriaeae at a Veterans Medial Center: Seek and You May Find. J Clin Mirobiol. ; (0):-. [] Bhattaharya S. Extended spetrum β-latamases from petridish to the patient. Indian J Med Mirobial. 00;():0- [] Singhal S, Mathur T, Khan S, Upadhyay DJ. Evaluation of method for Amp C β-latamases in gram negative linial isolate from tertiary are hospital. Indian Journal of Medial Mirobiology. 00;():0- [] Blak JA, Moland ES, Thomson KS. Amp C disk test for detetion of plasmid mediated Amp C β-latamases. Journal of Clin Mirobiol. 00;():0- [0] M Sinha, H Srinivasa. Mehanisms of resistane to arbapenems in meropenem resistant Ainetobater isolates from linial samples. Indian Journal of Medial Mirobiology. 00;();- [] Leek, lim YS, Yong D, Yum JH, Chong Y. Evaluation of the Hodge test and the imipenem-edta double disk synergy test for differentiation of metallo- β- latamases produing linial isolates of Pseudomonas spp and Ainetobater spp. J Clin Mirobiol. 00;:- [] Mary VJ, Kandathi AJ, Balaji V. Comparison of methods to detet arbapenamase and metallo β-latamases prodution in linial isolates. Indian J Med es. 00;:0- Copyright 0 BioMedSiDiret Publiations IJBM - ISSN: 0:. All rights reserved. [] Bush K, Jaoby GA, Medeiros AA. A funtional lassifiation sheme for β- latamases and its orrelation with moleular struture. Antimirob Agent Chemother. ;:- [] Jaoby GA. Extended-spetrum b-latamases and other enzymes providing resistane to oxyimino--latams. Infet Dis Clin North Am. ; ():-. [] Livermore DM, Yuan M. Antibioti resistane and prodution of extended spetrum b-latamases amongst Klebsiella spp. from intensive are units in Europe. J Antimirob Chemother. ; ():0-. [] Albertini MT. Surveillane of methiillin-resistant Staphyloous aureus (MSA) and Enterobateriaeae produing extended-spetrum betalatamase (ESBLE) in Northern Frane: a five-year multientre inidene study. J Hosp Infet. 00; (): 0-. [] Kumar MS, Lakshmi V, ajagopalan. Ourrene of extended spetrum β- latamases among Enterobateriaeae speies isolated at a tertiary are institute. Indian J Med Mirobial. 00;():0- [] Zorgani A., Zaidi M., anka., Shahen A.:The pattern and outome of septiaemia in a burns intensive are unit.ann.burns Diasters. 00;: -. [] Ananthakrishnan AN, Kanungo, Kumar A, Badrinath S. Detetion of extended spetrum β-latamase produers among surgial wound infetions and burn patients in JIPME. Indian Journal of Medial Mirobiology. 00;:0-. [0] Chu, Y.-W., M. Afzal-Shah, E. T. S. Houang, M.-F. I. Palepou, D. J. Lyon, N. Woodford, and D. M. Livermore (0) IMP-, a novel metallo-β-latamase from nosoomial Ainetobater spp. olleted in Hong Kong between and. Antimirob. Agents Chemother. 0;: