ࡱ>  Oh+'0p   , 8 DPX`hssBrankaran Normal.dotBranka Bedeni91nMicrosoft Word 8.0@nGx@~5@&T[i@h Ailes 0Microsoft Office0TemplatesDB<@ ՜.+,D՜.+,0 hp  Zagrebl!P   Title 6> _PID_GUIDAN{A21FBD7A-E12E-11D2-8E8D-CBCBFEBEB77C}0TemplatesDB<@ SELECTION OF KLEBSIELLA PNEUMONIAE MUTANTS WITH HIGH LEVEL CEFOTAXIME RESISTANCE DURING GROWTH IN SERUM, CONTAINING THERAPEUTIC CONCENTRATIONS OF CEFOTAXIME Branka Bedeni}, A. [tampar School of Public Health, Zagreb, Croatia RUNNING HEADLINE: CEFOTAXIME RESISTANCE IN KLEBSIELLA PNEUMONIAE Correspondence: Branka Bedeni}, MD, PhD, Department of Microbiology, A. [tampar School of Public Health, Medical School, University of Zagreb, Rockefeller Street 4, 10000 Zagreb, Croatia, PHONE: + 385 1 46 84 443, FAX: + 385 1 46 84 441 ABSTRACT Background In the previous investigation on genetic characterization of extended-spectrum (-lactamases in Klebsiella pneumoniae from Zagreb, Croatia, 20 strains were found to produce SHV-2 (-lactamase. Those strains displayed a varying degree of (-lactam resistance and a wide range of (-lactamase activity. We concluded that more resistant isolates were hyperproducers of SHV-2 (-lactamase. Methods In this investigation, we tried to develop the hyperproducing variants from 8 low level SHV-2 (-lactamase producing Klebsiellae by subculturing them in the serum containing therapeutic concentrations of cefotaxime. Results In most cases there was a moderate increase in cefotaxime resistance (two fold to three fold) except in one strain which displayed 16 fold increase in cefotaxime MIC after incubation in the serum. That strain showed a marked increase in enzyme activity as well. The strains with moderate increase in cefotaxime MIC did not produce more enzyme after exposure to the serum except of one strain which had a three fold rise in (-lactamase activity after exposure to serum. Conclusions In this investigation, it was established that the mutants with high level cefotaxime resistance developed very quickly in the biological fluids containing therapeutic concentrations of cefotaxime. It is likely to expect that a similar process occurs in the patient infected with an ESBL producing K. pneumoniae strains during antibiotic treatment. Since most of the high level cefotaxime mutants did not have a marked rise in (-lactamase activity after exposure to serum it is possible that the elevated resistance was due to some other mechanism like reduced PBP affinity, changes in outer membrane proteins or efflux by multidrug efflux pumps. Key words: extended-spectrum (-lactamases, cefotaxime, SHV-2 (-lactamase, hyperproduction, Klebsiella pneumoniae INTRODUCTION In the previous investigation on genetic characterization of extended-spectrum (-lactamases in Klebsiella pneumoniae from Zagreb, Croatia, 20 strains were found to produce SHV-2 (-lactamase (1, unpublished data(. Those strains displayed a varying degree of (-lactam resistance and a wide range of (-lactamase activity. We concluded that more resistant isolates were hyperproducers of SHV-2 (-lactamase. A similar phenomenon was previously observed for SHV-5 SYMBOL 98 \f "Symbol"-lactamase producing strains during a nosocomial outbreak (2(. Different isolates of the epidemic strain exhibited a wide range of SYMBOL 98 \f "Symbol"-lactamase acitivites and varying degrees of SYMBOL 98 \f "Symbol"-lactam resistance. It was proved that the strains hyperproducing SHV-5 SYMBOL 98 \f "Symbol"-lactamase could be selected from low SYMBOL 98 \f "Symbol"-lactamase producing strains in vitro by subculturing them in the presence of cefotaxime in the medium. The hyperproduction was due to an increased number of gene copies on the plasmid (3(. It was demonstrated that the genetic mutations leading to hyperproduction of SHV-5 SYMBOL 98 \f "Symbol"-lactamase could be easily gained and lost in vitro, and it was assumed that hyperproducers could also be selected in vivo in patients on SYMBOL 98 \f "Symbol"-lactam therapy. It is well known that hyperproduction of plasmid (-lactamases can be due to an increased number of plasmid copies (4-5(, multiple gene copies on the same plasmid (3( or due to the changes in the promoter region as was previously described for TEM group of enzymes (6-7 ( and for SHV-2 SYMBOL 98 \f "Symbol"-lactamase (8(. Such hyperproducing strains may cause serious therapeutic problems in the hospitals because they are often resistant not only to SYMBOL 98 \f "Symbol"-lactams but also to other antibiotics such as aminoglycosides, chloraphenicol or chinolons (2(. In this investigation, we tried to develop the hyperproducing variants from the low level SHV-2 (-lactamase producing Klebsiellae by subculturing them in the serum containing therapeutic concentrations of cefotaxime.The genetic mechanism od the hyperproduction was not determined in this study. MATERIALS AND METHODS Bacteria The test organisms were 8 clinical isolates of K. pneumoniae producing SHV-2 (-lactamase with low level cefotaxime (CTX) resistance (CTX MIC < 32 mg/L). The strains were obtained from two hospitals in Zagreb, during 1994-1995., from various clinical specimens. (-lactamase characterization and sequencing of blaSHV genes was performed as described previously (1, unpublished results(. Selection of mutants with high level cefotaxime resistance The strains were inoculated in 1 ml of serum obtained from a healthy volunteer who received 1 g cefotaxime iv bolus. 250 ml of blood was taken after 2 hours. The blood was centrifuged in aseptic conditions and the serum was used in the experiments. The cefotaxime concentation in serum was estimated to be 8 mg/L by using E. coli A 15 R+ strain with know cefotaxime MIC. A heavy inoculum (107 CFU/ml) was applied to abrogate the inhibitory activity of cefotaxime. Sequential subcultures were performed on each sample (10 SYMBOL 109 \f "Symbol"l 106 dilution in 1 ml of serum) eight times (8 subcultures). The original low level CTX R (resistant) SHV-2 producers and their high level CTX R mutants obtained after 8 subcultures in the serum were used for antibiotic susceptibility testings and determination of enzyme activity. Determination of minimum inhibitory concentrations (MICs) MICs of amoxycillin, cephalexin, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, cefoperazone, cefepime, cefpirome and aztreonam were determined by a twofold broth microdilution technique using microtiter plates and Mueller-Hinton broth inoculated with 105 cells/ml (9(. Dilutions were prepared for each antibiotic, ranging from 1-1024 mg/l. Clavulanic acid was added to ceftazidime in a fixed concentration (20 mg/L). Antibiotic susceptibilities to cefoxitin and imipenem were determined by disk-diffusion test as described previously (10(. SYMBOL 98 \f "Symbol"-lactamase preparation Microorganisms were grown for 18 h at 37SYMBOL 176 \f "Symbol"C in a shaking incubator. After centrifugation for 15 minutes at 10 000 g, the cells were washed in 0.1 M phosphate buffer, pH 7, and resuspended in the same buffer to give 20 fold concentrated suspensions. The cells were disrupted by sonication in an ice bath and the resulting preparations were centrifuged at 20 000 g for 20 minutes. The supernatans were used as crude enzyme preparations. SYMBOL 98 \f "Symbol"-lactamase assay Enzyme activity was measured from crude extracts by macroiodometric method. Cephaloridine (substrate) was prepared as 5 mM in 0.05 M phosphate buffer pH 7.0. Test and control flasks containing 5 ml substrate were equilibrated at 37 SYMBOL 176 \f "Symbol"C in a shaking water bath before adding 1 ml of enzyme to each of the test flasks. Following a reaction period of 30 minutes, 10 ml of iodine reagent (0.0166 M iodine, 0.06 M potassium iodide in 1.75 M sodium acetate buffer, pH 4.0) was added to stop the enzymatic reaction. A further incubation period of 20 minutes was required for completion of the reaction between the hydrolysis products and iodine. 1 ml of enzyme was added in the control flask after incubation with iodine. The flasks were then removed from the water bath and titrated with sodium thiosulphate (0.0166 M) using a starch indicator. Under those conditions, 1 ml of 0.0166 M iodine reduced was equivalent to 4 SYMBOL 109 \f "Symbol"mol of cephaloridine substrate (11(. (-lactamase activity was standardized against the protein content of the sample. Protein concentration was determined by the method of Lowry (12(. Transfer of high level cefotaxime resistance. K. pneumoniae isolates with low and high level CTX resistance were investigated for the transferability of their resistance determinants. Conjugation experiments were set up employing E. coli A15 RSYMBOL 45 \f "Symbol" strain resistant to rifampicin. Logarithmic phase Mueller-Hinton broth cultures of the donor and recipient strains were mixed in a ratio of 1:2 and incubated without shaking at 37SYMBOL 176 \f "Symbol" C for 18 h. Transconjugant clones were selected on Mac Conkey agar containing cefotaxime (2 mg/L) and rifampicin (256 mg/L). Frequency of transconjugation was expressed relative to the number of donor cells (13(. Investigation of kinetics of development of high level CTX resistance 100 SYMBOL 109 \f "Symbol"l of 106-fold diluted culture of each of 8 subcultures were spread on plates containing doubling dilutions of cefotaxime (0.5-64 mg/L) plus one plate with no antibiotics and incubated at 37SYMBOL 176 \f "Symbol"C overnight. The number of colonies was then counted (3(. RESULTS Antibiotic susceptibilities The antibiotic susceptibilities of low level CTX resistant SHV-2 producers and their high level resistant variants are shown in Table 1. One strain was killed after exposure to serum concentration of cefotaxime and one strain did not show any changes in antibiotic susceptibilities. In most cases, there was a moderate increase in CTX resistance (two fold to three fold), except in strain 4559 which displayed 16 fold increase in CTX MIC after incubation in the serum. The susceptibility to cefoxitin and imipenem, determined by disk-diffusion test, remained unchanged. MICs of ceftazidime were strongly reduced in the presence of clavulanic acid. All strains with the low level cefotaxime resistance (7/7) were resistant to amoxycillin, 85.7% (6/7) were resistant to cephalexin, 57.4% (4/7) to cefaloridine, 42.85 (3/7) to ceftazidime, 28.57% (2/7) to aztreonam, cefoperazone and ceftriaxone, and 14.28% (1/7) to cefpirome. There was no resistance observed to cefepime, cefoxitin, ceftazidime/clavulanate, and imipenem. All high level CTX resistant variants (7/7) were resistant to amoxycillin, cefaloridine, cephalexin, cefuroxime, cefoperazone, and cefpirome. 85.71% of the strains (6/7) were resistant to ceftazidime, ceftriaxone and aztreonam, and 14.28% (1/7) to cefepime. No resistance to ceftazidime/clavulanate, cefoxitin and imipenem was observed. Determnation of (-lactamase activity High level CTX resistance in this investigation was associated with the significant increase in enzyme activity only in the strain 4559 (table 1). Only one strain with a moderate increase in cefotaxime MIC displayed a marked increase in (-lactamase activity strain (59) in distinction from the rest of strains which had only minor elevation in the enzyme production after exposure to serum (less than two fold). Transfer of cefotaxime resistance CTX resistance was transferred to E. coli recipient from only one strain (4559) and only from the low level resistant strain. High level cefotaxime resistance was untransferable. Kinetics of development of high level cefotaxime resistance It was found that phenotypic changes contributing to the higher level of CTX resistance developed already after the first exposure to the serum containing therapeutic concentrations of cefotaxime. Low level CTX resistant strains gave a large number of colonies on the plates containing up to 1 mg/L cefotaxime but only occasional colonies on the higher concentrations; there was no change in behaviour after repeated subculturing in the serum without antibiotic. The strains with high level CTX resistance produced a gradually diminishing number of colonies as the CTX concentration increased, with only a few colonies at the concentrations of >4 mg/L. After repeated subculture in the serum, there were more colonies on the plates with higher concentration of CTX (Figures 1 and 2). DISCUSSION In this investigation, it was established that the mutants with high level CTX resistance developed from low level CTX resistant SHV-2 producers very quickly in the biological fluids containing therapeutic concentrations of CTX. It is likely to expect that a similar process occurs in the patient infected with an ESBL producing K. pneumoniae strains during antibiotic treatment and that subMIC concentrations of (lactam antibiotic which are present in the biological fluids between two doses can stimulate the genetic mutations leading to higher level of resistance to these antibiotics. In the species which possess a functional amp R gene, an increase in (-lactamase production can be due to induction or derepression (14(. There is no amp R gene in Klebsiella spp but the increase in enzyme production may result from the increased plasmid copy number, more gene copies on the same plasmid, or from mutations in the promoter sequence (3,5-6(. High level CTX resistance in this investigation was associated with the significant increase in enzyme activity only in one strain (Kl. 4559). Since most of the strains did not show a marked increase in (-lactamase activity after exposure to serum, it is possible that the elevated resistance was due to some other mechanisms such as reduced PBP affinity (15(, alterations in porin channels (16( or efflux by multidrug efflux pumps as described previously (17(. The lack of the outer membrane protein OmpK 36 was associated with high level resistance to ceftazidime, cefpirome and cefepime in K. pneumoniae strains producing different TEM and SHV ESBLs (16(. In the other publication (17(, it was stated that the presence of ArcAB pump was responsible for raising the MIC of lipophilic (-lactams to a high level. (-lactamases of all our strains were strongly inhibited by clavulanic acid. According to our results, there were different mechanisms involved in the development of high level cefotaxime resistance in ESBL producing organisms. Unfortunately, the transconjugants were not obtained in the mating experiment which would enable to study the resistance mechanism in an isogenic system. The mutants highly resistant to CTX, described in this investigation, were resistant to almost all (-lactams apart of imipenem and cefoxitin, bearing in mind that some of those strains displayed reduced susceptibility to cefoxitin as well. After exposure to serum most of them developed resistance not only to cefotaxime but also to other third generation cephalosporins like ceftazidime, ceftriaxone and aztreonam and to forth generation cephalosporin cefpirome. The activity of cefepime appeared to be affected by mutations leading to a high level cephalosporin resistance less than that of the other antibiotics. A marked elevation in cefepime MIC was observed only in two strains (8-16 fold). It was associated with hyperproduction of (-lactamase in one of those strains (4559). The older generation cephalosporins like cephaloridine, cephalexin and cefuroxime had high MICs even in low level CTX resistant Klebsiellae (before exposure to serum). Some of those mutants obtained after exposure to serum had the cefotaxime and ceftriaxone MICs below the resistance break-point, which was interpreted as susceptibility or intermediate susceptibility but the previous investigations proved that those antibiotics are often inefficient in vivo in spite of the fact that in vitro tests show susceptibility. For that reason, the most recent version of the NCCLS document recommends that all ESBL producing strains should be considered resistant to all (-lactams except for imipenem with independence of the MIC values (9(. Contrary to the previous investigations, the hyperproducers in this study retained susceptibility to inhibition by clavulanic acid (2(. Most of the test strains were resistant to aminoglycosides, tetracyclines, sulphonamides and chloramphenicol and some of them were resistant to ciprofloxacin as well (results not shown). For that reason such organisms pose a serious therapeutic problem in Croatian hospitals. Imipenem remains the antibiotic of choice for treatment of infections caused by ESBL producing strains, but since the (-lactamases capable of hydrolysing that antibiotic have already appeared in some countries of the world (18(, it is likely to expect that the organisms producing such enzymes will be a problem in Croatia in the future as well. REFERENCES Bedenic B, Zagar Z: Extended-spectrum SYMBOL 98 \f "Symbol"-lactamases in clinical isolates of Klebsiella pneumoniae from Zagreb, Croatia. J Chemother 1998SYMBOL 59 \f "Symbol" 10(6):449-459. French, G L, Shannon, K P, Simmons, N: Hospital outbreak of Klebsiella pneumoniae resistant to broad-spectrum cephalosporins and SYMBOL 98 \f "Symbol"-lactam-SYMBOL 98 \f "Symbol"-lactamase inhibitor combinations by hyperproduction of SHV-5 SYMBOL 98 \f "Symbol"-lactamase. J Clin Microbiol 1996; 34: 358-363. Xiang X, Shannon K, French G: Mechanisms and stability of hyperproduction of the extended-spectrum SYMBOL 98 \f "Symbol"-lactamase SHV-5 in Klebsiella pneumoniae. J Antimicrob Chemother 1997; 40: 525-532. Martinez JL, Vincente MF, Delgrado-Iribarren A, Perez-Diaz JC, Baquero F. Small plasmids are involved in amoxycillin-clavulanate resistance in Escherichia coli. Antimicrob Agents Chemother 1989;33:595. Wu PJ, Shannon KP, Phillips I: Mechanisms of hyperproduction of TEM-1SYMBOL 98 \f "Symbol"-lactamase by clinical isolates of Escherichia coli. J Antimicrob Chemother 1995; 36: 927-9. Chen ST, Clowes R C: Two improved promotor sequences for the SYMBOL 98 \f "Symbol"-lactamase expression arising from a single base-pair substitution. Nucleic Acid Research 1984;12:3219-34. Chen ST, Clowes RC: Variation between the nucleotide sequences of Tn1, Tn2 abd Tn3 and expression of SYMBOL 98 \f "Symbol"-lactamase in Pseudomonas aeruginosa and Escherichia coli. J Bacteriol 1987;169:913-6. Podbielski A, Schonling J, Melzer B, Haase G: Different promoters of SHV-2 and SHV-2a SYMBOL 98 \f "Symbol"-lactamase lead to diverse levels of cefotaxime resistance in their bacterial producers. Gen Microbiol 1991;137:1667-75. NCCLS: Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard-fifth edition. NCCLS document M7-A5.NCCLS:Wayne, Pennsylvania;2000. NCCLS: Performance standards for antimicrobial disk susceptibility tests - fifth edition; approved standard. In: NCCLS document M2-A5. NCCLS. Villanova, Pa: 1993. Perret CJ: Iodometric assay of Penicillinase. Nature 1954; 174: 1012-1013. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-275. Elwell LP, Falkow S: The characterization of R plasmids and the detection of plasmid-specified genes; In Lorian V (ed): Antibiotics in Laboratory Medicine. Baltimore, Williams and Wilkins, 1986, pp 683-721. Phillips I, Shannon K: Class I (-lactamases, induction and derepression. Drugs 1989; 37:402-407. Livermore DM: Mechanisms of resistance to cephalosporin antibiotics. Drugs 1987; 34 (Suppl 2): 64-88. Martinez-Martinez L, Pascual A, Hernandez-Alles S, Alvarez-Diaz D, Suarez AI, Tran J, Benedi Javier B, Jacoby GA: Roles of (-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrob Ag Chemother 1990; 43:1669-1673. Nikaido H: Crossing the envelope: how cephalosporins reach their targets. Clin Microbiol Infect 2000: 6 (Suppl 3):22-26. Bradford P A, Urban C, Mariano N, Projan S J, Rahal J J, Bush K: Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC SYMBOL 98 \f "Symbol"-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother 1997; 41(3): 563-569. FIGURES LEGEND 1. Figure 1. Kinetics of development of high level cefotaxime resistance in the strain 4559 with marked increase in enzyme activity after exposure to serum. 2. Figure 2. Kinetics of development of high level cefotaxime resistance in the strain 4667 without marked increase in enzyme activity after exposure to serum. PAGE  PAGE 9 #)*;<'2 GH/:A B o { & '   ? @ ] r 45?@ATϨ j[CJCJH* 6CJmH 5CJmH jbCJmHCJmH6CJ jbCJCJ5CJCJH*OJQJmHCJOJQJmH5CJ5CJOJQJ 56CJ5CJ=*+,'2o {  8Ad$7d$d*+,'2o {  8A<v#9$e%&-'Z(c((+,--//j00l13333:EEEGGHsI4JKKLIMMNN                                 FTU LMbc*+@A+,/0[\]^~YZoz"78Ap}FCJ5CJ6CJ j[CJ jCJU jbCJCJ j]CJVFGux{@GNO  $%<mxyz i#j#########3$4$6$7$9$u$ %'%-%.%C%jCJEHUj6CJUCJEHCJEH jCJUCJH* j]CJ j[CJCJH*6CJCJ jbCJJ<v#9$e%&-'Z(c((+,--//j00l13333:$7d$dC%D%E%%%&&&&&&&-'1'2'H'I'P'Q'((((T(U(V(W([(c((----..////j0033355c5d5Y6Z6666666r7s7x7y7F8G8ȺòòòæÞæÞò j]CJmH j[CJmH5CJ jbCJmH jb6CJmHCJmH 6CJmH5CJCJEH6CJ j]CJ j[CJ jCJUCJCJEHjCJEHU>G888889999D9E9G9H999 : : : :(:):+:,:::::<< ? ???AABBBBBBBB,D-DDDDDEEBECEXEYE}EEEEEEF3FcFdFyFzFFFFFFFFF G GGG5CJ6CJ jCJUCJ 5CJmH jbCJmH 6CJmH j]CJmH j[CJmHCJmHN:EEEGGHsI4JKKLIMMNNKOOP1]@1111 14]@1 1@120^@1Z0lb@1Hj@1\1p@11p@111111&1f1s@1h1Js@11110t@1&1^z@1(1z@1*1{@11~@1:1@1,@1B0P@0@0@10@00@GTimes New Roman5Symbol3& Arial7HRTimes"V hC3&IT&IT&[] A!$V ant to amoxycillin, 85.7% (6/7)T (three fold),yme activity only in one strain 9 $d [4@4NormalCJOJQJkHmH <A@<Default Paragraph Font,@,Header  9r &)@& Page NumberO'p()+,e-/6 AOOOOOOOOin association with production of TEM and SHV ESBLs induced high level of resistance to as described by Martinez et al 9 ant to amoxycillin, 85.7% (6/7)T (three fold)7 @@@@@ TFC%G8G2RUVXY[`:rRSWZ]fNST\UnknownBranka Bedeni M. StegiL b * @ ~i-!C!!"1#H#$$RAhAAAsBBBBBBCCE$EEEFFmGGNNO999999999999999999999999999999 !!Branka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni.C:\WINDOWS\TEMP\AutoRecovery save of HIPER.asd7   cnH) l%8 _pkP (1]@1111 14]@1 1@120^@1Z0lb@1Hj@1\1p@11p@111111&1f1s@1h1Js@11110t@1&1^z@1(1z@1*0{@0@0@10@00@GTimes New Roman5Symbol3& Arial70dPBrankaBranka BedenilCJOJQJkHmH <A@<Default Paragraph FontG Sbjbjَ O]?77777K8U<,K`&l"A!C!C!C!%h!""$N'B)R"t7"GGGGGGGGG}HHHHII8IHIVIWIIIIIJJJJJJJJ]K^KsKtK~MMMMNNNN O OJOLO.P/P~PPPP;Qzo p@1Table)WordDocument}SummaryInformation( e  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ~r_^[]sodfghijklmn|ctabuvwxyz{\BD7A-E12E-11D2-8E8D-CBCBFEBEB77C} Oh+'0p   , 8 DPX`hssBrankaran Normal.dotBranka Bedeni91nMicrosoft Word 8.0@nGx@~5@&T[i@h A  FMicrosoft Word Document MSWordDocWord.Document.89q ՜.+,D՜.+,0 hp  Zagrebl!P   Title 6> _PID_GUIDAN{A21Fhh.hh.hh.hh.hh.hh.p=}l%8c|_pkPcnH)7 (zoH߯K@1Table?WordDocumentSummaryInformation(  [4@4NormalCJOJQJkHmH <A@<Default Paragraph Font,@,Header  9r &)@& Page NumberO'p()+,e-/6,AOOOOOOOO@@@@@ TFC%G8G2bRUVXY[`:rRbSWZ]NST\UnknownBranka Bedeni M. StegiL b * @ ~i-!C!!"1#H#$$^AtAAABBBBB CCCE0EEEFFyGG N#NO999999999999999999999999999999 !!Branka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni'C:\My Documents\neobj. Radovi\HIPER.DOCBranka Bedeni.C:\WINDOWS\TEMP\AutoRecovery save of HIPER.asd7   cnH) l%8 _pkP (?@ABCDEFGHIJKLMNOPQRSTUVWXYZ~_^`]rsoptabuvwxyz{\c| p=} hh.hh.hh.hh.hh.hh.hh.hh.p=}l%8c|_pkPcnH)7 (1]@1111 14]@1 1@120^@1Z0lb@1Hj@111111&1f1s@1h1Js@11110t@1&1^z@1(1z@1*0{@0@0@1\0@0^0@GTimes New Roman5Symbol3& Arial7HRTimes"V hC3&DT%3TYO A!$V 0dP