7 7 9 99 9 Escherichia coli Klebsiella pneumoniae MIC9.5 g ml Enterobacter cloacae Citrobacter freundii Serratia marcescens meropenem MIC9. g ml ceftazidime 5.5 Double disk synergy test DDST 9 mercapto propionic acid test MP 5 extended spectrum lactamase ESBL CTX M 9 metallo lactamase MBL 5 IMP 99 K. pneumoniae SHV ESBL IVH ESBL MBL aztreonam Key words:blood culture, ESBL, metallo lactamase, Enterobacteriaceae, PCR extended spectrum lactamase ESBL metallo lactamase MBL 5 6 7 Escherichia coli Klebsiella spp National Committee for Clinical Laboratory Standard NCCLS ESBL ESBL E. coli Klebsiella pneumoniae 9 MBL 99 ESBL MBL 5 DNA I 99 9 VITEK GNI 9 NCCLS MIC MIC5 MIC MIC9 piperacillin PIPC aztreonam AZT cefpodoxime CPDX cefotaxime CTX ceftazidime CAZ cefepime CF- PM cefpirome CPR meropenem MEPM imipenem IPM panipenem PAPM sulbactam cefoperazone SBT CPZ amikacin AMK ciprofloxacin CPFX. ESBL MBL
E. coli Klebsiella spp NCCLS CAZ CTX MIC 6 ml double disk synergy test DDST ESBLs DDST NCCLS MacFarland.5. ml amoxicillin clavulanic acid AMPC CVA disk CPDX CTX CAZ CPR CFPM 5 mm : BBL5 PCR TEM SHV CTX M group CTX M CTX M CTX M9 MBL NCCLS MacFarland.5. ml SMA CAZ IPM MBL PCR IMP 5. Arbitrarily primed PCR AP PCR ESBL AP PCR Enterobacter cloacae 9 K. pneumoniae E. cloacae ERIC 5 AAGTAAGTGACTGGGGTGA- GCG 6 Serratia marcescens Hejajy 7 HLWL 7 5 CGTCTATGCA 5 5 AACCCACGCC DNA LB rpm 5 DNA Takara Taq Mg mm.5 5. ESBL MBL ESBL MBL II 99 9 9. 999 5 Table 5 MIC5 MIC MIC9 Table E. coli K. pneumoniae MIC9.5 ml Citrobacter freundii S. marcscens MIC9 ml MBL MEPM MIC9. ml Table ESBL CAZ 5.5 C. freundii 5. E. coli K. oxytoca Proteus mirabilis Table Detection of Enterobacteriaceae isolated from blood cultures 99 Year 99 99 99 99 995 996 997 99 999 Total Total no of strains 9 66 6 5 5 9 Enterobacteriaceae no of strains 5 9 55 9 E. coli K. pneumoniae K. oxytoca E. aerogenes C. freundii P. mirabilis M. morganii 5 6 5 5 9 7 6 5 7 5 6 9 5 76 66
Table Antimicrobial activity of agents in 9 strains of Enterobacteriaceae detected in blood cultures between 99 and Organism MIC MICs µg ml no of isolates 5 9 PIPC CPDX S C AZT CTX CAZ CFPM CPR MEPM IPM PAPM AMK CPFX E. coli 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 9 6 5 5 5 5 5 5 K. pneumoniae 5 5 5 5 5 5 5 5 76 5 5 5 5 5 5 5 5 5 9 5 5 5 5 5 5 5 5 5 5 5 5 5 5 66 6 5 9 5 5 5 5 5 5 5 6 9 6 C. freundii 5 5 5 5 5 5 5 5 6 5 5 5 9 5 5 All strains 5 5 5 5 5 5 5 5 9 6 5 5 5 5 5 9 6 PIPC: piperacillin, CPDX: cefpodoxime, S C: sulbactam cefoparazone, AZT: aztreonam, CTX: cefotaxime, CAZ: ceftazidime, CFPM: cefepime, CPR:cefpirome,MEPM: meropenem, IPM: imipenem, PAPM: panipenem, AMK: amikacin, CPFX: ciprofloxacin Table Antibiotic tolerance Organism no of isolates No of strains of intermediate and resistant a PIPC CPDX AZT CTX CAZ CFPM MEPM IPM AMK CPFX E. coli 5 5 5 6 7 6 7 K. pneumoniae 76 9 9 6 K. oxytoca 66 5 6 69 7 5 6 5 5 5 5 5 7 E. aerogenes 7 9 6 6 7 5 7 7 C. freundii 6 5 66 7 6 5 6 5 6 5 6 7 P. mirabilis M. morganii 55 6 5 5 9 9 7 9 5 65 7 9 5 7 9 5 6 9 9 All 9 7 55 6 7 7 6 5 5 5 5 9 9 a Strains of intermediate and resistant were befined according to NCCLS criteria PIPC: piperacillin, CPDX: cefpodoxime, CTX: cefotaxime, AZT: aztreonam, CAZ: ceftazidime, CFPM: cefepime, MEPM: meropenem, AMK: amikacin, CPFX: ciprofloxacin E. coli CPDX 7. C IPM. MEPM.5 AMK. CPFX.9. ESBL MBL DDST MP Table MIC Table 5 NCCLS ESBL E. coli Klebsiella spp 9.7 7. 79. DDST 9 5.7 5 K. pneumoniae C. freundii CTX M 9 C. freundii CTX M K. pneumoniae TEM SHV CTX M SHV ESBL MIC MEPM AMK CPFX 7 MP 5.5
Table Strains indicated rather than extraction conditions a for DDST and MP procedures Total no strains No of DDST b positive No of MP positive E. coli 5 K. pneumoniae 76 K. oxytoca 66 E. aerogenes C. freundii P. mirabilis M. morganii 6 7 9 6 5 5 6 6 5 9 9 5 7 5 6 All 9 79 9 5 7 5 5 a E. coli and Klebsiella spp : rather than µg ml of MIC of cefpodoxime, others Enterobacteriaceae: rather than 6 µg ml of MIC of ceftadime and or cefotaxime b DDST: double disk synergy test c MP: Inhibitory effects of mercaptopropionic acid 5 IMP AZT MIC IPM ESBL MBL 996 99 K. pneumoniae SHV ESBL AP PCR Fig ESBL No.7 6 No. 6 No.7 No.7 No.6 No. No.6 ESBL K. pneumoniae MBL 996 99 No. 7 999 No.6 9 No. 7 6 5 ESBL MBL 5 Table 6 ESBL 6 IVH 5 5 K. pneumoniae Case no. M 7 6 6 5 6 Case no. M 9 6 7 Case no. M 7 6 9 Fig. AP PCR patterns of ESBL producing Enterobacter cloacaeklebsiella pneumoniae and MBL producing Serratia marcescenslane: M bp ladder molecular weight marker. Others lanes: Correspond to strain number listed in Table 6.
Table 5 MICs and β lactamase gene types of DDST and MP positive strains Case Organism β lactamase gene no MICs µg ml PIPC CPDX S C AZT CTX CAZ CFPM CPR MEPM IPM PAPM AMK CPFX 7 9 6 6 5 6 CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M CTX M 6 6 6 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 6 6 5 9 6 7 K. pneumoniae K. pneumoniae K. pneumoniae TEM, SHV, CTX M TEM, SHV, CTX M SHV 6 5 6 6 6 5 5 5 C. freundii TEM, CTX M 6 7 6 9 IMP IMP IMP IMP IMP 6 6 6 6 6 6 6 6 6 6 6 6 PIPC: piperacillin, CPDX: cefpodoxime, S C: sulbactam cefoparazone, AZT: aztreonam, CTX: cefotaxime, CAZ: ceftazidime, CFPM: cefepime, CPR: cefpirome, MEPM: meropenem, IPM: imipenem, PAPM: panipenem, AMK: amikacin, CPFX: ciprofloxacin
Table 6 Information on patients infected by ESBL and MBL producing organisms Case no Organism β lactamase gene Detected year Antibiotics Clinical Age Underlined disease a before after effect Simultaneous detection Prognosis 7 CTX M 996 7 Stevens Johnson syndrome CAZ, IPM IPM PAPM S C LMOX unknown PIPC CFPM MRSA,, P. aeruginosa 6 CTX M 997 acute lymphocytic leukemia CPFX, FLCZ AZT, CLDM AMK, CLDM good good CTX M 997 69 hypoplastic leukemia CPFX, FLCZ PAPM CPR good MSSA good 6 CTX M 999 acute cholecystitis no therapy CFPM PAPM good good MRSA, CTX M 999 5 acute myelocytic leukemia CAZ, TEIC CAZ,TEIC C. albicans CTX M 999 6 myocardial infarction no therapy PIPC GM good good 5 CTX M 79 renal failure CPR, MINO MINO PAPM 6 CTX M 7 aortic dissedction TEIC TEIC unknown MRSA good CTX M 77 gastric cancer CTM, PAPM PAPM good good 9 K. pneumoniae TEM, SHV, CTX M 995 7 intracerebral bleeding CAZ, AMPC CAZ good 6 K. pneumoniae TEM, SHV, CTX M 99 7 neck tumor no therapy IPM CAZ good good 7 K. pneumoniae SHV 99 ARDS ABPC, TOB CTX, TOB good good C. freundii TEM, CTX M 9 brain tumor no therapy AMPC CAZ hepatic cirrhosis IMP 996 66 CFPM PAPM AZT good good polycythemia IMP 997 7 acute renal failure PIPC PIPC unknown heart failure 7 IMP 99 head injury CTM CAZ AZT good good 6 IMP 999 5 spinal cord injury CTM FMOX chronic renal failure 9 IMP 5 CMZ GM good renal transplantation renal failure a ARDS: acute respiratory distress syndrome CAZ: ceftazidime, IPM: imipenem, PAPM: panipenem, S C: sulbactam cefoparazone, LMOX: latamoxef, PIPC: piperacillin, CFPM: cefepime, CPFX: ciprofloxacin, FLCZ: fluconazole, AZT: aztreonam CLDM: clindamycin, AMK: amikacin, CPR: cefpirome, TEIC: teicoplanin, GM:gentamicin, MINO: minocycline, CTM: cefotiam, AMPC: amoxicillin, ABPC: ampicillin, TOB: tobramycin, CTX: cefotaxime, FMOX: flomoxel, CMZ: cefmetazole
6 7 9 PAPM AMK gentamicin tobramycin CAZ No.9 No.6 CTX M K. pneumoniae CAZ MIC ml No.5 minocycline No. CAZ MIC ml No.6 No. CPFX CPFX translocation MBL ICU GVHD 5 AZT MBL PIPC GM Cefotiam flomoxef III E. coli K. pneumoniae C. freundii MIC9 ml C C ESBL C. freundii S. marcescens C ESBL MEPM MIC9. ml MBL ESBL Jarlier DDST MBL Arakawa PCR ESBL 9 9 5.7 ESBL 9 5.7 9 CTX M 9 MBL 5. IMP ESBL 9 Knoche 99 K. pneunoniae SHV ESBL ESBL 996 ESBL MBL ESBL
MBL 996 997 99 999 996 997 99 999 R Pulsed field gel electrophoresis AP PCR 5 PCR ESBL MBL IVH MRSA ESBL ESBL CAZ CAZ MIC ml CTX M CAZ NCCLS ESBL NCCLS MBL AZT MBL AZT AZT MIC ml AZT AZT AZT MIC blaimp aac aad Palucha A, Mikiewict B, Hryniewicz W, et al.: Concurrent outbreaks of extended spectrum lactamase producing of the family Enterobacteriaceae in a Warsaw hospital. J Antimicrob Chemother : 999 999 Rebuck J A Olsen K M Fey P D et al.: Characterization of an outbreak due to extended spectrum lactamase producing klebsiella pneumoniae in a pediatic intensive care unit transplant population. Clin infect Dis : 6 7 Livermore D M Yuan M: Antibiotic resistance and production of extended spectrum lactamases amongst Klebsiella spp. from intensive care unit in Europe. J Antimicrob Chemother : 9 996 : Escherichia coli SHV extended spectrum lactamase SHV 7: 9 5 : Serratia marcescens 9: 5 999 6 Komatsu M Ikeda N Aihara M et al.: Hospital outbreak of MEN derived extended spectrum beta lactamase produsing Klebsiella pneumoniae JinfectChemother 7: 9 7 : 99 Serratia marcescens IMP
76: 6 5 National Committee for Clinical Laboratory Standards: Performance standards for antimicrobial disk susceptibility testing. Eleventh informational supplement M S : National Committee for Clinical Laboratory Standards Villanova Pa., 9 : Escherichia coli Klebsiella spp extended spectrum lactamase 5: 5 : 7: 7 5 : 5: 5 Jarlier V Nicolas M H Fournier G et al.: Extended broad spectrum beta lactamases conferring transferable resistance to newer lactum agents in Enterobacteriaceae: hospital prevalence and susceptibility patterns. Rev Infect Dis : 67 7 9 : Extended spectrum lactamase 7: 5 5 Arakawa Y Shibata N Shibayama K et al.: Convenient test for screening metallo beta lactamase producing gram negative bacteria by using thiol compounds. J Clin Microbiol : 5 Senda K Arakawa Y Nakashima K et al.: Multifocal outbreaks of metallo lactamase producing Pseudomonas aeruginosa resistant to broad spectrum beta lactams including carbapenems. Antimicrob Agents Chemother. : 9 5 996 6 Versalovic J Koeuth T Lupski J R: Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 9: 6 6 99 7 Hejazi A Keane C T Falkiner F R et al.: The use of RAPD PCR as a typing method for Serratia marcescens.jmedmicrobiol 6: 9 99 997 Balis E Vatopoulos A C Kanelopoulou M et al.: Indications of in vivo transfer of an epidemic R plasmid from Salmonella enteritidis to Escherichia coli of the normal human gut flora. J Clin Microbiol : 977 979 996 9 Leblebicioglu H Gunaydin M Esen S et al.: Surveillance of antimicrobial resistance in gram negative isolates from intensive care units in Turkey: analysis of data from the last 5 years. J Chemother : 6 Perilli M Amico E D Segatore B et al.: Molecular characterization of extended spectrum lactamases produced by nosocomial isolates of Enterobacteriacea from an Italian nationwide survey. J Clin Microbiol : 6 Bell J M Turnidge J D Gales A C et al.: Prevalence of extended spectrum lactamase ESBL producing clinical isolates in the Asia Pacific region and South Africa: regional results from SENTRY antimicrobial surveillance progrum 99 99 Diag Micro Infect Dis : 9 9 Knothe H Shah P Krcmery V et al.: Transferable resistance to cefotaxime cefoxitin cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection : 5 7 9 : 5 Patton T G Katz S Sobieski R J et al.: Genotyping of clinical Serratia marcescens isolates: a comparison of PCR based methods. FEMS microbial Lett 9: 9 5 5 Liu P Y Lau Y Hu B et al.: Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection. J Clin Microbiol : 95 9 99
Susceptibility and analysis of resistance to lactum agents of Enterobacteriaceae detected in blood cultures Tatsuya Nakamura Nobuaki Matsuo and Hakuo Takahashi Clinical Central Laboratory Kansai Medical University Hospital 5 Fumizono cho Moriguchi Osaka 57 57 Japan Department of Emergency and Critical Care Medicine Kansai Medical University Hospital We studied resistance to lactum agents in 9 strains of Enterobacteriaceae detected in blood cultures in the years between 99 and. Drug sensitivity tests showed good sensitivity MIC9:.5 g ml in third generation cephem and in carbapenem to Escherichia coli and Klebsiella pneumoniae. We found relatively good sensitivity of fourth generation cephem the sensitivity of third generation cephem was slightly lower and carbapenem to Enterobacter cloacae Citrobacter freundii and Serratia marcescens For all Enterobacteriaceae meropenem was the lowest MIC9:. ml Resistance among bacterial species using ceftazidime was the highest in 5.5Double disk synergy tests DDST were positive in 9 strains and mercapto propionic acid tests MP were positive in 5 strains. As resistance genes the extended spectrum lactamase ESBL producing gene CTX M was detected in of 9 strains. The metallo lactamase MBL producing gene was detected in 5 strains which were IMP. In K. pneumoniae detected in 99 the presence of SHV ESBL producing bacteria was confirmed. A clinical background survey showed a high incidence of serious conditions such as malignant tumors and cerebrovascular diseases as underlying diseases and IVH catheters had been inserted in all patients. Clinical symptoms were improved after administration of carbapenem and aminoglycosides in patients with ESBL producing bacteria infection and by administration of aztreonam in patients with MBL producing bacteria infection.