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Enterobacter cloaceae
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Papers 148Most cited
Enterobacter cloacae :: isolation & purification
Latest Paper:
Rev Esp Anestesiol Reanim. 2009 Apr ;56 (4):239-44 19537264
[Severe perioperative thrombocytosis in a patient undergoing lung resection]
B Fernández Torres, E Ramos Martínez, R Rosendo Ríos, R Rodríguez Mejías, A Gutiérrez Guillén, M de las Mulas Béjar
Departamento de Anestesiología y Reanimación, Hospital Virgen Macarena, Sevilla. barfertor@tiscali.es
Severe thrombocytosis (platelet count > 1,000,000 microL(-1)) is a rare, usually reactive, process and few perioperative cases have been reported. We describe the management of a patient who developed severe reactive thrombocytosis in the preoperative period before undergoing segmentectomy to remove a malignant nodule. A platelet count of 2,086,000 microL(-1) was observed during the first few days after surgery; we therefore started antiplatelet therapy to prevent thrombotic complications. We analyze the factors that might have contributed to the development of severe thrombocytosis in this case and discuss the different treatment options that may affect perioperative outcomes in these patients.
Most cited papers:
Proc Natl Acad Sci U S A. 1995 Mar 14;92 (6):2081-5 7892228 Cit:129
FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.
C H Jones, J S Pinkner, R Roth, J Heuser, A V Nicholes, S N Abraham, S J Hultgren
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Type 1 pili are heteropolymeric mannosebinding fibers produced by all members of the Enterobacteriaceae family. The bulk of the fiber is composed of FimA. Two macromolecular complexes responsible for mediating an interaction with mannose-containing receptors were purified from fimA- Escherichia coli by mannose affinity chromatography and ion-exchange chromatography. One complex contained only the mannose-binding adhesin, FimH, associated with FimG, a minor component of the type 1 pilus. In the other complex the FimG-FimH moiety was loosely associated with a chaperone-minor subunit complex (FimC-FimF), possibly representing an intermediate in tip fibrilla assembly. The FimC chaperone has also been shown to form a preassembly complex with FimH that has been purified and characterized previously. Purified FimC did not bind to the FimG-FimH complex but did recognize FimH dissociated from the FimG-FimH complex. Quick-freeze deep-etch electron microscopy revealed that the FimG-FimH complex had a thin fibrillar architecture. High-resolution electron microscopy of type 1 pili revealed that a 16-nm fibrillar tip structure with an architecture identical to that of the FimG-FimH complex was joined end-to-end to the pilus rod. In a fimH- deletion mutant, the tip fibrillae joined to pilus rods were approximately 3 nm in length. The full-length tip fibrilla was restored by complementation with the fimH gene in trans. The bipartite nature of the type 1 pilus was also demonstrated on pili purified from clinical isolates of members of the Enterobacteriaceae family arguing that it is a conserved feature of the type 1 pilus.
J Infect Dis. 1995 Sep ;172 (3):886-91 7658090 Cit:57
Antibiotic-induced endotoxin release in patients with gram-negative urosepsis: a double-blind study comparing imipenem and ceftazidime.
J M Prins, M A van Agtmael, E J Kuijper, S J van Deventer, P Speelman
Department of Internal Medicine, Academic Medical Center, Amsterdam, Netherlands.
The clinical significance of differences between antibiotics in endotoxin-liberating potential is unknown. Thirty patients with gram-negative urosepsis were randomized between imipenem and ceftazidime, which have, respectively, a low and a high endotoxin-liberating potential in vitro. In patients treated with ceftazidime, a slower defervescence was noticed. After 4 h of treatment, the blood endotoxin level decreased in all 3 endotoxemic patients receiving imipenem, whereas it increased in 2 of the 4 endotoxemic patients receiving ceftazidime, and in ceftazidime-treated patients, the endotoxin level in urine decreased less than in imipenem-treated subjects. Serum and urine cytokine levels increased 10%-40% after 4 h of ceftazidime treatment compared with no increase in the imipenem-treated patients (P >.05). Endotoxin release during antibiotic killing in vitro, assessed for all microorganisms, was 10-fold higher with ceftazidime (P <.001). These results indicate that differences between antibiotics in endotoxin release may affect the inflammatory response during treatment.
J Clin Microbiol. 2002 Apr ;40 (4):1237-43 11923338 Cit:47
Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period.
Rafael Cantón, Antonio Oliver, Teresa M Coque, María del Carmen Varela, José Claudio Pérez-Díaz, Fernando Baquero
Servicio de Microbiología Hospital Ramón y Cajal, Madrid, Spain. rcanton@hrc.insalud.es
Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.
J Clin Microbiol. 1994 Mar ;32 (3):596-602 8195364 Cit:34
Arbitrarily primed PCR, ribotyping, and plasmid pattern analysis applied to investigation of a nosocomial outbreak due to Enterobacter cloacae in a neonatal intensive care unit.
F Grattard, B Pozzetto, P Berthelot, I Rayet, A Ros, B Lauras, O G Gaudin
Laboratoire de Bactériologie-Virologie, Faculté de Médicine J. Lisfranc, Saint-Etienne, France.
In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.
J Hosp Infect. 1994 Dec ;28 (4):273-86 7897189 Cit:31
Enterobacter cloacae in a neonatal intensive care unit: account of an outbreak and its relationship to use of third generation cephalosporins.
D Acolet, Z Ahmet, E Houang, R Hurley, M E Kaufmann
Hillingdon Hospital, London, UK.
After uneventful use of cefotaxime and ceftazidime as first line therapy for three years in our neonatal intensive care unit we isolated cephalosporin-resistant Enterobacter cloacae (CREC) strains which caused clusters of cases or colonization and/or serious neonatal infection. By using two or more typing methods, at least five different strains with similar patterns of antimicrobial sensitivities were identified. The results of a case-control study did not support the notion that the use of third generation cephalosporins was associated with colonization and infection by CREC. The outbreak was brought under control by interrupting the transmission of the epidemic strain D, by measures such as cohort nursing, diligent handwashing before and after procedures, and thorough environmental cleaning as well as by decontamination with glutaraldehyde after dismantling of the blood gas analyser believed to have acted as a persistent reservoir. Our experience highlights the danger of inadequate supervision and maintenance of equipment used for near-patient testing and the need to monitor such equipment not only in terms of its calibration and analytical performance but also microbiologically.
J Biol Chem. 1995 Mar 17;270 (11):5729-35 7890700 Cit:29
Molecular evolution of a class C beta-lactamase extending its substrate specificity.
M Nukaga, S Haruta, K Tanimoto, K Kogure, K Taniguchi, M Tamaki, T Sawai
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.
Clin Infect Dis. 1992 Jul ;15 (1):30-2 1352150 Cit:26
Molecular analysis provides evidence for the endogenous origin of bacteremia and meningitis due to Enterobacter cloacae in an infant.
N Lambert-Zechovsky, E Bingen, E Denamur, N Brahimi, P Brun, H Mathieu, J Elion
Laboratoire de Microbiologie, Hôpital Robert Debré, Paris, France.
We analyzed the restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) to document the occurrence of endogenous, systemic bacteremia and meningitis due to Enterobacter cloacae in a newborn. Five strains of E. cloacae were isolated from this newborn. Three of these strains were recovered from stool at counts of 10(8), 10(9), and 10(9) organisms/g of feces, respectively; one strain was isolated from blood; and one strain was isolated from cerebrospinal fluid. In addition, five epidemiologically unrelated strains of E. cloacae were studied for comparison. Our study clearly shows the genetic relatedness of the strains isolated sequentially from cultures of stool, blood, and cerebrospinal fluid. RFLP analysis of total DNA and ribotyping seem particularly well suited to the study of the epidemiology of nosocomial E. cloacae strains.
J Hosp Infect. 1992 Jun ;21 (2):95-101 1353097 Cit:24
Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections.
E Bingen, E Denamur, N Lambert-Zechovsky, N Brahimi, M el Lakany, J Elion
Laboratoire de Bactériologie, Hôpital Robert Debré, Paris, France.
Restriction fragment length polymorphism analysis (RFLP) of total DNA and rDNA regions was used for the epidemiological evaluation of 10 Enterobacter cloacae nosocomial isolates obtained from nine patients in our hospital. Five of these patients were hospitalized during overlapping periods, thus raising the question of cross-contamination. A single biochemical pattern and antibiotic susceptibility profile was observed for all isolates but one. In contrast, based on the results of total DNA and rDNA RFLP patterns, the genetic unrelatedness of the isolates was clearly shown, thus excluding a common source of contamination or patient-to-patient transfer.
J Antimicrob Chemother. 2002 Oct ;50 (4):503-11 12356794 Cit:21
Metallo-beta-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii.
Jing-Jou Yan, Wen-Chien Ko, Chin-Luan Chuang, Jiunn-Jong Wu
Department of Pathology, College of Medicine, National Cheng Kung University, No. 1 University Road, Tainan 70101, Taiwan.
A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp. collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- beta-lactamases (MBLs). Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry bla(IMP-8) and bla(VIM-2), respectively, by colony hybridization, PCR and sequence analysis. The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E. cloacae among different healthcare settings. Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured bla(SHV-12), which was on the same transferable plasmids with bla(IMP-8). The bla(VIM-2)-positive isolate and all non-repetitive bla(IMP-8)-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing. Ribotyping of the IMP-8-producing E. cloacae isolates indicated that the dissemination of bla(IMP-8) was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.
Antimicrob Agents Chemother. 1999 Aug ;43 (8):2051-5 10428935 Cit:21
Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.
S Brisse, D Milatovic, A C Fluit, J Verhoef, N Martin, S Scheuring, K Köhrer, F J Schmitz
Eijkman-Winkler Institute, Utrecht University, 3584 CX, Utrecht, The Netherlands. sbrisse@lab.azu.nl
The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.
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Papers 148Most cited
Enterobacter cloacae :: isolation & purification
Latest Paper:
Rev Esp Anestesiol Reanim. 2009 Apr ;56 (4):239-44 19537264
[Severe perioperative thrombocytosis in a patient undergoing lung resection]
B Fernández Torres, E Ramos Martínez, R Rosendo Ríos, R Rodríguez Mejías, A Gutiérrez Guillén, M de las Mulas Béjar
Departamento de Anestesiología y Reanimación, Hospital Virgen Macarena, Sevilla. barfertor@tiscali.es
Severe thrombocytosis (platelet count > 1,000,000 microL(-1)) is a rare, usually reactive, process and few perioperative cases have been reported. We describe the management of a patient who developed severe reactive thrombocytosis in the preoperative period before undergoing segmentectomy to remove a malignant nodule. A platelet count of 2,086,000 microL(-1) was observed during the first few days after surgery; we therefore started antiplatelet therapy to prevent thrombotic complications. We analyze the factors that might have contributed to the development of severe thrombocytosis in this case and discuss the different treatment options that may affect perioperative outcomes in these patients.
Most cited papers:
Proc Natl Acad Sci U S A. 1995 Mar 14;92 (6):2081-5 7892228 Cit:129
FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.
C H Jones, J S Pinkner, R Roth, J Heuser, A V Nicholes, S N Abraham, S J Hultgren
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Type 1 pili are heteropolymeric mannosebinding fibers produced by all members of the Enterobacteriaceae family. The bulk of the fiber is composed of FimA. Two macromolecular complexes responsible for mediating an interaction with mannose-containing receptors were purified from fimA- Escherichia coli by mannose affinity chromatography and ion-exchange chromatography. One complex contained only the mannose-binding adhesin, FimH, associated with FimG, a minor component of the type 1 pilus. In the other complex the FimG-FimH moiety was loosely associated with a chaperone-minor subunit complex (FimC-FimF), possibly representing an intermediate in tip fibrilla assembly. The FimC chaperone has also been shown to form a preassembly complex with FimH that has been purified and characterized previously. Purified FimC did not bind to the FimG-FimH complex but did recognize FimH dissociated from the FimG-FimH complex. Quick-freeze deep-etch electron microscopy revealed that the FimG-FimH complex had a thin fibrillar architecture. High-resolution electron microscopy of type 1 pili revealed that a 16-nm fibrillar tip structure with an architecture identical to that of the FimG-FimH complex was joined end-to-end to the pilus rod. In a fimH- deletion mutant, the tip fibrillae joined to pilus rods were approximately 3 nm in length. The full-length tip fibrilla was restored by complementation with the fimH gene in trans. The bipartite nature of the type 1 pilus was also demonstrated on pili purified from clinical isolates of members of the Enterobacteriaceae family arguing that it is a conserved feature of the type 1 pilus.
J Infect Dis. 1995 Sep ;172 (3):886-91 7658090 Cit:57
Antibiotic-induced endotoxin release in patients with gram-negative urosepsis: a double-blind study comparing imipenem and ceftazidime.
J M Prins, M A van Agtmael, E J Kuijper, S J van Deventer, P Speelman
Department of Internal Medicine, Academic Medical Center, Amsterdam, Netherlands.
The clinical significance of differences between antibiotics in endotoxin-liberating potential is unknown. Thirty patients with gram-negative urosepsis were randomized between imipenem and ceftazidime, which have, respectively, a low and a high endotoxin-liberating potential in vitro. In patients treated with ceftazidime, a slower defervescence was noticed. After 4 h of treatment, the blood endotoxin level decreased in all 3 endotoxemic patients receiving imipenem, whereas it increased in 2 of the 4 endotoxemic patients receiving ceftazidime, and in ceftazidime-treated patients, the endotoxin level in urine decreased less than in imipenem-treated subjects. Serum and urine cytokine levels increased 10%-40% after 4 h of ceftazidime treatment compared with no increase in the imipenem-treated patients (P >.05). Endotoxin release during antibiotic killing in vitro, assessed for all microorganisms, was 10-fold higher with ceftazidime (P <.001). These results indicate that differences between antibiotics in endotoxin release may affect the inflammatory response during treatment.
J Clin Microbiol. 2002 Apr ;40 (4):1237-43 11923338 Cit:47
Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period.
Rafael Cantón, Antonio Oliver, Teresa M Coque, María del Carmen Varela, José Claudio Pérez-Díaz, Fernando Baquero
Servicio de Microbiología Hospital Ramón y Cajal, Madrid, Spain. rcanton@hrc.insalud.es
Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.
J Clin Microbiol. 1994 Mar ;32 (3):596-602 8195364 Cit:34
Arbitrarily primed PCR, ribotyping, and plasmid pattern analysis applied to investigation of a nosocomial outbreak due to Enterobacter cloacae in a neonatal intensive care unit.
F Grattard, B Pozzetto, P Berthelot, I Rayet, A Ros, B Lauras, O G Gaudin
Laboratoire de Bactériologie-Virologie, Faculté de Médicine J. Lisfranc, Saint-Etienne, France.
In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.
J Hosp Infect. 1994 Dec ;28 (4):273-86 7897189 Cit:31
Enterobacter cloacae in a neonatal intensive care unit: account of an outbreak and its relationship to use of third generation cephalosporins.
D Acolet, Z Ahmet, E Houang, R Hurley, M E Kaufmann
Hillingdon Hospital, London, UK.
After uneventful use of cefotaxime and ceftazidime as first line therapy for three years in our neonatal intensive care unit we isolated cephalosporin-resistant Enterobacter cloacae (CREC) strains which caused clusters of cases or colonization and/or serious neonatal infection. By using two or more typing methods, at least five different strains with similar patterns of antimicrobial sensitivities were identified. The results of a case-control study did not support the notion that the use of third generation cephalosporins was associated with colonization and infection by CREC. The outbreak was brought under control by interrupting the transmission of the epidemic strain D, by measures such as cohort nursing, diligent handwashing before and after procedures, and thorough environmental cleaning as well as by decontamination with glutaraldehyde after dismantling of the blood gas analyser believed to have acted as a persistent reservoir. Our experience highlights the danger of inadequate supervision and maintenance of equipment used for near-patient testing and the need to monitor such equipment not only in terms of its calibration and analytical performance but also microbiologically.
J Biol Chem. 1995 Mar 17;270 (11):5729-35 7890700 Cit:29
Molecular evolution of a class C beta-lactamase extending its substrate specificity.
M Nukaga, S Haruta, K Tanimoto, K Kogure, K Taniguchi, M Tamaki, T Sawai
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.
Clin Infect Dis. 1992 Jul ;15 (1):30-2 1352150 Cit:26
Molecular analysis provides evidence for the endogenous origin of bacteremia and meningitis due to Enterobacter cloacae in an infant.
N Lambert-Zechovsky, E Bingen, E Denamur, N Brahimi, P Brun, H Mathieu, J Elion
Laboratoire de Microbiologie, Hôpital Robert Debré, Paris, France.
We analyzed the restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) to document the occurrence of endogenous, systemic bacteremia and meningitis due to Enterobacter cloacae in a newborn. Five strains of E. cloacae were isolated from this newborn. Three of these strains were recovered from stool at counts of 10(8), 10(9), and 10(9) organisms/g of feces, respectively; one strain was isolated from blood; and one strain was isolated from cerebrospinal fluid. In addition, five epidemiologically unrelated strains of E. cloacae were studied for comparison. Our study clearly shows the genetic relatedness of the strains isolated sequentially from cultures of stool, blood, and cerebrospinal fluid. RFLP analysis of total DNA and ribotyping seem particularly well suited to the study of the epidemiology of nosocomial E. cloacae strains.
J Hosp Infect. 1992 Jun ;21 (2):95-101 1353097 Cit:24
Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections.
E Bingen, E Denamur, N Lambert-Zechovsky, N Brahimi, M el Lakany, J Elion
Laboratoire de Bactériologie, Hôpital Robert Debré, Paris, France.
Restriction fragment length polymorphism analysis (RFLP) of total DNA and rDNA regions was used for the epidemiological evaluation of 10 Enterobacter cloacae nosocomial isolates obtained from nine patients in our hospital. Five of these patients were hospitalized during overlapping periods, thus raising the question of cross-contamination. A single biochemical pattern and antibiotic susceptibility profile was observed for all isolates but one. In contrast, based on the results of total DNA and rDNA RFLP patterns, the genetic unrelatedness of the isolates was clearly shown, thus excluding a common source of contamination or patient-to-patient transfer.
J Antimicrob Chemother. 2002 Oct ;50 (4):503-11 12356794 Cit:21
Metallo-beta-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii.
Jing-Jou Yan, Wen-Chien Ko, Chin-Luan Chuang, Jiunn-Jong Wu
Department of Pathology, College of Medicine, National Cheng Kung University, No. 1 University Road, Tainan 70101, Taiwan.
A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp. collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- beta-lactamases (MBLs). Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry bla(IMP-8) and bla(VIM-2), respectively, by colony hybridization, PCR and sequence analysis. The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E. cloacae among different healthcare settings. Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured bla(SHV-12), which was on the same transferable plasmids with bla(IMP-8). The bla(VIM-2)-positive isolate and all non-repetitive bla(IMP-8)-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing. Ribotyping of the IMP-8-producing E. cloacae isolates indicated that the dissemination of bla(IMP-8) was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.
Antimicrob Agents Chemother. 1999 Aug ;43 (8):2051-5 10428935 Cit:21
Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.
S Brisse, D Milatovic, A C Fluit, J Verhoef, N Martin, S Scheuring, K Köhrer, F J Schmitz
Eijkman-Winkler Institute, Utrecht University, 3584 CX, Utrecht, The Netherlands. sbrisse@lab.azu.nl
The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.
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Klebsiella isolation from Blood
J Clin Microbiol. 2004 March; 42(3): 1337–1340.
doi: 10.1128/JCM.42.3.1337-1340.2004.
PMCID: PMC356840
Copyright © 2004, American Society for Microbiology
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Prathiba Kurupati,1 Carol Chow,2 Gamini Kumarasinghe,2 and Chit Laa Poh1*
Department of Microbiology, National University of Singapore, Singapore 117597,1 Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore2
*Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore. Phone: (65) 687-43674. Fax: (65) 677-66872. E-mail: micpohcl@nus.edu.sg.
Received July 14, 2003; Revised August 27, 2003; Accepted November 17, 2003.
This article has been cited by other articles in PMC.
ABSTRACT
A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.
Other Sections▼
Klebsiella pneumoniae is an important hospital-acquired pathogen that causes severe morbidity and mortality among the newborn, the elderly, and immunocompromised patients (3, 4, 15). K. pneumoniae infections acquired in hospitals can be difficult to treat, as many strains are highly resistant to treatment with broad-spectrum cephalosporins and aminoglycosides (1, 7, 8, 10, 13). The isolation of bacteria from blood culture bottles is usually indicative of a serious invasive infection (bacteremia) which requires urgent antimicrobial therapy. Microorganisms isolated from patients usually have different antimicrobial susceptibilities, and successful treatment is dependent on the prompt administration of the appropriate antibiotic (6, 9, 11, 14). K. pneumoniae is identified by various automated and manual products such as Vitek (BioMérieux, Inc.) and the Analytab Products (API) system (BioMérieux, Inc.) or by traditional biochemical tests, and the whole process requires at least 24 to 48 h.
In recent years, real-time PCR has emerged as a valuable tool for the rapid testing of various biological specimens and body fluids for the presence of microorganisms (2, 5, 12, 17). The development of highly sensitive and specific PCR assays has alleviated the problems typically associated with microorganisms that are found in low numbers in tissues or body fluids, that are difficult to culture, or that are serologically similar. The LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) offers two different fluorescence detection formats. The first employs SYBR Green I, which is a dye that binds nonspecifically to double-stranded DNA (16); the second uses hybridization probes which allow sequence-specific detection by using fluorescence energy transfer (FRET) between two fluorophores. This result is achieved by attaching the two fluorophores to two oligonucleotide probes designed to hybridize to a complementary region of the target gene, leaving a 1-nucleotide-wide gap. The LightCycler instrument has been developed for fast cycling and real-time monitoring of the amplification process and thus eliminates the need to carry out gel electrophoresis of the samples after PCR.
In this study, we describe a real-time PCR assay with specific primers and hybridization probes targeting the 16S rRNA gene for the direct detection of K. pneumoniae from positive blood culture bottles. The assay also provides specific and sensitive quantification of K. pneumoniae DNA.
We studied 142 blood cultures that were known to be positive controls for K. pneumoniae, 65 that were negative controls for K. pneumoniae, and 52 double-blind samples (Table 1) that showed positive growth in BACTEC Plus aerobic medium in the BACTEC 9240 system. DNA extractions were performed in all experiments by using the QIAamp blood and tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. DNA extracted from type strains which acted as positive and negative controls was stored at −20°C until use. Oligonucleotide primers were designed to amplify a 126-bp target sequence of the 16S rRNA gene spanning nucleotide positions 44 to 170 of the K. pneumoniae strain with EMBL databank accession no. X93214. The forward primer K16SF (15-mer; position 44 to 58) and reverse primer K16SR (16-mer; position 155 to 170) were used. Within the 16S rRNA gene, fluorescence-labeled probes were designed to hybridize to the species-specific region of K. pneumoniae. The sensor probe used was the K. pneumoniae sensor (19-mer; melting temperature [Tm] = 62.7°C) oligonucleotide labeled with LightCycler Red 640 at the 5′ end and phosphorylated at the 3′ end to block extension. The corresponding fluorescein isothiocyanate-labeled anchor probe at the 3′ end was the K. pneumoniae anchor (30-mer, Tm = 69.9°C) oligonucleotide that binds to the template strand at a distance of one base to the bound sensor probe.
TABLE 1.
Real-time PCR hybridization probe-based testing of microorganisms
REAL-TIME PCR SAMPLE PREPARATION.
The master mixture contained 2 μl of 10× LightCycler-FastStart DNA master hybridization probes (Roche Diagnostics), 3 mM MgCl2 (final concentration), 0.5 μM final concentration of K. pneumonia-specific primers, and 0.2 μM final concentration of hybridization probes. To complete the PCR mixtures, 18 μl of master mixture and 2 μl of a DNA preparation were added into the LightCycler glass capillaries. After a short centrifugation (700 × g for 10 s), the sealed capillaries were placed into the LightCycler.
Other Sections▼
REAL-TIME PCR AMPLIFICATION AND MELTING-CURVE ANALYSIS.
Thermocycling conditions were optimized to one cycle of denaturation at 95°C for 10 min, followed by 30 amplification cycles (temperature transition rate of 20°C/s), each comprising denaturation (95°C for 10 s), annealing (55°C for 15 s), and extension (74°C for 10 s). PCR amplification was followed by melting-curve analysis from 40 to 95°C (temperature transition rate of 0.1°C/s) with continuous fluorescence readings. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled sensor probe at the F2 channel. Fluorescence signals from F2 were plotted automatically in real time versus temperature (T) to produce melting curves for amplicons. Melting curves were then converted into melting peaks by plotting the negative derivative of fluorescence versus T (−dF2/dT versus T and −dF3/dT versus T). Water was used as the negative control and was included in each set of melting peak determinations. The entire process took approximately 40 min. The presence of the 126-bp amplicon was verified by agarose gel electrophoresis.
Other Sections▼
SENSITIVITY OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
The sensitivity of the real-time PCR assay was optimized to 10 fg of purified K. pneumoniae DNA. Agarose gel electrophoresis of the amplicons showed that the band intensity correlated well with the calculated concentrations of amplicons (Fig. (Fig.11 A), and the sequence was analyzed (data not shown). Real-time PCR sensitivity optimization performed by using the K. pneumoniae type strain (ATCC 13883) resulted in a linear-regression curve across 107 to 1 genomic equivalents (10 ng to 1 fg of DNA) with an error rate of <1% and a correlation coefficient at −1 (Fig. (Fig.1B).1B). The intraassay variation of the threshold/cycle (CT) values was evaluated by using K. pneumoniae DNA dilutions containing 107, 106, 105, 104, 103, 102, and 10 copies. CT values were established between 10 and 27. The CT values, which are inversely related to the quantity of organisms, ranged between 10 and 27 corresponded to 107 to 10 organisms (Fig. (Fig.1B1B).
FIG. 1.
Standard curve for K. pneumoniae DNA quantification. (A) Agarose gel electrophoresis of a 126-bp PCR product. Lane W represents the negative control (water). Lane M represents the 100-bp ladder DNA marker; lanes 1 to 8 represent the 10-fold-dilution series (more ...)
Other Sections▼
SPECIFICITY OF THE REAL-TIME PCR ASSAY.
The specificity of the real-time PCR assay for K. pneumoniae with primers specific for the 16S rRNA gene was investigated by testing 1 ng of purified DNA from each of the 65 negative-control organisms (Table 1). Amplification of Pseudomonas aeruginosa, Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae, and Neisseria meningitidis remained negative when tested at all concentrations (10 ng to 1 fg), whereas the CT values for Klebsiella oxytoca, Escherichia coli, and Enterococcus faecium were greater than 29 at a concentration of 1 ng/μl. By comparison, a CT value of ≤13 was achieved for K. pneumoniae at the same concentration of 1 ng/μl (Fig. (Fig.2).2). Upon agarose gel electrophoresis of PCR mixtures derived from negative-control organisms, no amplicon was ever observed. The target sequence was shown to be highly specific for K. pneumoniae, as it failed to detect any other bacteria that are known to cause bacteremia (Fig. (Fig.22).
FIG. 2.
Specificity of the real-time PCR assay for K. pneumoniae. Different bacterial species were subjected to DNA extraction and quantitation by the real-time PCR assay. Data for fluorescence versus cycle number plots for clinical samples are shown.
Other Sections▼
MELTING-CURVE ANALYSIS OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
When the temperature reached the Tm of the probes, a rapid loss of fluorescence was observed as the two adjacently bound probes dissociated from their complementary targets and FRET stopped. This was plotted as the negative derivative of fluorescence versus temperature to define the template-specific melting curves (Fig. (Fig.3).3). Without changing the MgCl2 concentration, we could still maintain a sensitivity of 10 organisms per sample, as shown by a 10-fold dilution series from 107 to 1 genome equivalent(s) of our reference strain. With the hybridization probes, the PCR products were found to reproducibly yield a melting peak of 66°C (Fig. (Fig.33).
FIG. 3.
Melting curve for the LightCycler KPS and KPA FRET hybridization probes. The specific melting peak can be observed with the quantified number of copies of K. pneumoniae DNA.
Other Sections▼
DOUBLE-BLIND TESTING OF BLOOD CULTURE BOTTLES.
Four of 52 double-blind samples tested positive for K. pneumoniae, with a CT value of <20, and 48 samples displayed no detectable fluorescence above the background level. Double-blind PCR-based testing results were concordant with those of biochemical identification.
PCR-based testing of all 259 blood samples by real-time PCR demonstrated a sensitivity of 100% (146 of 146) and a specificity of 100% (113 of 113). The molecular assay was completed within 2 h upon receiving BACTEC 9240 positive blood culture bottles. No contamination was observed at any time during the study. No false-positive signal was detected when the negative control (which comprised sterile water, primers, and probes) was amplified in the reaction capillaries. In contrast to conventional PCR methods, they are less prone to contamination because postamplification handling is eliminated (12). Our real-time PCR-based approach for identification of K. pneumoniae resulted in significant time saving in comparison to traditional biochemical identification of K. pneumoniae from BACTEC 9240 positive blood cultures.
In conclusion, real-time PCR offers a fast tool with high sensitivity and specificity for the identification of K. pneumoniae from positive blood cultures. If in the future it becomes possible to detect more pathogens by multiplex real-time PCR, this method could become a suitable technology for implementation in routine diagnostic laboratories. It is also suitable for laboratories which encounter high isolation rates for K. pneumoniae or in outbreak situations.
Other Sections▼
REFERENCES
1. Arlet, G., M. J. Sanson-Le Pors, M. Rouveau, G. Fournier, O. Marie, B. Schlemmer, and A. Philippon. 1990. Outbreak of nosocomial infection due to Klebsiella pneumoniae producing SHV-4 β-lactamase. Eur. J. Clin. Microbiol. Infect. Dis. 9:797-803. [PubMed]
2. Bellin, T., M. Pulz, A. Matussek, H. G. Hempen, and F. Gunzer. 2001. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 39:370-374. [PubMed]
3. Bingen, E. H., P. Desjardins, G. Arlet, F. Bourgeois, P. Marianikurkdjian, N. Y. Lambertzechovsky, E. Denamur, A. Philippon, and J. Elion. 1993. Molecular epidemiology of plasmid spread among extended broad-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital. J. Clin. Microbiol. 31:179-184. [PubMed]
4. Coovadia, Y. M., A. P. Johnson, R. H. Bhana, G. R. Hutchinson, R. C. George, and I. E. Hafferjee. 1992. Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks. J. Hosp. Infect. 22:197-205. [PubMed]
5. Corless, C. E., M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, and E. B. Kaczmarski. 2001. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J. Clin. Microbiol. 39:1553-1558. [PubMed]
6. Cunney, R. J., E. B. McNamara, N. Alansari, B. Loo, and E. G. Smyth. 1997. The impact of blood culture reporting and clinical liaison on the empiric treatment of bacteraemia. J. Clin. Pathol. 50:1010-1012. [PubMed]
7. De Champs, C., D. Sirot, C. Chanal, M. C. Poupart, M. P. Dumas, and J. Sirot. 1991. Concomitant dissemination of three extended-spectrum β-lactamases among different Enterobacteriaceae isolated in a French hospital. J. Antimicrob. Chemother. 27:441-457. [PubMed]
8. Jacoby, G. A. 1996. Antimicrobial-resistant pathogens in the 1990s. Annu. Rev. Med. 47:169-179. [PubMed]
9. Jamulitrat, S., U. Meknavin, and S. Thongpiyapoon. 1994. Factors affecting mortality outcome and risk of developing nosocomial bloodstream infection. Infect. Control Hosp. Epidemiol. 15:163-170. [PubMed]
10. Johnson, A. P., M. J. Weinren, B. Ayling-Smith, S. K. Du Bois, S. G. B. Amyes, and R. C. George. 1992. Outbreak of infection in two U.K. hospitals caused by a strain of Klebsiella pneumoniae resistant to cefotaxime and ceftazidime. J. Hosp. Infect. 20:97-103. [PubMed]
11. Lebovici, L. S., I. Shraga, M. Drucker, H. Konigsberger, Z. Samra, and S. D. Pitlik. 1998. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection. J. Intern. Med. 244:379-386. [PubMed]
12. Logan, J. M. J., K. J. Edwards, N. A. Saunders, and J. Stanley. 2001. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR. J. Clin. Microbiol. 39:2227-2232. [PubMed]
13. Nouvellon, M., J. L. Pons, D. Sirot, M. L. Combe, and J. F. Lemeland. 1994. Clonal outbreaks of extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae demonstrated by antibiotic susceptibility testing, β-lactamase typing, and multilocus enzyme electrophoresis. J. Clin. Microbiol. 32:2625-2627. [PubMed]
14. Pedersen, G., H. C. Schonheyder, and H. T. Sorensen. 1997. Antibiotic therapy and outcome of monomicrobial gram-negative bacteremia: a 3-year population-based study. Scand. J. Infect. Dis. 29:601-606. [PubMed]
15. Rice, L. B., S. H. Willey, G. A. Papanicolaou, A. A. Medeiros, G. M. Eliopoulos, R. C. Moellering, Jr., and G. A. Jacoby. 1990. Outbreak of ceftazidime resistance caused by extended-spectrum β-lactamases at a Massachusetts chronic-care facility. Antimicrob. Agents Chemother. 34:2193-2199. [PubMed]
16. Skeidsvoll, J., and P. M. Ueland. 1995. Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR Green I. Anal. Biochem. 231:359-365. [PubMed]
17. Tyagi, S., and F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303-308. [PubMed]
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Kurupati, P.
Chow, C.
Kumarasinghe, G.
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Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
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Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.
[J Clin Microbiol. 1993]
Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks.
[J Hosp Infect. 1992]
Outbreak of ceftazidime resistance caused by extended-spectrum beta-lactamases at a Massachusetts chronic-care facility.
[Antimicrob Agents Chemother. 1990]
Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV-4 beta-lactamase.
[Eur J Clin Microbiol Infect Dis. 1990]
See more articles cited in this paragraph
Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.
[J Clin Microbiol. 2001]
Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.
[J Clin Microbiol. 2001]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
Molecular beacons: probes that fluoresce upon hybridization.
[Nat Biotechnol. 1996]
Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.
[Anal Biochem. 1995]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
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doi: 10.1128/JCM.42.3.1337-1340.2004.
PMCID: PMC356840
Copyright © 2004, American Society for Microbiology
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Prathiba Kurupati,1 Carol Chow,2 Gamini Kumarasinghe,2 and Chit Laa Poh1*
Department of Microbiology, National University of Singapore, Singapore 117597,1 Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore2
*Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore. Phone: (65) 687-43674. Fax: (65) 677-66872. E-mail: micpohcl@nus.edu.sg.
Received July 14, 2003; Revised August 27, 2003; Accepted November 17, 2003.
This article has been cited by other articles in PMC.
ABSTRACT
A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.
Other Sections▼
Klebsiella pneumoniae is an important hospital-acquired pathogen that causes severe morbidity and mortality among the newborn, the elderly, and immunocompromised patients (3, 4, 15). K. pneumoniae infections acquired in hospitals can be difficult to treat, as many strains are highly resistant to treatment with broad-spectrum cephalosporins and aminoglycosides (1, 7, 8, 10, 13). The isolation of bacteria from blood culture bottles is usually indicative of a serious invasive infection (bacteremia) which requires urgent antimicrobial therapy. Microorganisms isolated from patients usually have different antimicrobial susceptibilities, and successful treatment is dependent on the prompt administration of the appropriate antibiotic (6, 9, 11, 14). K. pneumoniae is identified by various automated and manual products such as Vitek (BioMérieux, Inc.) and the Analytab Products (API) system (BioMérieux, Inc.) or by traditional biochemical tests, and the whole process requires at least 24 to 48 h.
In recent years, real-time PCR has emerged as a valuable tool for the rapid testing of various biological specimens and body fluids for the presence of microorganisms (2, 5, 12, 17). The development of highly sensitive and specific PCR assays has alleviated the problems typically associated with microorganisms that are found in low numbers in tissues or body fluids, that are difficult to culture, or that are serologically similar. The LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) offers two different fluorescence detection formats. The first employs SYBR Green I, which is a dye that binds nonspecifically to double-stranded DNA (16); the second uses hybridization probes which allow sequence-specific detection by using fluorescence energy transfer (FRET) between two fluorophores. This result is achieved by attaching the two fluorophores to two oligonucleotide probes designed to hybridize to a complementary region of the target gene, leaving a 1-nucleotide-wide gap. The LightCycler instrument has been developed for fast cycling and real-time monitoring of the amplification process and thus eliminates the need to carry out gel electrophoresis of the samples after PCR.
In this study, we describe a real-time PCR assay with specific primers and hybridization probes targeting the 16S rRNA gene for the direct detection of K. pneumoniae from positive blood culture bottles. The assay also provides specific and sensitive quantification of K. pneumoniae DNA.
We studied 142 blood cultures that were known to be positive controls for K. pneumoniae, 65 that were negative controls for K. pneumoniae, and 52 double-blind samples (Table 1) that showed positive growth in BACTEC Plus aerobic medium in the BACTEC 9240 system. DNA extractions were performed in all experiments by using the QIAamp blood and tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. DNA extracted from type strains which acted as positive and negative controls was stored at −20°C until use. Oligonucleotide primers were designed to amplify a 126-bp target sequence of the 16S rRNA gene spanning nucleotide positions 44 to 170 of the K. pneumoniae strain with EMBL databank accession no. X93214. The forward primer K16SF (15-mer; position 44 to 58) and reverse primer K16SR (16-mer; position 155 to 170) were used. Within the 16S rRNA gene, fluorescence-labeled probes were designed to hybridize to the species-specific region of K. pneumoniae. The sensor probe used was the K. pneumoniae sensor (19-mer; melting temperature [Tm] = 62.7°C) oligonucleotide labeled with LightCycler Red 640 at the 5′ end and phosphorylated at the 3′ end to block extension. The corresponding fluorescein isothiocyanate-labeled anchor probe at the 3′ end was the K. pneumoniae anchor (30-mer, Tm = 69.9°C) oligonucleotide that binds to the template strand at a distance of one base to the bound sensor probe.
TABLE 1.
Real-time PCR hybridization probe-based testing of microorganisms
REAL-TIME PCR SAMPLE PREPARATION.
The master mixture contained 2 μl of 10× LightCycler-FastStart DNA master hybridization probes (Roche Diagnostics), 3 mM MgCl2 (final concentration), 0.5 μM final concentration of K. pneumonia-specific primers, and 0.2 μM final concentration of hybridization probes. To complete the PCR mixtures, 18 μl of master mixture and 2 μl of a DNA preparation were added into the LightCycler glass capillaries. After a short centrifugation (700 × g for 10 s), the sealed capillaries were placed into the LightCycler.
Other Sections▼
REAL-TIME PCR AMPLIFICATION AND MELTING-CURVE ANALYSIS.
Thermocycling conditions were optimized to one cycle of denaturation at 95°C for 10 min, followed by 30 amplification cycles (temperature transition rate of 20°C/s), each comprising denaturation (95°C for 10 s), annealing (55°C for 15 s), and extension (74°C for 10 s). PCR amplification was followed by melting-curve analysis from 40 to 95°C (temperature transition rate of 0.1°C/s) with continuous fluorescence readings. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled sensor probe at the F2 channel. Fluorescence signals from F2 were plotted automatically in real time versus temperature (T) to produce melting curves for amplicons. Melting curves were then converted into melting peaks by plotting the negative derivative of fluorescence versus T (−dF2/dT versus T and −dF3/dT versus T). Water was used as the negative control and was included in each set of melting peak determinations. The entire process took approximately 40 min. The presence of the 126-bp amplicon was verified by agarose gel electrophoresis.
Other Sections▼
SENSITIVITY OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
The sensitivity of the real-time PCR assay was optimized to 10 fg of purified K. pneumoniae DNA. Agarose gel electrophoresis of the amplicons showed that the band intensity correlated well with the calculated concentrations of amplicons (Fig. (Fig.11 A), and the sequence was analyzed (data not shown). Real-time PCR sensitivity optimization performed by using the K. pneumoniae type strain (ATCC 13883) resulted in a linear-regression curve across 107 to 1 genomic equivalents (10 ng to 1 fg of DNA) with an error rate of <1% and a correlation coefficient at −1 (Fig. (Fig.1B).1B). The intraassay variation of the threshold/cycle (CT) values was evaluated by using K. pneumoniae DNA dilutions containing 107, 106, 105, 104, 103, 102, and 10 copies. CT values were established between 10 and 27. The CT values, which are inversely related to the quantity of organisms, ranged between 10 and 27 corresponded to 107 to 10 organisms (Fig. (Fig.1B1B).
FIG. 1.
Standard curve for K. pneumoniae DNA quantification. (A) Agarose gel electrophoresis of a 126-bp PCR product. Lane W represents the negative control (water). Lane M represents the 100-bp ladder DNA marker; lanes 1 to 8 represent the 10-fold-dilution series (more ...)
Other Sections▼
SPECIFICITY OF THE REAL-TIME PCR ASSAY.
The specificity of the real-time PCR assay for K. pneumoniae with primers specific for the 16S rRNA gene was investigated by testing 1 ng of purified DNA from each of the 65 negative-control organisms (Table 1). Amplification of Pseudomonas aeruginosa, Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae, and Neisseria meningitidis remained negative when tested at all concentrations (10 ng to 1 fg), whereas the CT values for Klebsiella oxytoca, Escherichia coli, and Enterococcus faecium were greater than 29 at a concentration of 1 ng/μl. By comparison, a CT value of ≤13 was achieved for K. pneumoniae at the same concentration of 1 ng/μl (Fig. (Fig.2).2). Upon agarose gel electrophoresis of PCR mixtures derived from negative-control organisms, no amplicon was ever observed. The target sequence was shown to be highly specific for K. pneumoniae, as it failed to detect any other bacteria that are known to cause bacteremia (Fig. (Fig.22).
FIG. 2.
Specificity of the real-time PCR assay for K. pneumoniae. Different bacterial species were subjected to DNA extraction and quantitation by the real-time PCR assay. Data for fluorescence versus cycle number plots for clinical samples are shown.
Other Sections▼
MELTING-CURVE ANALYSIS OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
When the temperature reached the Tm of the probes, a rapid loss of fluorescence was observed as the two adjacently bound probes dissociated from their complementary targets and FRET stopped. This was plotted as the negative derivative of fluorescence versus temperature to define the template-specific melting curves (Fig. (Fig.3).3). Without changing the MgCl2 concentration, we could still maintain a sensitivity of 10 organisms per sample, as shown by a 10-fold dilution series from 107 to 1 genome equivalent(s) of our reference strain. With the hybridization probes, the PCR products were found to reproducibly yield a melting peak of 66°C (Fig. (Fig.33).
FIG. 3.
Melting curve for the LightCycler KPS and KPA FRET hybridization probes. The specific melting peak can be observed with the quantified number of copies of K. pneumoniae DNA.
Other Sections▼
DOUBLE-BLIND TESTING OF BLOOD CULTURE BOTTLES.
Four of 52 double-blind samples tested positive for K. pneumoniae, with a CT value of <20, and 48 samples displayed no detectable fluorescence above the background level. Double-blind PCR-based testing results were concordant with those of biochemical identification.
PCR-based testing of all 259 blood samples by real-time PCR demonstrated a sensitivity of 100% (146 of 146) and a specificity of 100% (113 of 113). The molecular assay was completed within 2 h upon receiving BACTEC 9240 positive blood culture bottles. No contamination was observed at any time during the study. No false-positive signal was detected when the negative control (which comprised sterile water, primers, and probes) was amplified in the reaction capillaries. In contrast to conventional PCR methods, they are less prone to contamination because postamplification handling is eliminated (12). Our real-time PCR-based approach for identification of K. pneumoniae resulted in significant time saving in comparison to traditional biochemical identification of K. pneumoniae from BACTEC 9240 positive blood cultures.
In conclusion, real-time PCR offers a fast tool with high sensitivity and specificity for the identification of K. pneumoniae from positive blood cultures. If in the future it becomes possible to detect more pathogens by multiplex real-time PCR, this method could become a suitable technology for implementation in routine diagnostic laboratories. It is also suitable for laboratories which encounter high isolation rates for K. pneumoniae or in outbreak situations.
Other Sections▼
REFERENCES
1. Arlet, G., M. J. Sanson-Le Pors, M. Rouveau, G. Fournier, O. Marie, B. Schlemmer, and A. Philippon. 1990. Outbreak of nosocomial infection due to Klebsiella pneumoniae producing SHV-4 β-lactamase. Eur. J. Clin. Microbiol. Infect. Dis. 9:797-803. [PubMed]
2. Bellin, T., M. Pulz, A. Matussek, H. G. Hempen, and F. Gunzer. 2001. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 39:370-374. [PubMed]
3. Bingen, E. H., P. Desjardins, G. Arlet, F. Bourgeois, P. Marianikurkdjian, N. Y. Lambertzechovsky, E. Denamur, A. Philippon, and J. Elion. 1993. Molecular epidemiology of plasmid spread among extended broad-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital. J. Clin. Microbiol. 31:179-184. [PubMed]
4. Coovadia, Y. M., A. P. Johnson, R. H. Bhana, G. R. Hutchinson, R. C. George, and I. E. Hafferjee. 1992. Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks. J. Hosp. Infect. 22:197-205. [PubMed]
5. Corless, C. E., M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, and E. B. Kaczmarski. 2001. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J. Clin. Microbiol. 39:1553-1558. [PubMed]
6. Cunney, R. J., E. B. McNamara, N. Alansari, B. Loo, and E. G. Smyth. 1997. The impact of blood culture reporting and clinical liaison on the empiric treatment of bacteraemia. J. Clin. Pathol. 50:1010-1012. [PubMed]
7. De Champs, C., D. Sirot, C. Chanal, M. C. Poupart, M. P. Dumas, and J. Sirot. 1991. Concomitant dissemination of three extended-spectrum β-lactamases among different Enterobacteriaceae isolated in a French hospital. J. Antimicrob. Chemother. 27:441-457. [PubMed]
8. Jacoby, G. A. 1996. Antimicrobial-resistant pathogens in the 1990s. Annu. Rev. Med. 47:169-179. [PubMed]
9. Jamulitrat, S., U. Meknavin, and S. Thongpiyapoon. 1994. Factors affecting mortality outcome and risk of developing nosocomial bloodstream infection. Infect. Control Hosp. Epidemiol. 15:163-170. [PubMed]
10. Johnson, A. P., M. J. Weinren, B. Ayling-Smith, S. K. Du Bois, S. G. B. Amyes, and R. C. George. 1992. Outbreak of infection in two U.K. hospitals caused by a strain of Klebsiella pneumoniae resistant to cefotaxime and ceftazidime. J. Hosp. Infect. 20:97-103. [PubMed]
11. Lebovici, L. S., I. Shraga, M. Drucker, H. Konigsberger, Z. Samra, and S. D. Pitlik. 1998. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection. J. Intern. Med. 244:379-386. [PubMed]
12. Logan, J. M. J., K. J. Edwards, N. A. Saunders, and J. Stanley. 2001. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR. J. Clin. Microbiol. 39:2227-2232. [PubMed]
13. Nouvellon, M., J. L. Pons, D. Sirot, M. L. Combe, and J. F. Lemeland. 1994. Clonal outbreaks of extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae demonstrated by antibiotic susceptibility testing, β-lactamase typing, and multilocus enzyme electrophoresis. J. Clin. Microbiol. 32:2625-2627. [PubMed]
14. Pedersen, G., H. C. Schonheyder, and H. T. Sorensen. 1997. Antibiotic therapy and outcome of monomicrobial gram-negative bacteremia: a 3-year population-based study. Scand. J. Infect. Dis. 29:601-606. [PubMed]
15. Rice, L. B., S. H. Willey, G. A. Papanicolaou, A. A. Medeiros, G. M. Eliopoulos, R. C. Moellering, Jr., and G. A. Jacoby. 1990. Outbreak of ceftazidime resistance caused by extended-spectrum β-lactamases at a Massachusetts chronic-care facility. Antimicrob. Agents Chemother. 34:2193-2199. [PubMed]
16. Skeidsvoll, J., and P. M. Ueland. 1995. Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR Green I. Anal. Biochem. 231:359-365. [PubMed]
17. Tyagi, S., and F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303-308. [PubMed]
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Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.
[J Clin Microbiol. 1993]
Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks.
[J Hosp Infect. 1992]
Outbreak of ceftazidime resistance caused by extended-spectrum beta-lactamases at a Massachusetts chronic-care facility.
[Antimicrob Agents Chemother. 1990]
Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV-4 beta-lactamase.
[Eur J Clin Microbiol Infect Dis. 1990]
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Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.
[J Clin Microbiol. 2001]
Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.
[J Clin Microbiol. 2001]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
Molecular beacons: probes that fluoresce upon hybridization.
[Nat Biotechnol. 1996]
Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.
[Anal Biochem. 1995]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
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Thursday, March 4, 2010
Tuesday, March 2, 2010
Tuesday, February 23, 2010
Monday, February 22, 2010
Cefotaxime Dosage
H. Sumano1, Lilia Gutierrez2, L. Ocampo3
1Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
2Department of Physiology and Pharmacology, School of Veterinary Medicine, National Autonomous University of Mexico Mexico City 04510, Mexico
3Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
Abstract
Considering the already known pharmacological features of cefotaxime, a study with two approaches of pharmacokinetics and clinical efficacy in septicaemic dogs was carried out. Pharmacokinetic variables were defined for doses of 10 mg/kg, and 20 mg/kg, utilising a quantitative bacteriological analysis. Values for half-life (T½ß) at 10 mg/kg were 0.8, 1.48 and 1.52 h for the i.v., s.c. and i.m. routes, respectively. Corresponding values for the 20 mg/kg dose for the same routes were 0.8, 1.49 and 1.53 h, respectively. Relatively fast clearance (ranging from 0.58 to 0.64 L/kg/h) allowed a maximum dose interval of 12 h. The above-stated doses of cefotaxime were administered i.v. to 40 cases of septicaemia, clinically divided into 20 moderately severe cases treated with 10 mg/kg i.v., of cefotaxime bid, and 20 severe ones, treated with 20 mg/kgi.v. of cefotaxime bid. Injections continued until a previously defined criterion of 'clinically recovered' was obtained. Thereafter, a follow-up treatment was established using the same dose and dose-interval but through the s.c. route. Due to the apparent volumes of distribution obtained (ranging from 0.48 to 0.51 L/kg), considering the overall clinical efficacy obtained (90% for the 10 mg/kg dose and 75% for the 20 mg/kg dose), and due to the rapid improvement observed after a few doses of the drug (1.8 to 2.5 doses to 'clinical improvement'), it is safe to postulate such doses of cefotaxime as excellent choices for the treatment of septicaemia in dogs.
Keywords
pharmacokinetics, pharmacokinetics, pharmacokinetics, dogs, Cefotaxime, septicaemia, efficacy
References
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Billiet, J., Kuypers, S., Van Lierde, S. and Verhaegen, J. (1989): Plesiomonas shigelloides meningitis and septicaemia in a neonate: report of a case and review of the literature. J. Infect. 19, 267-271.
[CrossRef] [PubMed]
Berezhinskaia, V. V., Dolgova, G. V., Egorenko, G. G., Svinogeeva, T. P. and Zebrev, A. I. (1990): General toxic and organotropic properties of cefotaxime in acute and chronic experiments (in Russian). Antibiot. Khimioter. 35, 25-27. 94 SUMANO et al. Acta Veterinaria Hungarica 52, 2004.
[PubMed]
Bocazzi, A., Tonelli, P., Bellosta, C. and Careddu, P. (1998): Clinical and pharmacological evaluation of modified cefotaxime bid regimen vs.traditional tid in pediatric lower respiratory tract infections. Diagn. Microbiol. Infect. Dis. 32, 265-272.
[CrossRef] [PubMed]
Carli, S., Anfossi, P., Villa, R., Castellani, G., Mengozzi, G. and Montesissa, C. (1999): Absorption kinetics and bioavailability of cephalexin in the dog after oral and intramuscular. J. Vet. Pharmacol. Therap. 22, 308-313.
[CrossRef] [PubMed]
Chamberland, S., Blais, J., Hoang, M., Dinh, C., Cotter, D., Bond, E., Gannon, C., Park, C., Halovin, F. and Dudley. M. N. (2001): In vitro activities of RWJ-54428 (MC-02,479) against multiresistant Gram-positive bacteria. Antimicrob. Agents Chemother. 45, 1422-1430.
[CrossRef] [PubMed]
De Sarro, A., Ammendola, D., Zappala, M., Grasso, S. and De Sarro, G. B. (1995): Relationship between structure and convulsant properties of some beta-lactam antibiotics following intracerebroventricular microinjection in rats. Antimicrob. Agents Chemother. 39, 232-237.
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Diaz, A. J., Sumano, L. H., Ocampo, C. L. and Mateos, T. G. (1995): Evaluación de la nefrotoxicidad en la administración conjunta de cefalexina sódica y sulfato de gentamicina en perros. Vet. Méx. 26, 247-249.
Fernandez, G. M., Lumbreras, J. M., Sanchez, J. A. and Ordoñez, D. (1991): The interaction of cefotaxime with the serum albumin of several mammalian species. Comp. Biochem. Physiol. 100, 413-415.
[CrossRef]
Gardner, S. Y. and Papich, M. G. (2001): Comparison of cefepime pharmacokinetics in neonatal foals and adult dogs. J. Vet. Pharmacol. Therap. 24, 187-192.
[CrossRef] [PubMed]
Guerrini, V. H., English, P. B., Filippich, L. J., Schneider, J., Pharm, B. and Bourne, D. W. A. (1986): Pharmacokinetic evaluation of a slow-release cefotaxime suspension in the dog and in sheep. Am. J. Vet. Res. 47, 2057-2061.
[PubMed]
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1Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
2Department of Physiology and Pharmacology, School of Veterinary Medicine, National Autonomous University of Mexico Mexico City 04510, Mexico
3Department of Physiology and Pharmacology, College of Veterinary Medicine, National Autonomous University of Mexico 04510 Mexico City, Mexico
Abstract
Considering the already known pharmacological features of cefotaxime, a study with two approaches of pharmacokinetics and clinical efficacy in septicaemic dogs was carried out. Pharmacokinetic variables were defined for doses of 10 mg/kg, and 20 mg/kg, utilising a quantitative bacteriological analysis. Values for half-life (T½ß) at 10 mg/kg were 0.8, 1.48 and 1.52 h for the i.v., s.c. and i.m. routes, respectively. Corresponding values for the 20 mg/kg dose for the same routes were 0.8, 1.49 and 1.53 h, respectively. Relatively fast clearance (ranging from 0.58 to 0.64 L/kg/h) allowed a maximum dose interval of 12 h. The above-stated doses of cefotaxime were administered i.v. to 40 cases of septicaemia, clinically divided into 20 moderately severe cases treated with 10 mg/kg i.v., of cefotaxime bid, and 20 severe ones, treated with 20 mg/kgi.v. of cefotaxime bid. Injections continued until a previously defined criterion of 'clinically recovered' was obtained. Thereafter, a follow-up treatment was established using the same dose and dose-interval but through the s.c. route. Due to the apparent volumes of distribution obtained (ranging from 0.48 to 0.51 L/kg), considering the overall clinical efficacy obtained (90% for the 10 mg/kg dose and 75% for the 20 mg/kg dose), and due to the rapid improvement observed after a few doses of the drug (1.8 to 2.5 doses to 'clinical improvement'), it is safe to postulate such doses of cefotaxime as excellent choices for the treatment of septicaemia in dogs.
Keywords
pharmacokinetics, pharmacokinetics, pharmacokinetics, dogs, Cefotaxime, septicaemia, efficacy
References
Bennet, J. B., Brodie, J. L., Benner, E. J. and Kirby, W. M. (1966): Simplified accurate method for antibiotic assay of clinical specimens. Am. Soc. Microbiol. 14, 170-177.
Billiet, J., Kuypers, S., Van Lierde, S. and Verhaegen, J. (1989): Plesiomonas shigelloides meningitis and septicaemia in a neonate: report of a case and review of the literature. J. Infect. 19, 267-271.
[CrossRef] [PubMed]
Berezhinskaia, V. V., Dolgova, G. V., Egorenko, G. G., Svinogeeva, T. P. and Zebrev, A. I. (1990): General toxic and organotropic properties of cefotaxime in acute and chronic experiments (in Russian). Antibiot. Khimioter. 35, 25-27. 94 SUMANO et al. Acta Veterinaria Hungarica 52, 2004.
[PubMed]
Bocazzi, A., Tonelli, P., Bellosta, C. and Careddu, P. (1998): Clinical and pharmacological evaluation of modified cefotaxime bid regimen vs.traditional tid in pediatric lower respiratory tract infections. Diagn. Microbiol. Infect. Dis. 32, 265-272.
[CrossRef] [PubMed]
Carli, S., Anfossi, P., Villa, R., Castellani, G., Mengozzi, G. and Montesissa, C. (1999): Absorption kinetics and bioavailability of cephalexin in the dog after oral and intramuscular. J. Vet. Pharmacol. Therap. 22, 308-313.
[CrossRef] [PubMed]
Chamberland, S., Blais, J., Hoang, M., Dinh, C., Cotter, D., Bond, E., Gannon, C., Park, C., Halovin, F. and Dudley. M. N. (2001): In vitro activities of RWJ-54428 (MC-02,479) against multiresistant Gram-positive bacteria. Antimicrob. Agents Chemother. 45, 1422-1430.
[CrossRef] [PubMed]
De Sarro, A., Ammendola, D., Zappala, M., Grasso, S. and De Sarro, G. B. (1995): Relationship between structure and convulsant properties of some beta-lactam antibiotics following intracerebroventricular microinjection in rats. Antimicrob. Agents Chemother. 39, 232-237.
[PubMed]
Diaz, A. J., Sumano, L. H., Ocampo, C. L. and Mateos, T. G. (1995): Evaluación de la nefrotoxicidad en la administración conjunta de cefalexina sódica y sulfato de gentamicina en perros. Vet. Méx. 26, 247-249.
Fernandez, G. M., Lumbreras, J. M., Sanchez, J. A. and Ordoñez, D. (1991): The interaction of cefotaxime with the serum albumin of several mammalian species. Comp. Biochem. Physiol. 100, 413-415.
[CrossRef]
Gardner, S. Y. and Papich, M. G. (2001): Comparison of cefepime pharmacokinetics in neonatal foals and adult dogs. J. Vet. Pharmacol. Therap. 24, 187-192.
[CrossRef] [PubMed]
Guerrini, V. H., English, P. B., Filippich, L. J., Schneider, J., Pharm, B. and Bourne, D. W. A. (1986): Pharmacokinetic evaluation of a slow-release cefotaxime suspension in the dog and in sheep. Am. J. Vet. Res. 47, 2057-2061.
[PubMed]
Harpster, N. K. (1981): The effectiveness of the cephalosporins in the treatment of bacterial pneumonias in the dog. J. Am. Anim. Hosp. Assoc. 17, 766-772.
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[CrossRef] [PubMed]
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