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PubMed1993

In-vitro activities of cefepime against Enterobacter cloacae, Serratia marcescens, Pseudomonas aeruginosa and other aerobic gram-negative bacilli.

Infections caused by resistant bacterial pathogens such as Citrobacter freundii, Enterobacter cloacae, Serratia marcescens and Pseudomonas aeruginosa have become an increasing problem with respect to therapy in large medical centres in Korea. The MICs of cefepime for aerobic Gram-negative bacilli isolated during 1991, mostly from in-patients in a hospital in Seoul, were determined by the agar dilution method and compared with those of several other antimicrobials. Of the agents tested, cefepime had the lowest MIC90s for C. freundii and E. cloacae (0.12 and 8 mg/L, respectively). The MIC90s of cefepime and amikacin (both 8 mg/L) were the lowest for S. marcescens. The MIC90s of cefepime, ceftazidime and doxycycline (all 32 mg/L) were the lowest for Acinetobacter anitratus. For P. aeruginosa, the MIC90 was relatively high (32 mg/L) but still the lowest of the antimicrobials tested. Of the cefotaxime-resistant E. cloacae isolates studied, only 7% were resistant to cefepime, while 100%, 96% and 89% were resistant to ceftazidime, ceftizoxime and cefuzonam, respectively. Similarly, only 6% of gentamicin-resistant isolates were resistant to cefepime, compared with 91%, 72%, 69% and 63% to ceftazidime, ceftizoxime, cefuzonam and cefotaxime, respectively. In conclusion, isolates from Korean patients are often resistant to several antimicrobial agents. However, based on the results of this study, cefepime may be very useful as treatment for patients with nosocomial infections caused by aerobic Gram-negative bacilli, including those which are resistant to most of the third-generation cephalosporins and gentamicin.

Aminoglycosides↗
PubMed1997

Cefepime and amikacin synergy against a cefotaxime-susceptible strain of Enterobacter cloacae in vitro and in vivo.

We developed an experimental model of pneumonia to evaluate the efficacy of new antibiotic regimens against Enterobacter cloacae. Rats were infected by administering 8.5 log10 cfu E. cloacae intratracheally, and therapy was initiated 24 h later. At that time, animals' lungs showed bilateral pneumonia containing more than 7 log10 cfu/g of tissue. Because rats eliminate amikacin and cefepime much more rapidly than humans, renal impairment was induced in all animals to simulate the pharmacokinetic parameters in humans. Using this model, we compared the bactericidal activities of cefepime and amikacin alone or in combination against the same cefotaxime-susceptible E. cloacae strain. The MICs of cefepime and amikacin for this strain were 0.5 and 2 mg/L, respectively. In-vitro killing studies showed that antibiotic combinations were synergic only at intermediate concentrations. At peak concentrations, the combination was only as effective as amikacin alone. At trough concentrations, a non-significant trend towards the superiority of the combination over cefepime alone was found. In-vivo studies showed that each antibiotic alone failed to decrease bacterial counts in the lungs except at 6 h, whereas the combination of both antibiotics induced a significant decrease in the lung bacterial count 6, 12 and 24 h after the onset of therapy when compared with tissue bacterial numbers in untreated animals or animals treated with either antibiotic alone. In-vivo synergy between cefepime and amikacin was observed at the three time points studied. No resistant clones emerged during treatment with any of the antibiotic regimens studied.

Amikacin↗
PubMed1997

Detection of mutations in the gyrA and parC genes in quinolone-resistant clinical isolates of Enterobacter cloacae.

We have determined partial sequences of the gyrA and parC genes of Enterobacter cloacae type strain including the regions analogous to the quinolone resistance-determining region of the Escherichia coli gyrA gene. The deduced 65- and 49-amino acid sequences of the determined regions of the E. cloacae gyrA and parC genes were identical to the corresponding regions of the E. coli GyrA and ParC proteins, respectively. We examined 40 clinical strains of E. cloacae isolated from patients with urinary tract infection for susceptibilities to nalidixic acid and ciprofloxacin. Based on the nalidixic acid and ciprofloxacin MICs, these isolates were divided into 19 quinolone-susceptible strains (MICs of nalidixic acid, 3.13-25 mg/L; MICs of ciprofloxacin, < or = 0.025 mg/L) and 21 quinolone-resistant strains (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-100 mg/L). We analysed five quinolone-susceptible and 21 quinolone-resistant strains for alterations in GyrA and ParC. The five quinolone-susceptible strains had amino acid sequences in GyrA and ParC identical to those of type strain. Of the 21 quinolone-resistant isolates, three (MICs of nalidixic acid, 400 to > 800 mg/L; MICs of ciprofloxacin, 0.39-3.13 mg/L) had a single amino acid change at the position equivalent to Ser-83 in the E. coli GyrA protein and no alterations in ParC; one (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 3.13 mg/L) had a single amino acid change at Ser-83 in GyrA and a single amino acid change at the position equivalent to Glu-84 in the E. coli ParC protein; two (MIC of nalidixic acid, > 800 mg/L; MIC of ciprofloxacin, 25 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and no alterations in ParC; and 15 (MICs of nalidixic acid, > 800 mg/L; MICs of ciprofloxacin, 25-100 mg/L) had double amino acid changes at Ser-83 and Asp-87 in GyrA and a single amino acid change at Ser-80 or Glu-84 in ParC. This study suggests, that in clinical isolates of E. cloacae, DNA gyrase is a primary target of quinolones, that only a single amino acid change at Ser-83 in GyrA is sufficient to generate high-level resistance to nalidixic acid and to decrease susceptibility to ciprofloxacin, and that the accumulation of amino acid changes in GyrA and the simultaneous presence of the ParC alterations play a central role in developing high-level resistance to ciprofloxacin.

Amino Acid Sequence↗
PubMed1998

The effect of a combination of Iso-Sensitest agar and ampicillin on the detection of enterobacter beta-lactamase.

The ampicillin MICs for faecal strains of E. cloacae (n = 11) and Enterobacter species not identified to species level (n = 3) were noticed to be two-fold to four-fold lower on Iso-Sensitest than on Mueller-Hinton II agar. The levels of inducible beta-lactamase on Iso-Sensitest, Mueller-Hinton, and a selective MacConkey-based agar, with and without ampicillin, were determined. The level produced on Iso-Sensitest with ampicillin was comparable to that induced by Mueller-Hinton alone. We conclude that when routine procedures for the detection of beta-lactamase-based resistance are evaluated, not only should care be taken to choose the right antibiotics, but also different combinations of beta-lactams and agar should be tested.

Agar↗
PubMed1998

Cefepime and amikacin synergy in vitro and in vivo against a ceftazidime-resistant strain of Enterobacter cloacae.

The activities of cefepime and amikacin alone or in combination against an isogenic pair of Enterobacter cloacae strains (wild type and stably derepressed, ceftazidime-resistant mutant) were compared using an experimental model of pneumonia in non-leucopenic rats. Animals were infected by administering 8.4 log10 cfu of E. cloacae intratracheally, and therapy was initiated 12 h later. At that time, the animals' lungs showed bilateral pneumonia and contained more than 7 log10 E. cloacae cfu/g tissue. Because rats eliminate amikacin and cefepime much more rapidly than humans, renal impairment was induced in all animals to simulate the pharmacokinetic parameters of humans. In-vitro susceptibilities showed an inoculum effect with cefepime proportional to the bacterial titre against the two strains, but more pronounced with the stably derepressed mutant strain, whereas with bacterial concentrations of up to 7 log10 cfu/mL, no inoculum effect was observed with amikacin. In-vitro killing indicated that antibiotic combinations were synergic only at intermediate concentrations. At peak concentrations, the combination was merely as effective as amikacin alone. At trough concentrations, a non-significant trend towards the superiority of the combination over each antibiotic alone was noted. Moreover, cefepime was either bacteriostatic or permitted regrowth of the organisms in the range of antibiotic concentrations tested. Although each antibiotic alone failed to decrease bacterial counts in the lungs, regardless of the susceptibility of the strain used, the combination of both antibiotics was synergic and induced a significant decrease in the lung bacterial count 24 h after starting therapy when compared with tissue bacterial numbers in untreated animals or animals treated with either antibiotic alone. No resistant clones emerged during treatment with any of the antibiotic regimens studied.

Amikacin↗
PubMed1999

Markedly different rates and resistance profiles exhibited by seven commonly used and newer beta-lactams on the selection of resistant variants of Enterobacter cloacae.

Seven beta-lactam antibiotics (cefepime, cefoperazone, ceftazidime, ceftriaxone, cefamandole, imipenem and meropenem) were tested for their potential to select resistance in standard and clinical strains of Enterobacter cloacae (n = 9). The strains were subcultured daily with the test antibiotics at doubling concentrations starting at 0.125 x MIC. Development of resistance throughout the passages was detected by a disc diffusion test. Ceftazidime, ceftriaxone and cefamandole selected resistance at a faster rate than cefoperazone, cefepime and meropenem. Imipenem did not select resistance in the nine strains tested and was the only antibiotic that eradicated all the strains during selection. The resistance patterns of strains selected by meropenem, cefepime and the other cephalosporins were markedly different, although cross-resistance to the early generation cephalosporins was common. The resistance phenotypes of most strains remained stable upon serial passages in antibiotic-free medium. The findings of this study highlight the importance of the choice of antibiotic for therapy not only on the basis of its antibacterial activity, but also on its potential to select resistance to itself and other antibiotics.

Anti-Bacterial Agents↗
PubMed2002

Increase in MICs of ciprofloxacin in vivo in two closely related clinical isolates of Enterobacter cloacae.

The mechanisms of fluoroquinolone resistance in two isolates of Enterobacter cloacae, Ecl#1 and Ecl#2, from the same patient and with identical pulsed-field gel electrophoresis patterns, have been analysed. MICs of ciprofloxacin were 0.25 and 1 mg/L for Ecl#1 and Ecl#2, respectively. Ecl#2 was also more resistant to chloramphenicol and organic solvents. The quinolone resistance determining regions of gyrA/B and parC/E, and the marORA and acrB genes, were sequenced. Expression of marR, acrB, soxS, robA, ramA and fis was analysed by northern blotting. The activity of a 90 bp E. cloacae mar promoter fragment was examined with the reporter plasmid pIGJ-1mar. Sequencing the gyrAB and parCE genes revealed a single amino acid substitution in GyrA (corresponding to position 83 in GyrA of Escherichia coli) in Ecl#1 and Ecl#2 (Phe83) compared with reference strain E. cloacae DSMZ 3264 (Thr83). Ecl#2 accumulated significantly less norfloxacin and displayed higher levels of expression of marR and acrB than Exl#1, indicative of greater fluoroquinolone efflux activity. Sequencing gyrB, parC/E and marORA, and northern blotting of robA, ramA and fis, did not reveal any further differences between the two strains. No homologue of soxRS was detected in E. cloacae. Expression of GFP from pIGJ1-mar in Ecl#2 was higher than in Ecl#1. In these two closely related clinical isolates of E. cloacae, a target mutation in GyrA (Ecl#1 and Ecl#2) and increased fluoroquinolone efflux by AcrAB (Ecl#2) contribute to the resistance phenotypes, corroborating findings in vitro and in vivo about the sequential development of fluoroquinolone resistance.

Anti-Infective Agents↗
PubMed2002

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.

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.

Citrobacter freundii↗
PubMed2003

Characterization of a new integron containing VIM-2, a metallo- beta-lactamase gene cassette, in a clinical isolate of Enterobacter cloacae.

We report the first description of a new integron containing bla(VIM-2), a metallo-beta-lactamase gene from Enterobacter cloacae KU680, which was isolated from peritoneal fluid of a liver cirrhosis patient in South Korea. Antibiotic susceptibility testing, and modified Hodge and EDTA-disc synergy tests, were carried out to screen for metallo-beta-lactamase-producing strains. PCR and sequence analysis were used to identify and analyse the bla(VIM-2)-containing integron. The isolate was resistant to most beta-lactams, including imipenem, and demonstrated a positive modified Hodge and EDTA-disc synergy test, which are findings suggesting a metallo-beta-lactamase. Preliminary PCR-based experiments detected the metallo-beta-lactamase gene bla(VIM-2). Sequencing of the 4392 bp cloned PCR amplicon, containing the gene cassette bla(VIM-2), revealed the structure of the class 1 integron. The integron also contained additional insert gene cassettes, aadA, and unknown open reading frames 'orfII' and 'orfIII'. To the best of our knowledge, this is the first time that this metallo-beta-lactamase gene has been detected in E. cloacae.

Amino Acid Sequence↗
PubMed2004

Relationship between ceftriaxone use and resistance to third-generation cephalosporins among clinical strains of Enterobacter cloacae.

OBJECTIVE: To investigate the potential correlation between the use of extended-spectrum cephalosporins (ESCs) and resistance to this antibiotic class among clinical isolates of Enterobacter cloacae in a university-affiliated hospital. MATERIALS AND METHODS: Data on antimicrobial resistance and antimicrobial use concerning E. cloacae and ESCs were collected over a 4 year period. Various statistical tools were used to explore the potential relationship. RESULTS: From 1999 to 2002, the proportion of E. cloacae isolates resistant to ESCs increased from 24.3% to 29.6%. (P=0.04), and the quantity of ESCs prescribed and given did not change. Within the subclass constituted by first-line ESCs, the proportion of ceftriaxone increased from 64.3% to 77.6% and the proportion of cefotaxime decreased accordingly, from 35.7% to 22.4%. Statistical analyses showed that E. cloacae resistance to ESCs correlated with ceftriaxone use regardless of the other ESCs. For every defined daily dose of ceftriaxone per 1000 patient days used in our hospital, resistance of E. cloacae isolates to ESCs increased by 1.36%. CONCLUSION: This study demonstrates a specific correlation between ceftriaxone use and the development of resistance in E. cloacae clinical isolates. The high biliary elimination of ceftriaxone compared with other ESCs may be responsible for a greater impact of this antibiotic on the digestive flora.

Cefotaxime↗
PubMed2005

Characterization of a new integron containing bla(VIM-1) and aac(6')-IIc in an Enterobacter cloacae clinical isolate from Greece.

OBJECTIVES: A clinical isolate of Enterobacter cloacae exhibiting reduced susceptibility to imipenem and a positive EDTA-disc synergy test was studied for carbapenemase production. MATERIALS AND METHODS: MICs were determined with standard procedures as well as using a higher inoculum. Isoelectric focusing of cell extracts was used for detection of beta-lactamases. PCR assays with primers specific for the bla(VIM) gene and the conserved segments of class 1 integrons and sequence analyses were carried out to identify the gene and to map the metallo-beta-lactamase encoding integron. Transferability of the gene was assessed with conjugation experiments using the filter mating technique. To identify the location of the bla(VIM-1) gene, Southern hybridization was carried out in genomic DNA using an internal fragment of the bla(VIM-1) gene as a probe, amplified by PCR. RESULTS: The isolate was resistant to extended-spectrum beta-lactams. The MICs of carbapenems were below the resistance breakpoints but rose above resistance breakpoints when an inoculum of 10(8) cfu/mL was used. Isoelectric focusing detected a beta-lactamase with a pI of 6.1, which exhibited imipenem-hydrolysing activity in a microbiological assay. Ceftazidime and imipenem resistance were not transferable by conjugation. PCR assays identified the bla(VIM-1) gene in the variable region of a class 1 integron which also carried the aac(6')-IIc gene. The bla(VIM-1) probe hybridized with an approximately 130 kb fragment of genomic DNA, suggesting a chromosomal location of the gene. CONCLUSION: We describe a novel class 1 integron containing bla(VIM-1) and aac(6')-IIc genes in an E. cloacae clinical isolate.

Acetyltransferases↗
PubMed2006

Production of the cryptic EefABC efflux pump in Enterobacter aerogenes chloramphenicol-resistant mutants.

OBJECTIVES: AcrAB-TolC is the major tripartite multidrug efflux pump in Enterobacter aerogenes while EefABC is a cryptic efflux system. This study was conducted to identify and characterize E. aerogenes mutants producing the EefABC efflux pump. METHODS: Four spontaneous chloramphenicol-resistant (CMR) mutants were isolated. The expression level of the eefABC promoter and the production of the EefA and B proteins were analysed in the mutants. Antibiotic susceptibilities were compared for wild-type and mutant strains. Efflux activity was investigated using an efflux pump inhibitor. RESULTS: The activation of the eefABC promoter was detected in four CMR mutants. These mutants showed increased resistance to erythromycin and ticarcillin, but not to fluoroquinolones, ketolides and detergents. Two additional efflux proteins were detected in the mutants. The CMR mutants bear no mutation in hns, which encodes a repressor of eefABC. No alteration of porin expression, a phenotype observed in marA or ramA multidrug-resistant mutants, was detected in the mutants. CONCLUSIONS: These observations suggest that eefABC activation can occur in vitro independently of the H-NS, MarA or RamA global regulators.

Anti-Bacterial Agents↗
PubMed2006

Intracellular accumulation of linezolid in Escherichia coli, Citrobacter freundii and Enterobacter aerogenes: role of enhanced efflux pump activity and inactivation.

OBJECTIVES: The oxazolidinone class of antibiotics such as linezolid have a narrow spectrum of activity that targets Gram-positive bacteria. We hypothesized that the poor activity of linezolid in Gram-negative bacteria is in part caused by relatively low intracellular concentration due to efflux. METHODS: Using whole cell accumulation assays we estimated the intracellular concentration of linezolid in Escherichia coli and other Enterobacteriaceae. We included test strains with enhanced RND-type multidrug efflux pump activity and with genetic inactivation of the pump or functional inhibition by carbonyl cyanide m-chlorophenylhydrazone as inhibitor of the proton motive force or 1-(1-naphthylmethyl)-piperazine (NMP), an efflux pump inhibitor. RESULTS: Consistent with susceptibility studies, enhanced pump activity caused decreased accumulation, and pump inactivation and inhibition caused increased accumulation, of linezolid. The accumulation levels in test strains of E. coli, Citrobacter freundii and Enterobacter aerogenes with functional pumps were lower than in control strains of Staphylococcus aureus and Enterococcus faecium, but were higher after pump inactivation and correlated with ethidium bromide and pyronin Y accumulation. CONCLUSIONS: The intracellular concentration of linezolid is comparatively low owing to efficient efflux of the drug and could be increased substantially by inhibition of RND-type efflux pumps.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1981

Restriction endonuclease EcaI from Enterobacter cloacae.

Restriction endonuclease EcaI obtained from Enterobacter cloacae DSM30056 recognizes the group of heptanucleotide palindromes 5'-G[unk]G-T-N-A-C-C-3', and on cleavage (arrow) produces fragments with 5'-terminal pentanucleotide extensions. It is identical in specificity with restriction endonuclease BstEII from Bacillus stearothermophilus ET.

Base Sequence↗
PubMed2002

Mutation of rpiA in Enterobacter cloacae decreases seed and root colonization and biocontrol of damping-off caused by Pythium ultimum on cucumber.

Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.

Cucumis sativus↗
PubMed1978

Resistant Enterobacter cloacae in a burn center: the ineffectiveness of silver sulfadiazine.

Enterobacter cloacae sepsis was found in 15 burn center patients in 1976, of whom 13 died. Nine of the deaths occurred in the first 60 days. The Burn Center isolates were resistant to silver sulfadiazine (AgSD) in agar cup-plate tests and confirmed by tube dilution tests. Hospital, non-burn isolates of E. cloacae were sensitive to AgSD. All E. cloacae isolates were sensitive to mafenide acetate (MA) in the agar cup-plate tests, but this was not confirmed by the tube dilution tests. The agar cup-plate susceptibility test is a simple, rapid and effective technique for determining resistant and sensitive isolates of E. cloacae. Patients who were changed from AgSD to MA because of resistant E. cloacae infection did not have improved survival. An animal study showed that AgSD was ineffective against this strain of E. cloacae and that MA was more effective than AgSD when applied 24 hr postburn but neither were effective at 48 hr postburn. MA was bacteriostatic but not bactericidal with this E. cloacae strain.

Animals↗
PubMed1984

Treatment of invasive Enterobacter cloacae burn wound sepsis with topical nitrofurazone.

Nine patients with invasive Enterobacter cloacae burn wound sepsis were identified in a series of 400 admissions to our Burn Center. In vitro antibacterial testing of topical agents revealed sensitivity to nitrofurazone cream. Treatment of this series of patients with nitrofurazone resulted in an overall 66% survival compared with an 86% mortality reported in the literature. We recommend that topical antibacterial sensitivity testing be routinely performed in the microbiology laboratory for isolates from burn patients, and that nitrofurazone cream be included among the agents tested for effectiveness.

Adult↗
PubMed2002

Enterobacter hormaechei bloodstream infection at three neonatal intensive care units in Brazil.

Enterobacter hormaechei was defined as a unique species in 1989. We describe six case patients of E. hormaechei bloodstream infection in three neonatal intensive care units in Rio de Janeiro, Brazil. E. hormaechei identification was performed on the Vitek system and confirmed by conventional testing. Strain relatedness was evaluated by pulsed field gel electrophoresis. All children recovered completely. Chart review for previous procedures revealed parenteral nutrition as the only common procedure.

Brazil↗