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Enterobacter spp.: pathogens poised to flourish at the turn of the century.

Knowledge of the genus Enterobacter and its role in human disease has expanded exponentially in recent years. The incidence of infection in the hospital and the community has increased. New clinical syndromes have been recognized. Enterobacter spp. have also been implicated as causes of other syndromes that traditionally have been associated almost exclusively with more easily treatable pathogens, such as group A streptococci and staphylococci. Rapid emergence of multiple-drug resistance has been documented in individual patients during therapy and in populations and environments with strong selective pressure from antimicrobial agents, especially the cephalosporins. Therapeutic options for patients infected with multiply resistant strains have become severely limited. Carbapenems or, alternatively, fluoroquinolones are the most predictively active options, although resistance to both classes has been observed on rare occasions. Enterobacter spp. appear well adapted for survival and even proliferation as the turn of the century approaches.

Anti-Bacterial Agents↗

Enterobacter cloacae endophthalmitis: report of four cases.

Members of the genus Enterobacter are commensal organisms of the gastrointestinal tract and are considered pathogenic only for patients with lowered resistance to infection (e.g., chronic infection, cancer, or diabetes mellitus) or those with impaired immunity (congenital, acquired, or impaired immunity secondary to therapy). We report on four cases of endophthalmitis caused by Enterobacter cloacae: two in patients with acute postoperative endophthalmitis, one in a patient with delayed bleb-related endophthalmitis, and one in a patient presenting with presumed posttraumatic endophthalmitis. Each patient presented with severe disease many days after the onset of ocular symptoms, and two patients had systemic risk factors accounting for a reduced resistance to infection. Endophthalmitis caused by gram-negative bacilli is characterized by acute onset, rapid progression, and poor final visual outcome. Each of these patients was treated by a standard protocol with intravitreal, systemic, and topical antibiotics and systemic steroids. Despite treatment, the final visual outcomes for three of these patients was no perception of light, and that for one patient remained perception of hand movements only. In common with endophthalmitis caused by other gram-negative organisms, intraocular infection secondary to Enterobacter cloacae infection is a devastating disease which, despite treatment, results in extensive ocular damage and severe visual loss. Since 1966, only four cases of endophthalmitis secondary to infection with members of this genus have been reported. This report presents four cases which occurred over a period of 14 months and, to the best of our knowledge, the first case of bleb-related endophthalmitis secondary to E. cloacae infection.

Adult↗

Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan.

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.

Enterobacter cloacae↗

Infectious discitis caused by Enterobacter cloacae.

The case is reported of a patient who developed a vertebral osteomyelitis caused by Enterobacter cloacae. The organism was isolated in cultures of blood and vertebral puncture biopsy samples. The patient was satisfactorily treated with trimethroprim and sulphamethoxazole. Enterobacter cloacae, a Gram negative organism, has been confirmed as the cause of bacteremia in patients with burns, urinary infections, in adults with pneumonia, and in children with joint infections. Spondylodiscitis caused by Enterobacter cloacae has not previously been described.

Aged↗

Identification of DNA sequences that regulate the expression of the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylic acid deaminase gene.

Analysis of the DNA sequence upstream of the previously isolated Enterobacter cloacae UW4 ACC deaminase gene (Shah et al. 1998) suggests that this segment contains several features that are thought to be involved in the transcriptional regulation of this gene. These features include half of a CRP (cAMP receptor protein) binding site, an FNR (fumarate-nitrate reduction) regulatory protein binding site, an LRP (leucine responsive regulatory protein) binding site, and an LRP-like protein coding region. ACC deaminase activity was measured following growth of either various Escherichia coli strains carrying a plasmid that contained the Enterobacter cloacae UW4 ACC deaminase gene, or of Enterobacter cloacae UW4. Variables that were compared include aerobic versus anaerobic conditions, the presence and absence of ACC in the growth medium, addition of leucine to the medium, and bacterial strains that did or did not contain either lrp or fnr genes. The data reported are consistent with the involvement of most, if not all, of the above mentioned potential regulatory regions in the expression of ACC deaminase.

Aerobiosis↗

Alanine dehydrogenase from Enterobacter aerogenes: purification, characterization, and primary structure.

Alanine dehydrogenase [EC 1. 4. 1. 1] was purified to homogeneity from a crude extract of Enterobacter aerogenes ICR 0220. The enzyme had a molecular mass of about 245 kDa and consisted of six identical subunits. The enzyme showed maximal activity at about pH 10.9 for the deamination of L-alanine and at about pH 8.7 for the amination of pyruvate. The enzyme required NAD+ as a coenzyme. Analogs of NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide served as coenzymes. Initial-velocity and product inhibition studies suggested that the deamination of L-alanine proceeded through a sequential ordered binary-ternary mechanism. NAD+ bound first to the enzyme, followed by L-alanine, and the products were released in the order of ammonia, pyruvate, and NADH. The Km were 0.47 mM for L-alanine, 0.16 mM for NAD+, 0.22 mM for pyruvate, 0.067 mM for NADH, and 66.7 mM for ammonia. The Km for L-alanine was the smallest in the alanine dehydrogenases studied so far. The enzyme gene was cloned into Escherichia coli JM109 cells and the nucleotides were sequenced. The deduced amino acid sequence was very similar to that of the alanine dehydrogenase from Bacillus subtilis. However, the Enterobacter enzyme has no cysteine residue. In this respect, the Enterobacter enzyme is different from other alanine dehydrogenases.

Alanine Dehydrogenase↗

Purification, characterization and gene analysis of N-acetylglucosaminidase from Enterobacter sp. G-1.

Enterobacter sp. G-1 is a bacterium isolated previously as a chitinase-producing bacterium. We found this bacterium also produced N-acetylglucosaminidase and characterized that in this study. Extracellular N-acetylglucosaminidase of 92.0 kDa was purified near homogeneity by 8.57-fold from Enterobacter sp. G-1. The optimum temperature and the optimum pH of the purified N-acetylglucosaminidase was 45 degrees C and 6.0, respectively. The N-terminal amino acid sequence of 23 residues of N-acetylglucosaminidase was identified. Based on the N-terminal sequence, we amplified pieces of the DNA fragments by PCR. Using these PCR products as probes, we screened the genomic library and successfully isolated the entire N-acetylglucosaminidase gene (designated nag1) from Enterobacter sp. G-1. The nucleotide sequence of the nag1 gene was found to consist of 2,655 bp encoding a protein of 885 amino acid residues. Comparison of the deduced amino acid sequence from the nag1 gene found 97.3% identity with chitobiase from Serratia marcescens, 54.4% identity with N,N'-diacetylchitobiase from Vibrio harveyi, and 42.7% identity with N-acetylglucosaminidase (ExoI) from Vibrio furnissii. Enzymatic activity assay of N-acetylglucosaminidase indicated stronger activity toward PNP-GlcNAc than PNP-(GlcNAc)2 or PNP-(GlcNAc)3.

Acetylglucosaminidase↗

The effect of casein derivatives on glycine enhancement of beta-lactamase production in Enterobacter cloacae ATCC.13047.

Our earlier work showed that glycine enhancement of class I beta-lactamase production in Enterobacter cloacae ATCC.13047 was greater on Isosensitest Agar than on other laboratory media. In the present study it was demonstrated that Casein Hydrolysate was the constituent of Isosensitest Agar which was necessary for glycine enhancement of beta-lactamase production. Also, a comparison of the effect of casein and casein derivatives on glycine enhancement of beta-lactamase production showed that acid hydrolysis of casein was a prerequisite for glycine enhancement of beta-lactamase production. Enterobacter cloacae ATCC.13047 failed to grow on a medium containing casein, and glycine enhancement of beta-lactamase production did not occur on a medium containing tryptic hydrolysate of casein. The effect on beta-lactamase production of variations in the concentrations of both Casein Hydrolysate and glycine was examined by titration of both components in a chequerboard fashion. Beta-lactamase production was influenced by the concentrations of both Casein Hydrolysate and glycine. This inter-relationship indicated that there was a balance between the concentrations of Casein Hydrolysate and glycine and that alterations in this balance modified beta-lactamase production. The significance of the observations is discussed in relation to the factors which influence glycine enhancement of beta-lactamase production in Enterobacter cloacae ATCC.13047.

Caseins↗

A hospital outbreak of high-level beta-lactam-resistant Enterobacter spp.: association more with ampicillin and cephalosporin therapy than with nosocomial transmission.

We studied an 8 month outbreak of a 7-fold increased isolation rate of high-level beta-lactam-resistant Enterobacter spp. from clinical infections (20 patients, 22 isolates: 20 E. cloacae, 2 E. aerogenes). In a case-control analysis the occurrence of resistant Enterobacter spp. was found to be associated with treatment with multiple antibiotics (p = 0.03), broad-spectrum beta-lactam agents (p = 0.0001) including ampicillin (p = 0.04), and cephalosporins (cefuroxime and cefotaxime, p = 0.004). Biochemical fingerprinting and pulsed-field gel electrophoresis (PFGE) typing showed no identity between the resistant isolates, indicating that neither cross-infection nor nosocomial transmission from a common source was the immediate cause of the problem. The outbreak was not paralleled by the overall Enterobacter spp. isolation rate or the antibiotic usage pattern in the hospital. Thus, the underlying cause of the outbreak remained obscure.

Aged↗

[Occurrence and mechanisms of resistance to beta-lactam antibiotics in clinically important species of Enterobacter].

Extended-spectrum beta-lactam antibiotics have found great medical importance, but their wide use in clinical practice leads to increasing resistance to them. The more frequent occurrence of infections caused by Bush group 1 beta-lactamase producing organisms, including species of the genus Enterobacter, is a serious problem in this field. Resistance to beta-lactams in this important nosocomial pathogens can be due to 1) reduction in outer membrane permeability to antibiotics caused by alterations in outer membrane lipopolysacharides or proteins (porins); 2) production of beta-lactamases, which inactivate beta-lactams and can also lead to resistance by non-hydrolytic mechanism called trapping. Production of plasmid-mediated extended-spectrum beta-lactamases, but especially chromosomally-mediated inducible cephalosporinase AmpC, which can be synthesized constitutively in large amounts as consequence of spontaneous chromosomal mutations, are of great clinical importance. Fourth-generation cephalosporins and carbapenems are the most effective in the treatment of infections caused by species belonging to the genus Enterobacter, but combination of high level beta-lactamase production and decreased outer membrane permeability, which is not rare in Enterobacter spp., leads to resistance even to these drugs.

Enterobacter↗

The status of drug resistance and ampC gene expression in Enterobacter cloacae.

OBJECTIVE: To investigate the status of the drug resistance and the ampC gene expression of Enterobacter cloacae. METHODS: Disk diffusion tests were made for detecting the susceptibility of antimicrobial agents against Enterobacter cloacae. AmpC gene was amplified by polymerase chain reaction (PCR) and verified by DNA sequencing. AmpC gene expression was analyzed according to antimicrobial agent sensitive phenotype. RESULTS: The sensitivity rates of 144 strains to imipenam, cefepime and cefoperazone/sulbactam were 98.61%, 65.97% and 63.89%, respectively. The sensitivity rates of 144 strains to other antimicrobial agents were lower. Among the 144 strains 120 were found to be positive by PCR for ampC. The PCR product showed high homology to the GenBank ampC sequence. Stably derepressed strains, hyperinducible strains and unexpressing or lower level expressing strains accounted for 30.0% (36/120), 37.5% (45/120), and 32.5% (39/120), respectively. Fifty-six out of 120 strains (46.67%) also produced extended spectrum beta-lactamases (ESBLs). The hyperinducible strains were highly sensitive to all the antimicrobial agents except amoxicillin/clavulanic acid and cefuroxime, while the stably derepressed strains were only sensitive to imipenam and cefepime. However, sensitivity to cefepime decreased if the strains also produced ESBLs. CONCLUSIONS: The drug resistant status of Enterobacter cloacae is severe. Clearing out the expressive status of ampC gene will be helpful in selection of antimicrobial agents in the treatment of clinical infection.

Cefoperazone↗

The biological activity of antibacterial substance produced by Enterobacter cloacae B8.

An antibacterial substance was extracted and purified from the culture of Enterobacter cloacae B8 and the antagonistic activities of the extract to 229 clinical pathogen strains was tested by the K-B paper-dish method. The extract of Enterobacter cloacae B8 showed strongly antagonistic activity to all 146 tested stains of Staphylococcus spp.; the antagonistic diameter was about 18-32 mm, averaging about 26.5 mm; showing especially strong antagonistic activity to all 87 tested strains of Methicillin-resistant Staphylococcus aureus (MRSA), the average antagonistic diameter was about 22.6 mm. The average antagonistic diameter of the extract of Enterobacter cloacae B8 to 3 tested strains of Escherchia coli and 8 tested strains of Klebsiella pneumoniae was about 14 mm and 13 mm, respectively; showing only antagonistic activities to 3 strains of Acinetobacter spp. in 26 tested strains, and showing no antagonistic activities to all 25 tested strains of Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) of partially purified extract against MRSA was about 9.4 micrograms/ml determined by disc-diffusion method, and the MIC of Vancomycin was about 3.1 micrograms/ml as the control in this study.

Actinobacteria↗

[Typing by isoelectric focusing of beta-lactamase enzymes in Klebsiella and Enterobacter strains resistant to the new beta-lactam antibiotics].

Resistance to new generation beta-lactam antibiotics in Gram-negative bacteria is increasing worldwide. This resistance is due to EBS enzymes in Klebsiella spp., and Type-I chromosomal enzymes in Enterobacter spp., Citrobacter, P. aeruginosa, Providencia, M. morganii and S. marcescens. In this study, the types of beta-lactamases in 16 Klebsiella spp. and 15 Enterobacter spp. which are resistant to newer beta-lactam antibiotics was investigated by isoelectric focusing. The results of the study showed that TEM-1 enzymes are prevalent in these isolates. pI (isoelectric point) values and antibiotic susceptibility test results suggest that there are EBS enzymes in Klebsiella spp., and Type-I enzymes in Enterobacter spp. These results have to be supported by further investigations.

Anti-Bacterial Agents↗

[Characteristics of selected virulence factors of Enterobacter cloacae strains isolated from clinical specimens].

We demonstrated that Enterobacter cloacae possesses a selective haemolytic activity on sheep erythrocytes. All the screened strains showed a haemolytic activity on sheep erythrocytes when cultures were preincubated with beta-mercaptoethanol. The investigation circulation of the genes encoding extended spectrum beta-lactamases (ESBL) shows that beta-lactamase producers can be ascribed to specific patterns of plasmids. We also demonstrated that genetic material from E. coli can be transferred and established in selected Enterobacter cloacae strains. In a survival tests we demonstrated that similarly to Salmonella or Vibrio clinical isolates Enterobacter cloacae doesn't demonstrate acid tolerance.

Enterobacter cloacae↗

Antibiotic susceptibility and beta-lactamase production in clinical isolates of Enterobacter spp.

The in vitro susceptibility of 237 clinical isolates of Enterobacter spp. (E. aerogenes, E. agglomerans and E. cloacae; 41, 64 and 132 respectively) to 16 different antibiotics is described. Four quinolones (ciprofloxacin, lomefloxacin, norfloxacin and ofloxacin), two new cephalosporins (cefpirome and cefepime) and imipenem, all showed high activity against the three Enterobacter species tested (MIC50 less than or equal to 0.125 mg/l, MIC90 less than or equal to 0.5 mg/l). Also the aminoglycosides gentamicin and tobramycin were highly active antibiotics (MIC50 less than or equal to 0.5 mg/l, MIC90 less than or equal to 1.0 mg/l). The susceptibility of beta-lactam-antibiotics to beta-lactamase produced by Enterobacter spp. was evaluated, and imipenem and cefepime were found to be most stable. Different methods for detection of inducible beta-lactamases were used, the agar dilution method being more sensitive than the double-disc diffusion test. Elevated beta-lactamase production was detected, via induction, in 83% of E. aerogenes strains and 70% of E. cloacae strains, with cefamandole used as the substrate and cefoxitin as the inducer. Constitutive, high level enzyme production was detected in 7 and 13% respectively of the E. aerogenes and the E. cloacae strains. In all the strains of E. agglomerans, 10% of E. aerogenes and 13% of E. cloacae, no beta-lactamases could be detected with the methods studied.

Aminoglycosides↗

[Epidemiologic study on the prevalence of Enterobacter, Serratia and Pseudomonas strains, producers of cefoxitin-inducible beta-lactamases].

Resistance of Enterobacter, Serratia and pseudomonas strains to newer cephalosporins is often associated with stable derepression of synthesis of the chromosomal betalactamases. Similar resistance is developed by enzyme inducible strains in response to betalactamases inducers. This finding poses many clinical problems including emergence of resistance during therapy with the drugs. In this study we evaluated the MICs of several new betalactam compounds against 76 Enterobacter, Serratia and Pseudomonas strains before and after cefoxitin-induction of betalactamases. The MICs against several Enterobacter strains (45%) after cefoxitin induction were elevated four fold or more. Serratia strains showed no significant variations of the MICs after cefoxitin induction. The MICs of piperacillin against many Pseudomonas strains (78%) after cefoxitin induction were elevated four fold or more. These data were confirmed using cefoxitin disk approximation test. Outbreaks of nosocominal infection with these multiresistant bacteria and spread of the strains throughout the hospital are already being seen. Control of these problems can only be achieved through the judicious and restricted use of these new antibiotics.

Cefoxitin↗

[Study of the species "Enterobacter gergoviae" by DNA/DNA hybridization (author's transl)].

A DNA/DNA hybridization study was carried out with 13 strains of Enterobacter gergoviae and 123 strains of Enterobacteriaceae, Mostly Enterobacter and Klebsiella species. E. gergoviae strains were very highly related (relative binding ratios: 88-96%) to the type strain CIP 76-01, as previously reported by Brenner and co-workers. No close genomic relationship was found with other species of Enterobacter and Klebsiella (mean relative binding ratio: 39%) and other Enterobacteriaceae.

DNA, Bacterial↗

[3 new cases of Enterobacter taylorae infection].

BACKGROUND: Enterobacter taylorae is a recently identified microorganism pertaining to the enterobacterian family. To date few infections by this organism have been reported with their clinical significance being unknown. Three patients with Enterobacter taylorae infection are described with the possible pathogenic capacity of this microorganism in concurrence with opportune epidemiologic features being commented upon in addition to the different sources of infection. METHODS: Biochemical identification of the microorganisms was performed by the automatized MicroScan system (Baxter). The sensitivity to the different antimicrobians was determined by automatized reading (Baxter) of the seried microdilution to the last point. RESULTS: The two first patients presented infection of an open wound with E. taylorae being isolated from the culture of the exudate of the wound and from a culture of the drainage point, respectively. The third patient developed nosocomial bacteremia following prolonged hospital stay with growth of E. taylorae being found in two different blood culture series. Different biotypes of the E. taylorae species were isolated in the three patients. Specific treatment was administered to the three patients according to the antibiogram with improvement in the clinical manifestations being observed. CONCLUSIONS: The three clinical cases herewith described suggest that E. taylorae is a potentially pathogenic microorganism which, in determined circumstances (empiric antibiotic treatment, open wounds, diagnostic or therapeutic instruments) may produce clinical pictures similar to those described for other Enterobacter species.

Aged↗