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PubMed1989

Evaluation of numerical analysis of SDS-PAGE of protein patterns for typing Enterobacter cloacae.

Twenty cultures comprising 13 clinical isolates of Enterobacter cloacae from two hospitals, the type and another reference stain of E. cloacae and the type strains of four other Enterobacter sp. and of Escherichia coli, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. The protein patterns were highly reproducible and were used as the basis of a numerical analysis which divided the clinical isolates into nine clearly defined protein types. Comparison with established typing methods indicated that the discrimination of SDS-PAGE was similar to that achieved with conventional typing methods and all strain groups recognized by combined sero/phage typing were also found by SDS-PAGE. In addition, protein typing sub-divided a group of four serotype O3 isolates that were difficult to distinguish by phage typing. We conclude that high-resolution SDS-PAGE of proteins provides an effective method of typing isolates of E. cloacae.

Bacterial Proteins↗

PubMed1996

Kinetics and mechanism of the hydrolysis of depsipeptides catalyzed by the beta-lactamase of Enterobacter cloacae P99.

The steady-state kinetics and mechanism of the hydrolysis and aminolysis of a series of acyclic depsipeptides, catalyzed by the class C beta-lactamase of Enterobacter cloacae P99, have been studied in order to more firmly establish the nature of the transition states involved. The class C beta-lactamase of Enterobacter cloacae P99 was employed. The depsipeptide substrates contained a constant acyl group, (phenylacetyl)glycyl, and chemically different leaving groups, m-carboxyphenoxide, m-carboxythiophenoxide, 3-carboxyl-4-nitrophenoxide, lactate, and thiolactate. Evaluation of the steady-state kinetic parameters and the effect of the alternative nucleophile methanol on these parameters and on the product distribution showed that deacylation was largely rate-determining to turnover of the aryl esters under conditions of substrate saturation, while acylation was rate-determining to the alkyl esters. The earlier conclusion [Govardhan & Pratt (1987) Biochemistry 26, 3385-3395] that acylation largely limited the turnover of the aryl esters was shown to be an artifact of phosphate buffer inhibition. The aminolysis of both the aryl the alkyl esters by D-phenylalanine was influenced by binding of the substrate at a second binding site on the acyl-enzyme intermediate. A study of inhibiton of the hydrolysis of (phenylacetyl)-glycyl-D-thiolactate by the aminolysis product (phenylacetyl)glycyl-D-phenylalanine indicated that the second binding site is also available for ligands to bind the free enzyme and to the noncovalent Michaelis complex with this substrate. It is likely that penicillin-recognizing enzymes in general, both beta-lactamases and DD-peptidases, possess an extended substrate-binding site into which a variety of small ligands may bind at any point along the reaction coordinate and, to a greater or lesser extent depending on circumstances, affect catalysis.

Acylation↗

PubMed2001

Molecular epidemiological typing of Enterobacter cloacae isolates from a neonatal intensive care unit: three-year prospective study.

Since 1992, there has been an increase in the incidence of Enterobacter sepsis in the neonatal intensive care unit (NICU) of the authors' hospital. From 1995 to 1997, a prospective molecular epidemiological survey of the colonizing and infecting strains isolated from neonates was conducted. Enterobacter cloacae was the most frequent cause of neonatal sepsis, accounting for 19.2% of all neonatal infections, reaching a peak incidence of 2.2/1000 during 1996. Fifty isolates from the NICU and four epidemiologically unrelated strains were characterized by pulse-field gel electrophoresis (PFGE), ribotyping, enterobacterial repetitive intergenic consensus (ERIC)-PCR and plasmid profiling. PFGE was the most discriminatory technique and identified 13 types (two of them classified into two and three subtypes) compared with ERIC-PCR, plasmid profiling and ribotyping that identified 11, 11 and seven types, respectively. A good correlation was found between all techniques. Five different clones caused 15 cases of sepsis. Clones A and B were prevalent in 1995 and 1996, but they were not isolated in 1997. An outbreak caused by clone G in 1997 was controlled by cohort nursing and hygienic measures, without changing the antibiotic policy. Strains were characterized by their antibiotic resistance pattern and divided into three groups. Group I correlated with PFGE types A, B1 and B2, which hyperproduced Bush type 1 chromosomal beta-lactamase and expressed extended-spectrum ?-lactamases (ESBLs). Group II only hyperproduced Bush type 1 chromosomal beta-lactamase and correlated with PFGE-types D1, D2, D3 and I. Finally, Group III, with inducible beta-lactamases, correlated with the rest of PFGE types. The sudden disappearance of E. cloacae after reinforcement of hygienic measures confirms the importance of patient-to-patient transmission.

Bacterial Typing Techniques↗

PubMed2003

Purification and characterization of PrbA, a new esterase from Enterobacter cloacae hydrolyzing the esters of 4-hydroxybenzoic acid (parabens).

The esterase PrbA from Enterobacter cloacae strain EM has previously been shown to confer additional resistance to the esters of 4-hydroxybenzoic acid (parabens) to two species of Enterobacter. The PrbA protein has been purified from E. cloacae strain EM using a three-step protocol resulting in a 60-fold increase in specific activity. The molecular mass of the mature enzyme was determined to be 54,619 +/- 1 Da by mass spectrometry. It is highly active against a series of parabens with alkyl groups ranging from methyl to butyl, with K(m) and V(max) values ranging from 0.45 to 0.88 mM and 0.031 to 0.15 mM/min, respectively. The K(m) and V(max) values for p-nitrophenyl acetate were 3.7 mM and 0.051 mM/min. PrbA hydrolyzed a variety of structurally analogous compounds, with activities larger than 20% relative to propyl paraben for methyl 3-hydroxybenzoate, methyl 4-aminobenzoate, or methyl vanillate. The enzyme showed optimum activity at 31 degrees C and at pH 7.0. PrbA was able to transesterify parabens with alcohols of increasing chain length from methanol to n-butanol, achieving 64% transesterification of 0.5 mm propyl paraben with 5% methanol within 2 h. PrbA was inhibited by 1-chloro-3-tosylamido-4-phenyl-2-butanone and 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), with K(i) values of 0.29 and 0.20 mM, respectively, and was irreversibly inhibited by Diisopropyl fluorophosphate (DFP) or diethyl pyrocarbonate. The stoichiometry of addition of DFP to the enzyme was 1:1 and only 1 TLCK molecule was found in TLCK-modified enzyme, as measured by mass spectrometry. Analysis of the tryptic digest of the DFP-modified PrbA demonstrated that the addition of a DFP molecule occurred at Ser-189, indicating the location of the active serine.

Amino Acid Sequence↗

PubMedDate not supplied

Heterogeneity in ampR-ampC gene interaction in Enterobacter cloacae.

The ampR gene and its regulation of AmpC beta-lactamase synthesis were investigated for Enterobacter cloacae 1194E, a wild-type strain producing a group A (pI 8.7) enzyme. Expression of the cloned E. cloacae 1194E ampR-ampC region was examined initially in Escherichia coli HB101. However, transformants showed only constitutive beta-lactamase expression. For study of enzyme expression in a more closely related host, the cloned E. cloacae 1194E ampR-ampC region was transformed into E. cloacae 55, a wild-type strain producing a group B (pI 7.8) enzyme. Results indicated a functional E. cloacae 1194E ampR gene that could not be transcomplemented by E. cloacae 55. A comparative analysis of ampR nucleotide and amino acid-sequence data from E. cloacae 1194E and E. cloacae MHN1 revealed related but divergent genes. Thermal induction studies of AmpC beta-lactamase also indicated a difference between E. cloacae 1194E and E. cloacae 55 in ampR-ampC interaction. Thus, it appears that, in at least some strains of Enterobacter, significant intraspecies divergence of ampR has occurred. This heterogeneity in ampR would not have been detected with beta-lactamase expression studies conducted exclusively in E. coli.

Amino Acid Sequence↗

PubMed1996

Efficacy of cefepime in the treatment of infections due to multiply resistant Enterobacter species.

Cefepime is a new cephalosporin with an enhanced antibacterial potency and spectrum. More rapid penetration into many gram-negative bacilli, targeting of multiple penicillin-binding proteins, and resistance to inactivation by many beta-lactamases account for its activity against organisms that have developed resistance to agents such as ceftazidime, cefotaxime, or ceftriaxone. This study identified 16 patients with 17 infections due to Enterobacter species organisms with reduced susceptibility or resistance to ceftazidime. Most isolates were multiply resistant to other beta-lactam drugs as well, but all were susceptible to cefepime. All 17 infections, which included pneumonia, urinary tract infection, intraabdominal infection, and bacteremia, responded clinically to intravenous cefepime. In particular, cefepime was successfully used in the management of cases of chronic infection that had responded poorly to repeated therapy with imipenem, aminoglycosides, or ciprofloxacin. Eradication of Enterobacter species organisms occurred at 15 (88.2%) of the 17 sites of infection. No emergence of resistance to cefepime was noted.

Cefepime↗

PubMed1978

Cefamandole in the treatment of infections due to Enterobacter and indole-positive Proteus.

Clinical and bacteriologic results are reported for 80 patients treated with 1.5--12 g of cefamandole daily for a variety of infections caused by Enterobacter and indole-positive Proteus, organisms that have been resistant to most available cephalosporins. Of 45 patients with infections due to Enterobacter, 41 (91%) had satisfactory clinical responses; 36 were bacteriologic successes, and six cases of complicated urinary tract infections relapsed. Of 37 patients with infections due to indole-positive Proteus, 28 (88%) were clinical successes and 30 (81%) were bacteriologic successes. Fourteen cases of complicated urinary tract infection relapsed. Of 104 patients in whom the drug was evaluated for safety, use of cefamandole was discontinued in five; nine adverse reactions were considered drug-related. A summary of published in vitro data shows that the majority of strains of these organisms were susceptible to cefamandole at concentrations achievable in the serum. Minimal inhibitory concentrations are variable, and there is a significant inoculum effect, the clinical significance of which has not been determined.

Adult↗

PubMed1987

Development of beta-lactam-resistant Enterobacter cloacae in mice.

We compared the ability of four newer beta-lactam compounds to produce resistance in an experimental model of Enterobacter cloacae infection. Mice infected intraperitoneally developed resistance depending on antibiotic treatment and the dose given. Percentages of mice in which resistance was observed were as follows: 100% after ceftriaxone (50 mg/kg, two doses); 87% after ceftriaxone (50 mg/kg, one dose); 35% after ceftriaxone (500 mg/kg, one dose); and 21% after carumonam (25 mg/kg, two doses). No resistance occurred after therapy with either BMY 28142 (25 mg/kg, two doses) or Sch 34343 (50 mg/kg, two doses). Heterogeneous resistance to beta-lactams among the cells within a given Enterobacter population accounted for these differences. The minimal concentration inhibiting the growth of the preexisting resistant variants, together with the antibiotic concentrations obtained in the peritoneal fluid, were associated with further emergence of resistance in the mouse treated with this antibiotic.

Animals↗

PubMed1983

Broad-spectrum beta-lactam resistance in Enterobacter: emergence during treatment and mechanisms of resistance.

Of three patients with Enterobacter mediastinitis treated with cefamandole, two failed initial treatment due to emergence of strains resistant to cefamandole and third generation cephalosporins. Extracts of the sensitive strains showed inducible hydrolysis of cephalothin; resistant strains (wild and laboratory derived) showed constitutive hydrolysis of cephalothin and cefamandole and bound but did not hydrolyze cefotaxime and latamoxef (moxalactam). In one mutant, increased latamoxef resistance appeared due to non-enzymatic mechanism(s). The Enterobacter strain from the patient who was treated successfully with cefamandole yielded only low level resistant mutants with lower cephalosporin binding than obtained with the other strains.

Anti-Bacterial Agents↗

PubMed1989

How predictable is development of resistance after beta-lactam therapy in Enterobacter cloacae infection?

Certain non-fastidious Gram-negative bacilli, notably Enterobacter cloacae, although classified as susceptible by usual in-vitro susceptibility testing, often become resistant in patients treated with newer beta-lactam antibiotics. Here various in-vitro tests were carried out together with an animal model allowing the quantification of resistance that emerges after short term therapy. Mice were challenged (10(8) cfu plus talcum) intraperitoneally with one each of four strains of Ent. cloacae. Two hours later, a single beta-lactam dose was administered subcutaneously. The following day, the peritoneal bacterial population was analysed by using antibiotic-containing gradient plates. Development of resistance after therapy varied according to the compound considered. Imipenem (50 mg/kg) produced no resistance, and piperacillin (200 mg/kg) only a few, while resistance occurred frequently after therapy with aztreonam (50 mg/kg), ceftazidime (50 mg/kg), cefotaxime (50 mg/kg) and cefpirome (50 mg/kg). MICs increased by at least 16-fold when resistance developed. No simple correlations were found between these in-vivo results and initial MICs, killing kinetics, frequency of resistant variants within the bacterial populations before therapy, initial MIC of these variants or antibiotic concentrations assayed in peritoneal fluid 60 min after dosing. The most reliable predictive in-vitro test appeared to be the determination of resistance emerging in broth containing at least 16 times the MIC of the antibiotic tested. Such a test is unlikely to be used on a routine basis. When a beta-lactam compound seems appropriate for treating an Enterobacter infection, it may be advisable to avoid drugs that are prone to produce resistance in experimental or clinical infections, whatever the results of conventional in-vitro susceptibility tests.

Animals↗

PubMed1993

beta-Lactam resistance amongst Enterobacter species.

Following the introduction of extended-spectrum cephalosporins into clinical use, the prevalence of species belonging to the genus Enterobacter has increased because of their natural resistance to earlier cephalosporins and their ability to develop resistance rapidly to the newer drugs. beta-Lactam resistance in this genus is due, for the most part, to the presence of a Bush group 1 chromosomal cephalosporinase. This enzyme is normally inducible and resistance to older cephalosporins, cephamycins and aminopenicillins results from either the extreme lability of the drugs to the enzyme or from their inducer activities. Resistance to newer penicillins, cephalosporins and monobactams is attributable to the selection of mutants which express large amounts of the enzyme. Such mutants arise as the result of a spontaneous mutation in one of the regulatory genes responsible for suppressing enzyme expression. Since the enzyme has very high affinity for the newer cephalosporins, this, coupled with the slow penetration of the drugs into the cell, provides a very efficient mechanism of resistance. Recent surveys in the USA and elsewhere have shown that the increased prevalence of multi-beta-lactam-resistant strains of enterobacter is due to the increased use of the newer cephalosporins. Attempts to prevent these problems include the more judicious use of newer beta-lactam antibiotics and the development of enhanced-potency cephalosporins which are able to avoid resistance because they have lower enzyme affinity and permeate more rapidly into the cell.

Anti-Bacterial Agents↗

PubMed1996

Quantitative comparison in vitro of mutational antibiotic resistance of Enterobacter spp. using a spiral plater.

The presence of spontaneous mutational antibiotic resistance among 18 bacteremic isolates of Enterobacter spp. to cefotaxime, ceftazidime, gentamicin, amikacin, ciprofloxacin, and trimethoprim-sulfamethoxazole was determined quantitatively in vitro using a spiral plater. Each drug was delivered using the device and the agar plates were inoculated in radial streaks. The degree of resistance was estimated by dividing the antimicrobial concentration required to inhibit 90% of the colonies growing in the area beyond the MIC by the MIC itself. The degree of resistance to third-generation cephalosporins and aztreonam was statistically significantly greater than that to co-trimoxazole, imipenem, and ciprofloxacin (P < 0.01); the latter three antibiotics showed virtually no mutational resistance. An intermediate level of resistance was induced by aminoglycosides, and mutational resistance to piperacillin varied between this and the higher levels observed for the cephalosporins. By providing a simple and efficient means of detecting spontaneous mutational resistance, the spiral plater may prove useful in identifying those antimicrobial agents which induce few or no mutants and therefore may be more likely to be successful in treating infections due to Enterobacter spp.

Bacteriological Techniques↗

PubMed2006

Enterobacter gergoviae and the prevalence of efflux in parabens resistance.

OBJECTIVES: In order to characterize the mechanism involved in parabens resistance, we studied 13 Enterobacter gergoviae collected from diverse cosmetic formulations containing parabens as preservatives and 10 isolates from clinical or industrial sources. METHODS: RAPD and ERIC-PCR were employed and compared for the epidemiological typing. To study antibiotic and paraben susceptibility, the standard disc diffusion method and the 2-fold dilution method in Luria-Bertani medium were used. Characterization of porins was performed using immunodetection with polyclonal antibodies. Resistance mechanisms against parabens membrane permeabilization were evaluated by measuring K(+) efflux using a specific electrode. mar regulon identification and comparison were carried out. RESULTS: Epidemiological typing confirmed that most of the cosmetic formulations were contaminated by unrelated strains. All of the E. gergoviae strains presented high methylparaben MICs, ranging from 1 to 3.8 g/L, values that were 2-5 times higher than for Escherichia coli or Enterobacter aerogenes, even in strains overexpressing MarA. These MICs decreased in the presence of phenylalanine arginine beta-naphthylamide, pinpointing efflux as a major mechanism of parabens resistance even in E. gergoviae clinical strains. CONCLUSIONS: This is the first report showing the role of active efflux in the parabens resistance in E. gergoviae, a mechanism that may explain its frequent isolation in parabens-containing cosmetics compared with other enterobacterial species. Paraben efflux seems to be regulated by a mar-independent process in E. gergoviae.

Anti-Bacterial Agents↗

PubMed1983

Fimbrial haemagglutinins in Enterobacter species.

Fifty-two strains from seven species of Enterobacter, grown under a variety of conditions, were examined in rocked-tile tests for production of haemagglutinins and with the electron microscope for fimbriae. Thirteen non-haemagglutinating strains were non-fimbriate. Most (33) of the 39 haemagglutinating strains produced only one kind of haemagglutinin, either the mannose-sensitive haemagglutinin associated with type -1 Fimbriae or, the mannose-resistant, Klebsiella-like haemagglutinin associated with type-3 fimbriae. Multiply haemagglutinating strains were most common in E. aerogenes, in which species a third kind of haemagglutinin, also mannose-resistant, was found. The findings are discussed briefly in the light of the current taxonomy of Enterobacter.

Enterobacter↗

PubMed1986

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases.

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.

Acid Phosphatase↗

PubMed1993

Isolation, classification and molecular characterization of bacteriophages for Enterobacter species.

Out of 22 Enterobacter phages investigated, nine were found to be suitable for phage typing based on their different lytic spectra on 398 strains of Enterobacter spp. isolated from milk powder and other foods. These phages were compared on the basis of morphology, protein composition, restriction endonuclease patterns and DNA-DNA hybridization. Two phages (WS-EP19, WS-EP13) belonged to the Podoviridae family (morphotype C1), and three (WS-EP20, WS-EP26, WS-EP28) were classified as Siphoviridae (morphotype B1). The other four phages were Myoviridae of the morphological groups A1 (WS-EP57) and A2 (WS-EP32, WS-EP94, WS-EP96). SDS-PAGE revealed individual protein profiles for each phage, which corresponded to different restriction enzyme fragment patterns. DNA-DNA hybridization demonstrated the close relationship of phages WS-EP20 and WS-EP26, and of WS-EP94 and WS-EP96. In general, a good correlation was found between groupings obtained with the various methods. The nine phages could be attributed to existing enterobacterial phage species although some differences to the described type phages were observed.

Bacteriophage Typing↗

PubMed1998

Antimicrobial susceptibility of oral isolates of Enterobacter cloacae and Klebsiella pneumoniae from a southern Chinese population.

The antibiotic susceptibilities of 59 Enterobacter cloacae and 39 Klebsiella pneumoniae human oral isolates collected from a southern Chinese population in Hong Kong were investigated for their susceptibility to eight antibiotics: ampicillin, cephalothin, cefuroxime, ceftazidime, ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole using the E-Test method for direct quantification of minimum inhibitory concentrations. Most strains were sensitive to all antibiotics except ampicillin and cephalothin. Ampicillin resistance was exhibited by 82% of K. pneumoniae and 69% of E. cloacae isolates. Eighty-eight percent of E. cloacae isolates were resistant to cephalothin. Several strains fell within the intermediate category of sensitivity for ampicillin (E. cloacae and K. pneumoniae), cefuroxime (E. cloacae) and tetracycline (K. pneumoniae). Comparison with other Hong Kong data suggests that resistance rates to cefuroxime, ceftazidime, ciprofloxacin, gentamicin, tetracycline and trimethoprim/sulfamethoxazole exhibited by the oral isolates are generally lower than in enterobacters and Klebsiella spp. isolated from urine, skin and soft tissues in Hong Kong populations.

Adult↗

PubMed1995

Ascorbate as an induction inhibitor of beta-lactamase in a strain of Enterobacter cloacae.

The effect of ascorbate and anaerobiosis of beta-lactamase content (constitutive and inducible) in relation to the susceptibility of a standard strain of Enterobacter cloacae to ampicillin was studied. Enterobacter cloacae ATCC 13047 showed increasing susceptibility to ampicillin when incubated anaerobically in the presence of increasing concentrations of ascorbic acid. The inducible beta-lactamase activity in the cell-free extracts of Ent. cloacae decreased when the bacterium was grown aerobically in the presence of ascorbic acid. Under anaerobic growth conditions, however, ascorbic acid abrogated the induction of the enzyme completely. On the other hand, the constitutive enzymatic activity was markedly decreased as the bacterium was grown anaerobically. Thus under these growth conditions ascorbate-anaerobiosis, the total beta-lactamase level in the presence of ampicillin as inducer fell below the basal constitutive activity observed in the absence of ampicillin.

Aerobiosis↗