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PubMed2003

Characterization of blaCMY-10 a novel, plasmid-encoded AmpC-type beta-lactamase gene in a clinical isolate of Enterobacter aerogenes.

AIMS: We report the description of a novel plasmid-encoded AmpC beta-lactamase gene (blaCMY-10) from Enterobacter aerogenes K9911729 that was isolated from a patient suffering from pneumonia in South Korea. METHODS AND RESULTS: Using antibiotic susceptibility testing, plasmid analysis, transconjugation and Southern blot analysis, the cefoxitin resistance phenotype reflects the presence of a large plasmid [pYMG-1 (130 kb)] in Ent. aerogenes K9911729. One beta-lactamase with the pI of 8.0 from transconjugant of Ent. aerogenes K9911729 was identified by isoelectric focusing on a gel. A 1475 bp DNA fragment containing the blaCMY-10 gene, identified on pYMG-1 of Ent. aerogenes K9911729, was sequenced and an open reading frame coding for 382 amino acid, CMY-10, was found. The 37 class C beta-lactamases were subclassified into 1a to 1j and CMY-10 into 1a by phylogenetic analysis. A sequence identical to the common regions in In6, In7 and a novel integron from pSAL-1 was found upstream from blaCMY-10 gene at nucleotide 1-71. CONCLUSIONS: These results clearly show that blaCMY-10 gene belongs to the group of ampC-related bla genes. Homology analysis among AmpC enzymes or ampC genes implied that integration of the chromosomal ampC gene into a large resident plasmid, followed by transconjugation, was involved in the evolution of blaCMY-10 gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The first identification of the blaCMY-10 gene is of concern as chromosomal beta-lactamases may cause serious therapeutic problems if their genes are translocated onto plasmids.

Base Sequence↗
PubMed2003

Crystal structure of thiamindiphosphate-dependent indolepyruvate decarboxylase from Enterobacter cloacae, an enzyme involved in the biosynthesis of the plant hormone indole-3-acetic acid.

The thiamin diphosphate-dependent enzyme indolepyruvate decarboxylase catalyses the formation of indoleacetaldehyde from indolepyruvate, one step in the indolepyruvate pathway of biosynthesis of the plant hormone indole-3-acetic acid. The crystal structure of this enzyme from Enterobacter cloacae has been determined at 2.65 A resolution and refined to a crystallographic R-factor of 20.5% (Rfree 23.6%). The subunit of indolepyruvate decarboxylase contains three domains of open alpha/beta topology, which are similar in structure to that of pyruvate decarboxylase. The tetramer has pseudo 222 symmetry and can be described as a dimer of dimers. It resembles the tetramer of pyruvate decarboxylase from Zymomonas mobilis, but with a relative difference of 20 degrees in the angle between the two dimers. Active site residues are highly conserved in indolepyruvate/pyruvate decarboxylase, suggesting that the interactions with the cofactor thiamin diphosphate and the catalytic mechanisms are very similar. The substrate binding site in indolepyruvate decarboxylase contains a large hydrophobic pocket which can accommodate the bulky indole moiety of the substrate. In pyruvate decarboxylases this pocket is smaller in size and allows discrimination of larger vs. smaller substrates. In most pyruvate decarboxylases, restriction of cavity size is due to replacement of residues at three positions by large, hydrophobic amino acids such as tyrosine or tryptophan.

Amino Acid Sequence↗
PubMed2003

Studies on structure-function relationships of indolepyruvate decarboxylase from Enterobacter cloacae, a key enzyme of the indole acetic acid pathway.

Enterobacter cloacae, isolated from the rhizosphere of cucumbers, produces large amounts of indole-3-acetic acid. Indolepyruvate decarboxylase, the key enzyme in the biosynthetic pathway of indole-3-acetic acid, catalyses the formation of indole-3-acetaldehyde and carbon dioxide from indole-3-pyruvic acid. The enzyme requires the cofactors thiamine diphosphate and magnesium ions for catalytic activity. Recombinant indolepyruvate decarboxylase was purified from the host Escherichia coli strain JM109. Specificity of the enzyme for the substrates indole-3-pyruvic acid, pyruvic acid, benzoylformic acid, and seven benzoylformic acid analogues was investigated using a continuous optical assay. Stopped-flow kinetic data showed no indication for substrate activation in the decarboxylation reaction of indole-3-pyruvic acid, pyruvic acid or benzoylformic acid. Size exclusion chromatography and small angle X-ray solution scattering experiments suggested the tetramer as the catalytically active state and a pH-dependent subunit association equilibrium. Analysis of the kinetic constants of the benzoylformic acid analogues according to Hansch et al. [Hansch, C., Leo, A., Unger, S.H., Kim, K.H., Nikaitani, D & Lien, E.J. (1973) J. Med. Chem.16, 1207-1216] and comparison with indole-3-pyruvic acid conversion by pyruvate decarboxylases from Saccharomyces cerevisiae and Zymomonas mobilis provided some insight into the catalytic mechanism of indolepyruvate decarboxylase.

Binding Sites↗
PubMed2000

TEM-24 extended-spectrum beta-lactamase-producing Enterobacter aerogenes: long-term clonal dissemination in French hospitals.

OBJECTIVE: To investigate interstrain relatedness of TEM-24-producing Enterobacter aerogenes clinical strains isolated between 1993 and 1998 in 10 French hospitals from nine areas by pulsed-field gel electrophoresis (PFGE) and plasmid patterns. METHODS: Fifteen TEM-24-producing strains and a set of 16 control strains having various other antibiotic resistance phenotypes were genotyped by PFGE. Plasmid DNA from TEM-24-producing strains and transconjugants was analyzed. RESULTS: Analysis of XbaI macrorestriction patterns revealed only minor variations, and showed that all 15 TEM-24-producing strains were closely related. Some isolates originating from distant areas had indistinguishable patterns. According to their clustering correlation coefficients, they were also genomically distant from the control strains. Two plasmid patterns were observed in TEM-24-producing strains, one of them in 13 of the strains. Large plasmids of 85 kb encoding TEM-24 beta-lactamase were present in all isolates and, in all except one strain, could be transferred with high frequency by conjugation. CONCLUSIONS: These results confirm that the spread of the TEM-24 extended-spectrum beta-lactamase in France was essentially due to the dissemination of a single clone.

Bacterial Proteins↗
PubMed1997

Thermal resistance of Enterobacter sakazakii in reconstituted dried-infant formula.

Enterobacter sakazakii, designated a unique species in 1980, has been implicated in a rare but severe form of neonatal meningitis, with dried-infant formula being implicated as the mode of transmission. The high mortality rate (40-80%) and the lack of information about this organism led to a study of the heat resistance of Ent. sakazakii in reconstituted dried-infant formula. Ten Canadian Ent. sakazakii strains (5 clinical and 5 food isolates) were used to determine the heat resistance of this organism at 52, 54, 56, 58 and 60 degrees C in reconstituted dried-infant formula. D-values of 54.8, 23.7, 10.3, 4.2 and 2.5 min were obtained for each temperature, respectively. The overall calculated z-value was 5.82 degrees C. In a comparison of the D-values of several members of the Enterobacteriaceae in dairy products, Ent. sakazakii appeared to be one of the most thermotolerant organisms. The importance of process control during manufacture and the use of aseptic procedures during the preparation, use and storage of dried-infant formula is discussed.

Animals↗
PubMed2000

Enterobacter cloacae outbreak in the NICU related to disinfected thermometers.

In the first week ot December 1997, an increasing incidence of neonates colonized with multi-drug resistant Enterobacter cloacae (MR-E. cloacae) was observed in the neonatal Intensive care unit of our 950-bed university hospital. Initially, re-enforcement of infection control practices including hand disinfection and cohort isolation seemed to be sufficient to control the outbreak. Nevertheless, an increasing number of newly admitted patients was paralleled by another rise in the incidence of colonized neonates. Since E. cloacae was initially found in urine specimens of the patients, surveillance and environmental cultures were aimed at procedures and instruments that might colonize the gastro-intestinal and/or urinary tract. E, cloacae was isolated from a single cap of an electronic digital thermometer. Despite banning of this possible source, newly admitted neonates still became colonized. The unit was closed for further admissions and a second round of extensive screening was started; this time including all available thermometers and continuous rectal temperature probes. Ready-to-use 'disinfected thermometers and probes were found to be colonized with MR-E. cloacae. Observation of disinfection procedures and a laboratory investigation revealed that 'rushed disinfection with alcohol 80% led to a 1 in 10 chance of thermometers still being contaminated. Furthermore, alcoholic hand rub used for convenience disinfection failed to disinfect thermometers in 40% and 20% of the cases when done in a 'rushed' or 'careful' fashion, respectively. Adequate disinfection of the thermometers led to the control of the outbreak, with no new occurrence of MR-E. cloacae in the following months.

Cross Infection↗
PubMed2002

Management of an outbreak of Enterobacter cloacae in a neonatal unit using simple preventive measures.

Enterobacter cloacae is becoming an increasingly important nosocomial pathogen. Outbreaks of E. cloacae in intensive care units and burns units have been described frequently. In December 1999, a neonate with line sepsis was transferred from a university hospital to the neonatal unit of the Diakonessen Hospital. Blood culture yielded E. cloacae. An outbreak of E. cloacae was occurring in the university hospital at that time. In February 2000, a second neonate in our hospital developed line sepsis caused by E. cloacae. Direct measures taken included cohorting of infected children, disinfection of incubators, thermometers and wards, and screening patients. Of nine neonates, seven were colonized with E. cloacae. Despite these measures, the outbreak continued. Forty-one patients were screened; 15 were colonized. Environmental searches yielded E. cloacae in a sink and on two thermometers. Sixteen isolates were typed by arbitrarily primed PCR using four primers. All the patient isolates and the two isolates from thermometers were identical. The strain isolated from the sink was unrelated. Amplified fragment length polymorphism typing showed that the outbreak clone was identical to that in the university hospital. After the introduction of disposable thermometer covers, E. cloacae colonization slowly decreased.

Cross Infection↗
PubMed2002

Urinary tract infections caused by endemic multi-resistant Enterobacter cloacae in a dialysis and transplantation unit.

In the period 1994-1998, 42 multi-resistant Enterobacter cloacae isolates responsible for urinary tract infections (UTIs) were obtained from children hospitalized in our dialysis and transplantation unit. E. cloacae isolates obtained between 1994 and 1996 were susceptible to aminoglycosides, carbapenems and fluoroquinolones, whereas E. cloacae isolates obtained between 1997 and 1998 were susceptible to carbapenems and fluoroquinolones, only. All isolates were characterized by use of the polymerase chain reaction-based random amplification of polymorphic DNA and pulsed-field gel electrophoresis. It was found that the majority of multiresistant E. cloacae isolates obtained during 1997-1998, and all isolates obtained during 1994-1996, belonged to predominant clones A or B. These strains were responsible for gastrointestinal tract colonization and UTI of renal transplant recipients for several years, and persisted as endemic E. cloacae strains in the dialysis and transplantation unit from 1994 to 1998.

Adolescent↗
PubMed2002

DNA analysis of nosocomial infection by Enterobacter aerogenes in three cases of septicaemia in Japan.

Ceftazidime-resistant Enterobacter aerogenes was isolated from blood cultures of three patients with fever. DNA analysis using pulsed-field gel electrophoresis and ribosomal RNA gene restriction digest pattern analysis revealed that the strains were clonally similar to each other with a 79.3-96.0% homology. The same strain of E. aerogenes was isolated from a three-way stopcock connected to the indwelling catheter in one of the patients at a concentration of 45 cfu/mL. A similar strain was also isolated from the urine of one other patient on the same floor. The data suggest that E. aerogenes caused septicaemia via low bacterial contamination of a three-way stopcock in a peripheral drip intravenous infusion system in at least one patient, and that the outbreak of E. aerogenes infections was due to clonally-related strains.

Adult↗
PubMed2002

Study of an outbreak of Enterobacter cloacae sepsis in a neonatal intensive care unit: the application of epidemiologic chromosome profiling by pulsed-field gel electrophoresis.

OBJECTIVE: From October 1996 to March 1997, a cluster of 11 cases of neonatal sepsis caused by Enterobacter cloacae with similar antimicrobial susceptibility patterns occurred in a neonatal intensive care unit. This outbreak prompted an investigation. METHOD: Twelve isolates obtained from 6 neonatal patients who developed E cloacae sepsis during the outbreak were analyzed. Four E cloacae isolates from 2 preterm neonates without E cloacae infection on the same ward, and 1 isolate from the hands of a nurse, were also examined. No E cloacae were isolated from the environment. Bacterial DNA digested with XbaI or NotI was analyzed with pulsed-field gel electrophoresis. RESULT: Three distinct banding patterns were identified by pulsed-field gel electrophoresis. Of the 6 preterm infants with sepsis, strain I was identified in 1, strain II in 2, a mixed infection of strains I and II in 2, and strain III was found in only 1 infant. An isolate from the hands of a nurse was identified as strain II, as were the 4 isolates from the 2 preterm neonates without E cloacae infection. Thus, this outbreak of sepsis was caused by 2 genotypes of E cloacae. CONCLUSION: This study demonstrates that pulsed-field gel electrophoresis with restriction enzyme digestion is a valuable tool for genetic characterization of multidrug-resistant E cloacae strains during outbreaks.

Bacterial Typing Techniques↗
PubMed1993

Evolution of an enzyme activity: crystallographic structure at 2-A resolution of cephalosporinase from the ampC gene of Enterobacter cloacae P99 and comparison with a class A penicillinase.

The structure of the class C ampC beta-lactamase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-A resolution and compared to a class A beta-lactamase (penicillinase) structure. The binding site for beta-lactam (penicillinase) structure. The binding site for beta-lactam antibiotics is generally more open than that in penicillinases, in agreement with the ability of the class C beta-lactamases to better bind third-generation cephalosporins. Four corresponding catalytic residues (Ser-64/70, Lys-67/73, Lys-315/234, and Tyr-150/Ser-130 in class C/A) lie in equivalent positions within 0.4 A. Significant differences in positions and accessibilities of Arg-349/244 may explain the inability of clavulanate-type inhibitors to effectively inactivate the class C beta-lactamases. Glu-166, required for deacylation of the beta-lactamoyl intermediate in class A penicillinases, has no counterpart in this cephalosporinase; the nearest candidate, Asp-217, is 10 A from the reactive Ser-64. A comparison of overall tertiary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral beta-lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two beta-lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C beta-lactamase to evolve to the class A beta-lactamase having an improved ability to catalyze the deacylation step of beta-lactam hydrolysis.

Binding Sites↗
PubMed1994

Analysis of a carbapenem-hydrolyzing class A beta-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein.

Carbapenems such as imipenem are extended-spectrum beta-lactam antibiotics, which are not hydrolyzed by the beta-lactamases commonly found in Enterobacteriaceae. Here we report a gene encoding a carbapenemase, which was cloned from the chromosome of a clinical isolate of Enterobacter cloacae, strain NOR-1, into pACYC184 plasmid in Escherichia coli. Unlike all the sequenced carbapenemases, which are class B metallo-beta-lactamases, the mature protein (NmcA) is a class A serine beta-lactamase. NmcA shares the highest amino acid identity (50%) with the extended-spectrum class A beta-lactamase MEN-1 from E. coli. In the opposite orientation from the nmcA promoter, an overlapping and divergent promoter was detected, along with an open reading frame, which encoded a 33.5-kDa protein (NmcR). The NmcR amino acid sequence displays homology with LysR-type transcriptional regulatory proteins, including the conserved residues near its N terminus within a helix-turn-helix motif. Deletion of nmcR resulted in decreased carbapenem resistance and a loss of beta-lactamase inducibility, demonstrating a positive role of NmcR in NmcA expression.

Amino Acid Sequence↗
PubMed2002

Cloning and sequencing of Enterobacter aerogenes OmpC-type osmoporin linked to carbapenem resistance.

Using outbreak-related strains of Enterobacter aerogenes, we cloned and sequenced ompK39, the structural gene coding for outer membrane protein OmpK39. Its lack of expression was closely associated with a phenotype exhibiting low-level carbapenem resistance. Detailed alignment of the predicted amino acid sequence revealed that OmpK39 is a member of the OmpC subclass of enterobacterial porins, with the highest degree of homology to Klebsiella pneumoniae OmpK36. Based on a computerized alignment including Escherichia coli PhoE and OmpF, the 3D structures of which are known from X-ray studies, OmpK39 can be assumed to form the typical beta-barrel structure which is common to all enterobacterial porins. Since no inhibitory DNA sequences could be detected in ompk39 in the resistant strains, porin deficiency leading to carbapenem resistance seems to involve alterations in key regulatory genes and/or the promotor sequence rather than a direct mutation in the structural gene.

Amino Acid Sequence↗
PubMed2000

Outbreak investigation of nosocomial enterobacter cloacae bacteraemia in a neonatal intensive care unit.

Over a period of 7 months, 23 patients hospitalized in a neonatal intensive care unit (NICU) developed nosocomial Enterobacter cloacae bacteraemia. Contaminated saline for preparing heparin solution was initially identified as the common source of E. cloacae bacteraemia. Although environmental sanitation was enforced, the outbreak continued. E. cloacae has always been isolated from various cultures of the environmental specimens, from the hands of personnel and from the faeces of patients. All of the 23 bacteraemic isolates and 8 stool isolates from infected infants, as well as the 17 isolates from environmental specimens were found to be of the same genotype using the polymerase chain reaction-based DNA fingerprinting method. After various infection control methods were instituted, the outbreak eventually came under control. For epidemiological investigation, 23 neonates without E. cloacae bacteraemia were matched for case-control study. Nineteen (83%) of the case-patients were premature. The significant risk factors leading to E. cloacae bacteraemia in the NICU included small gestation age, low birthweight, exposure to personnel with contaminated hands and the presence of E. cloacae in the stool carriage (p=0.003, 0.007, 0.018 and 0.040, respectively). The gastrointestinal tracts of the patients and environmental surfaces appeared to be the principal sites of bacterial reservoir. In conclusion, the outbreak of E. cloacae bacteraemia was caused by a particular strain and possibly via multiple modes of transmission, including a bottle of contaminated saline as an initial common source, endogenous spread from the gastrointestinal tract and successive cross-infections between patients, hands of personnel and the environment. Effective infection control requires a multidisciplinary approach and reinforcement of infection control procedures, including aseptic technique, hand washing, proper isolation and disinfection of environmental surfaces.

Bacteremia↗
PubMed1998

Control of an outbreak of a highly beta-lactam-resistant Enterobacter cloacae strain in a neonatal special care unit.

Two successive outbreaks of colonization and infection with Enterobacter cloacae resistant to third generation cephalosporins (cephalosporin-resistant E. cloacae, CREC) and involving 15 infants occurred within 12 months in a neonatal special care unit. Isolates of clinical significance were obtained from four infants (urine 2 cases, blood, pleural drainage). According to epidemiological typing using computerized biochemical fingerprinting and pulsed-field gel electrophoresis (PFGE) the same CREC strain was found in both outbreaks. The origin of the strain and its reservoir between the two outbreaks remained unknown. Emphasizing strict barrier nursing of the infants had little or no impact on the presence and transmission of the strain in the unit. In contrast, replacing ampicillin plus cefotaxime as standard empiric therapy with penicillin G plus netilmicin plus consequent cohorting of newborns and staff promptly halted both the outbreaks. During a 5-y follow-up after the last episode, the choice of antibiotics for empirical treatment has varied, and no further outbreaks of CREC have been seen, with the exception of two sporadic cases.

Ampicillin↗
PubMed1991

Beta-lactamase induction antagonizes beta-lactam susceptibilities in Citrobacter diversus and Enterobacter cloacae clinical isolates.

Inducible beta-lactamases were obtained after exposure to several beta-lactams in clinical isolates of Enterobacter cloacae and Citrobacter diversus. Enzyme production was related to the inducer and medium composition. beta-lactamase is able to inactivate only labile compounds, thus generating minimum inhibitory concentrations higher than in the absence of the inducer; imipenem susceptibilities usually were not changed.

Citrobacter↗
PubMed1992

Plasmid profiles of Acinetobacter and Enterobacter species of hospital origin: restriction endonuclease analysis of plasmid DNA and transformation of Escherichia coli by R plasmids.

A total of 37 multi-resistant strains (20 Acinetobacter calcoaceticus and 17 Enterobacter cloacae) were isolated from patients of the Intensive Care Units. All the isolates were examined for resistance to a battery of antimicrobial agents by the disk diffusion method. Plasmid profiles and restriction endonuclease analysis of plasmid DNA by EcoR1 revealed the spread of one A. calcoaceticus and two E. cloacae endemic strains. Transformation experiments on Escherichia coli competent cells by three plasmids established the presence of R plasmids in the multi-resistant isolates.

Acinetobacter↗
PubMed2002

Chemical modification of specific active site amino acid residues of Enterobacter aerogenes glycerol dehydrogenase.

Enterobacter aerogenes glycerol dehydrogenase (G1DH EC 1.1.1.6), a tetrameric NAD+ specific enzyme catalysing the interconversion of glycerol and dihydroxyacetone, was inactivated on reaction with pyridoxal 5-phosphate (PLP) and o-phthalaldehyde (OPA). Fluorescence spectra of PLP-modified, sodium borohydride-reduced G1DH indicated the specific modification of epsilon-amino groups of lysine residues. The extent of inhibition was concentration and time dependent. NAD+ and NADH provided complete protection against enzyme inactivation by PLP, indicating the reactive lysine is at or near the coenzyme binding site. Modification of G1DH by the bifunctional reagent OPA, which reacts specifically with proximal epsilon-NH2 group of lysines and -SH group of cysteines to form thioisoindole derivatives, inactivated the enzyme. Molecular weight determinations of the modified enzyme indicated the formation of intramolecular thioisoindole formation. Glycerol partially protected the enzyme against OPA inactivation, whereas NAD+ was ineffective. These results show that the lysine involved in the OPA reaction is different from the PLP-reactive lysine, which is at or near the coenzyme binding site. DTNB titration showed the presence of only a single cysteine residue per monomer of G1DH. This could be participating with a proximal lysine residue to form a thioisoindole derivative observed as a result of OPA modification.

Amino Acids↗