Purification and characterization of a cytotoxin from Enterobacter cloacae

OBJECTIVE: To document our evolving surgical management of colonoscopic perforation and examine factors crucial to the improvement of patient care. DESIGN: We conducted a computer-based retrospective analysis of medical records (1980 through 1995). MATERIAL AND METHODS: Among 57,028 colonoscopic procedures performed, 43 patients (0.075%, or 1 perforation in 1,333 procedures) had a colonic perforation. Two additional patients were treated after colonoscopy performed elsewhere. The outcomes analyzed included surgical morbidity and mortality. RESULTS: Twenty-six women and 19 men who ranged in age from 28 to 85 years (median, 69) were treated for colonic perforation. More than 80% of perforations occurred during the latter half of the study period because of the increased volume of colonoscopic procedures (8 perforations among 12,581 examinations from 1980 through 1987 versus 35 perforations among 44,447 colonoscopies from 1988 through 1995). Emergency laparotomy was performed in 42 patients (93%). Perforations occurred throughout the colon: right side = 10; transverse = 9; and left side = 23. Three patients without evidence of peritoneal irritation fared well with nonoperative management. Most patients underwent primary repair or limited resection in conjunction with end-to-end anastomosis. In 14 patients (33%), an ostomy was created. One patient underwent laparotomy without further treatment. Intra-abdominal contamination ranged from none (31%) to local soiling (48%) to diffusely feculent (21%). Postoperative complications occurred in 12 patients and were associated with older age (P = 0.01), large perforations (P = 0.03), and prior hospitalization (P = 0.04). No postoperative deaths occurred. CONCLUSION: Despite a consistently low risk of colonic perforation, the increasing use of colonoscopy in our practice has resulted in an increased number of iatrogenic colonic perforations. In order to minimize morbidity and mortality, prompt operative intervention is the best strategy in most patients. Non-operative management is warranted in carefully selected patients without peritoneal irritation.     

Steady-state kinetic mechanism, stereospecificity, substrate and inhibitor specificity of Enterobacter cloacae nitroreductase

Enterobacter cloacae nitroreductase (NR) is a flavoprotein which catalyzes the pyridine nucleotide-dependent reduction of nitroaromatics. Initial velocity and inhibition studies have been performed which establish unambiguously a ping-pong kinetic mechanism. NADH oxidation proceeds stereospecifically with the transfer of the pro-R hydrogen to the enzyme and the amide moiety of the nicotinamide appears to be the principal mediator of the interaction between NR and NADH. 2,4-Dinitrotoluene is the most efficient oxidizing substrate examined, with a kcat/KM an order of magnitude higher than those of p-nitrobenzoate, FMN, FAD or riboflavin. Dicoumarol is a potent inhibitor competitive vs. NADH with a Ki of 62 nM. Several compounds containing a carboxyl group are also competitive inhibitors vs. NADH. Yonetani-Theorell analysis of dicoumarol and acetate inhibition indicates that their binding is mutually exclusive, which suggests that the two inhibitors bind to the same site on the enzyme. NAD+ does not exhibit product inhibition and in the absence of an electron acceptor, no isotope exchange between NADH and 32P-NAD+ could be detected. NR catalyzes the 4-electron reduction of nitrobenzene to hydroxylaminobenzene with no optically detectable net formation of the putative two-electron intermediate nitrosobenzene.     

Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae

Enterobacter cloacae 8009 produced an inducible class A beta-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other beta-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. The bla gene was transferable to Escherichia coli by electroporation of plasmid DNA. The molecular mass of the beta-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A beta-lactamases like FEC-1. The gene encoding this beta-lactamase was cloned and sequenced. The deduced amino acid sequence of the beta-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A beta-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the beta-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal beta-lactamases from Citrobacter diversus (80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intact ampR produced a small amount of beta-lactamase constitutively, suggesting that AmpR works as an activator of ampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A beta-lactamase in gram-negative bacteria.     

Investigation of cell envelope damage to Pseudomonas aeruginosa and Enterobacter cloacae by dibromopropamidine isethionate

Electron micrographs of log-phase Pseudomonas aeruginosa and Enterobacter cloacae cultured for 4 h in the presence of subinhibitory concentrations of dibromopropamidine isethionate indicate that this antibacterial agent can cause marked damage to the cell envelope structures of both species. This result provides an explanation of how dibromopropamidine can enhance the uptake and thus the activity of a second antibacterial agent used in combination with it.     

Development of resistance by Enterobacter cloacae during therapy of pulmonary infections in intensive care patients

The emergence of resistance during therapy and the efficacy of different antibiotic therapy regimens were studied in 38 intensive care patients suffering from pulmonary infections caused by Enterobacter cloacae. Every three days a fresh isolate was obtained from each patient and tested in vitro for susceptibility to 16 antibiotics by determination of the minimal inhibitory concentrations. During therapy with cefotaxime and tobramycin the E. cloacae strains from 47% of the patients became resistant to cefotaxime within 6 days. In all cases resistance encompassed all other broad-spectrum penicillins and cephalosporins tested, as well as aztreonam. Development of resistance regularly led to persistence of bacteria. Resistance to tobramycin, ciprofloxacin or imipenem was not observed. Treatment of 25 patients with persisting E. cloacae infections was successful in 17 out of 18 patients treated with imipenem and in 6 out of 7 patients receiving ciprofloxacin.     

Metal accumulation and vanadium-induced multidrug resistance by environmental isolates of Escherichia hermannii and Enterobacter cloacae

Contaminated soils from an oil refinery were screened for the presence of microorganisms capable of accumulating either nickel, vanadium, or both metals. Three strains of bacteria that belonged to the family Enterobacteriaceae were selected. Two of them were Escherichia hermannii strains, and outer membrane profile (OMP) analysis showed that they were similar to a strain of clinical origin; the other one was an Enterobacter cloacae strain that differed from clinical isolates. The selected bacteria accumulated both nickel and vanadium. Growth in the presence of vanadium induced multidrug resistance phenotypes in E. hermannii and E. cloacae. Incubation with this metal changed the OMP profile of E. hermannii but did not produce variations in the expression of the major OMPs of E. cloacae.     

Specificity of the high-mannose recognition site between Enterobacter cloacae pili adhesin and HT-29 cell membranes

Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.     

Fingerprinting with the polymerase chain reaction: confirmation of a Enterobacter cloacae epidemic in a neonatal intensive care unit

Between end December 1993 and end January 1994 a cluster of children infected/colonized with Enterobacter cloacae was seen in the neonatal intensive care unit of the University Hospital Nijmegen. The results of the epidemiological investigation are reported, which was aimed to differentiate between a random cluster of endogenously acquired Enterobacter strains and those possibly acquired exogenously via cross-infection. 5 isolates of the 7 patients were available for fingerprinting using interrepeat PCR. According to the fingerprint pattern, 4 of these isolates were identical, thereby suggesting cross-infection among the children. 3 neonates were colonized/infected with genotypically different isolates, suggesting that the infection/colonization developed endogenously. A control strain isolated from a patient at another ward showed the same genotype as the outbreak isolates. The transmission took probably place through one of the surgeons who, among all possible health care workers, were the only professional group treating patients in both units.     

Inactivation of the Enterobacter cloacae P99 beta-lactamase by a fluorescent phosphonate: direct detection of ligand binding at the second site

The synthesis of a fluorescent beta-lactamase inhibitor, p-nitrophenyl [(dansylamido)methyl]-phosphonate is described. The compound inactivated the class C beta-lactamase of Enterobacter cloacae P99 with stoichiometric release of p-nitrophenol, presumably, as with other phosphonate inhibitors, by phosphonylation of the active site serine. The inhibited enzyme exhibited typical dansyl fluorescence emission at 533 nm with excitation maxima at 345 and 283 nm; the latter excitation peak probably arises from radiationless energy transfer to the dansyl group from aromatic chromophores on the protein-inspection of the crystal structure shows that the closest are tyrosines. The fluorescence of the p-nitrophenyl phosphonate and the inhibited enzyme varied with pH in a very similar fashion, reflecting dissociation of the dimethylammonium ion in the ground state at low pH and of the sulfonamide in the excited state above pH 6. No perturbation of the fluorescence of the inhibited enzyme due to active site functional groups was observed. This may reflect the distance between the dansyl fluorophore and the phosphonyl group and/or the high pKa's of the protonated active site functional groups in the presence of the phosphonate. The addition of certain small molecular weight N-acyl amino acids, of preferred structure D-RCONHCHR'CO2-, to the inhibited enzyme led to an enhancement of dansyl fluorescence intensity and a blue shift in the emission maximum. This suggested that these molecules bind to the beta-lactamase at a site other than the active site and supports previous kinetic data to this effect [Dryjanski, M., & Pratt, R. F., (1995) Biochemistry 34, preceding paper in this issue].(ABSTRACT TRUNCATED AT 250 WORDS)     

Antimicrobial activities of beta-lactam antibiotics and gentamicin against penicillin-susceptible and penicillin-resistant pneumococci

The MICs of penicillin and cefotaxime for a range of penicillin-susceptible and penicillin-resistant isolates of Streptococcus pneumoniae were unchanged by the addition of gentamicin. In time-kill studies the rate of killing was greater for 18 of 20 isolates in the presence of gentamicin. However, mean differences in killing after 6 h of incubation were modest, not exceeding 1 log10 unit.     

Molecular epidemiology of an outbreak of Enterobacter cloacae in the neonatal intensive care unit of a provincial hospital in Gauteng, South Africa

An outbreak of Enterobacter cloacae in the neonatal intensive care unit of a provincial hospital in Gauteng, South Africa, resulting in nine deaths was investigated. Macrorestriction analysis using pulsed-field gel electrophoresis revealed that three isolates of E. cloacae from blood cultures of patients, six from environmental sources, and one from the hands of a staff member belonged to the same genotypic cluster.     

Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.     

Efficacies of cefepime, ceftazidime, and imipenem alone or in combination with amikacin in rats with experimental pneumonia due to ceftazidime-susceptible or -resistant Enterobacter cloacae strains

The antibacterial activities of human regimens of cefepime, ceftazidime, and imipenem alone or in combination with amikacin against an isogenic pair of Enterobacter cloacae strains (wild type and its corresponding derepressed cephalosporinase mutant) were compared by using our nonlethal model of pneumonia with 180 immunocompetent rats. Compared with untreated animals, all beta-lactam-treated rats, except those inoculated with the mutant isolate and receiving ceftazidime, had significantly lower bacterial counts in their lungs 60 h after the onset of therapy. Although the combination of a beta-lactam and amikacin was more bactericidal than each corresponding antimicrobial agent alone, true synergy was noted only with cefepime and imipenem against the constitutive derepressed strain.     

The efficacy of ciprofloxacin and doxycycline against experimental tularaemia

A group of patients with balance complaints (n = 16) was compared with a group of normal subjects (n = 17) by means of posturography, subjective assessments of balance, anxiety and unsteadiness when standing on a force platform with eyes closed. Postural instability was induced by vibratory stimulation of the calf muscles (20, 40, 60, 80 and 100 Hz). As a control condition, the arm (biceps) was stimulated at similar frequencies. In order to control for arousal, blood pressure and heart beat were assessed. Furthermore, questionnaire responses on psychological measures were collected. Results showed clear differences between the groups in terms of imbalance and self-reports. However, the 2 groups displayed similar increases of imbalance during calf stimulation and no increase during arm stimulation. Patients generally rated less increase of unsteadiness when the calf was stimulated than did the controls. No differences in arousal were found between the groups or within conditions. Results are discussed in terms of the proposed desynchrony between symptoms and complaints.     

Initial high anti-emetic efficacy of granisetron with dexamethasone is not maintained over repeated cycles

We have reported previously that the anti-emetic efficacy of single agent 5HT3 antagonists is not maintained when analysed with the measurement of cumulative probabilities. Presently, the most effective anti-emetic regimen is a combination of a 5HT3 antagonist plus dexamethasone. We, therefore, assessed the sustainment of efficacy of such a combination in 125 patients, scheduled to receive cisplatin > or = 70 mg m(-2) either alone or in combination with other cytotoxic drugs. Anti-emetic therapy was initiated with 10 mg of dexamethasone and 3 mg of granisetron intravenously, before cisplatin. On days 1-6, patients received 8 mg of dexamethasone and 1 mg of granisetron twice daily by oral administration. Protection was assessed during all cycles and calculated based on cumulative probability analyses using the method of Kaplan-Meier and a model for transitional probabilities. Irrespective of the type of analysis used, the anti-emetic efficacy of granisetron/dexamethasone decreased over cycles. The initial complete acute emesis protection rate of 66% decreased to 30% according to the method of Kaplan-Meier and to 39% using the model for transitional probabilities. For delayed emesis, the initial complete protection rate of 52% decreased to 21% (Kaplan-Meier) and to 43% (transitional probabilities). In addition, we observed that protection failure in the delayed emesis period adversely influenced the acute emesis protection in the next cycle. We conclude that the anti-emetic efficacy of a 5HT3 antagonist plus dexamethasone is not maintained over multiple cycles of highly emetogenic chemotherapy, and that the acute emesis protection is adversely influenced by protection failure in the delayed emesis phase.     

Testosterone impairs efficacy of protective vaccination against P. chabaudi malaria

Vaccination with surface membranes isolated from Plasmodium chabaudi-infected erythrocytes can protect B10.A mice from the lethal outcome of P. chabaudi malaria. However, the efficacy depends on gender and testosterone levels. Thus, vaccination protects over 90% of female mice, but only about 55% of male mice and only about 34% of female mice when pretreated with testosterone for 4 weeks. The suppressive testosterone effect remains imprinted in females even at 10 weeks after the testosterone treatment. These data indicate that not only genetic but also environmental factors restrict the host's immune response to a malaria vaccine.     

Investigation into the efficacy of disinfectants against MRSA and vancomycin-resistant enterococci

In the study reported here, several disinfectants for surfaces, instruments, hands and mucous membrane were tested for their effectiveness against methicillin-resistant strains of S. aureus and vancomycin-resistant enterococci. The results show that after use of the recommended concentrations with the recommended contact times, which were determined on the basis of the criteria for disinfectants according to the DGHM guideline for the testing and evaluation of chemical disinfection procedures, an adequate effect was able to be proved. Increased resistance of the methicillin-resistant S. aureus and vancomycin-resistant E. faecium strains to the tested disinfectants was not found. Only after use of diluted solutions differences in the bacterial count reductions were observed. In further studies it is to be investigated whether these differences are due to resistance to antibiotics or to intrinsic resistance.     

In vitro experiments on the working of combinations of gentamicin and beta-lactam antibiotics against Pseudonomas aeruginosa (author's transl)

The M.B.C.'s of gentamicin and carbenicillin against Pseudonomas aeruginosa NCTC 10490 were measured under controlled conditions using a Biophotometer. The M.B.C. of gentamicin was 15 mug/ml but even in a concentration of 1,000 mug/ml carbenicillin was not bactericidal. In further experiments, subinhibitory concentrations of gentamicin (1 mug/ml) together with varying concentrations of carbenicillin were added to a log phase culture of the organism. Under these conditions the M.B.C. of carbenicillin was now 6 mug/ml. In tube dilution test the M.B.C. of carbenicillin alone was 15.6 mug/ml and for gentamicin 3.9 mug/ml. The M.B.C.'s of other beta-lactam antibiotics (ampicillin, penicillin G and cephalothin) were four to five times as great as for carbenicillin whereas that for ticarcillin was identical. Parallel to the "multiplication inhibition" test in the Biophotometer we investigated 51 strains of Pseudomonas aeruginosa freshly isolated from clinical material. Their M.B.C.'s were determined in tube dilution tests against doubling dilutions of beta-lactam antibiotics, with and without the addition of 1 mug/ml gentamicin. With this concentration of gentamicin, the M.B.C.'s of carbenicillin and ticarcillin were considerably lower than for these substances alone. In comparison to carbenicillin, ticarcillin was more effective against Pseudomonas aeruginosa. Our findings indicate that for Pseudomonas aeruginosa infections the combination of gentamicin with other beta-lactam antibiotics (ampicillin, penicillin G and cephalothin) is to be avoided. But the combination of gentamicin with either carbenicillin or ticarcillin appeared to be effective.     

Rapid monitoring method to assess efficacy of sanitizers against Pseudomonas putida biofilms

Biofilms of luminescent Pseudomonas putida were developed on rubber surfaces by incubation in brain heart infusion (BHI) broth. Scanning electron microscopy (SEM) and epifluorescence microscopy (EFM) were used to examine biofilm formation. To test the efficacy of two sanitizers commonly employed in dairy plants for CIP (cleaning in place) procedures, a novel bioluminescence method and aerobic plating were used to enumerate cells. Immediately after the sanitizer treatments an apparent 5-log reduction of biofilm-associated cells was determined. However, when the samples were resuscitated for 18 h in BHI broth, high numbers of cells were detected which reached levels close to those of nontreated controls. The results demonstrated that neither sanitizer could completely eliminate biofilm-associated P. putida. The microbial bioluminescence method proved to be the best way for assessing effectiveness of sanitizers against microbial biofilms.