Modulation of P-fimbriation by ciprofloxacin in uropathogenic Escherichia coli

BACKGROUND: The aim of this study was to determine the effect of ciprofloxacin at subinhibitory concentrations on the expression of P fimbriae of uropathogenic Escherichia coli. Thirty-nine strains of Escherichia coli isolated from out patients with urinary tract infection were studied. Thirty-nine of these strains had been previously characterized as P-fimbriated and the remaining non fimbriated strain was used as a negative control. METHODS: Fimbriation was quantitatively studied by electron microscope observation of the strains before and after treatment. To determine possible qualitative variations in the fimbrial proteins and in the external membrane (OMPs), extraction and electrophoretic separation was performed in polyacrylamide gels. RESULTS: No qualitative differences were observed in the OMPs profile and fimbrial proteins induced by ciprofloxacin in any of the strains studied. However, electron microscopic observation generally showed a decrease in the percentage fimbriated bacterial cells by the antimicrobial effect. CONCLUSIONS: The mechanism of action of ciprofloxacin at subinhibitory doses may correspond to a process of fimbrial protein synthesis inhibition secondary to the initiation of general repair mechanism of the cell exposed to the antimicrobial and not to a process of specific mutations which qualitatively affect fimbrial protein composition.     

Regulation of Staphylococcus aureus capsular polysaccharide type 5: CO2 inhibition in vitro and in vivo

Staphylococcus aureus capsular polysaccharide type 5 (CP5) expression was investigated in lung tissue and nasal polyps of two cystic fibrosis (CF) patients, in rats, and in vitro using ELISA and IFA. In CF tissues, S. aureus expressed protein A and teichoic acid but only 1%-5% of cells expressed CP5. When rats were challenged with CP5-positive S. aureus in the granuloma pouch model, only 1%-5% of CP5-positive cells were detectable in pouch exudates. CF and pouch isolates, however, reexpressed CP5 (70%-90% of cells) when grown in vitro with air. Addition of > or = 1% CO2 to air or to O2/N2 gas mixtures reduced CP5 expression significantly (P < .001) in a dose-dependent manner (6%-1% CP5-positive cells). The results show that S. aureus does not produce CP5 in CF airways and in rat granuloma pouches and that CO2 is an environmental signal that regulates CP5 expression.     

The lipase from Staphylococcus aureus. Expression in Escherichia coli, large-scale purification and comparison of substrate specificity to Staphylococcus hyicus lipase

The genes coding for the mature part of the lipases from Staphylococcus aureus NCTC8530 and Staphylococcus hyicus have been cloned and overexpressed in Escherichia coli as fusion proteins with an N-terminal hexa-histidine tag. The enzymes accumulated in the cytoplasm and were purified using sequential precipitation with protamine sulphate and ammonium sulphate, followed by metal-affinity and hydroxyapatite chromatography. The yield of pure lipase was 4.5 mg/g wet cells for S. aureus lipase and 13 mg/g for S. hyicus lipase. The purified enzymes need calcium for activity, albeit with different affinities, and a low residual activity was found in the absence of calcium. In contrast to S. hyicus lipase, not only strontium but also barium can replace calcium with full retention of activity of S. aureus lipase. Whereas S. hyicus lipase is optimally active at pH 8.5, the optimum pH for enzymatic activity for S. aureus lipase was found to be pH 6.5. The S. aureus lipase has a narrow substrate specificity: short-chain triacylglycerols and acyl esters of both p-nitrophenol and umbelliferone are readily degraded, whereas medium- and long-chain lipids, as well as phospholipids, are poor substrates. In contrast, S. hyicus lipase prefers phospholipids as substrate and hydrolyses neutral lipids irrespective of their chain length. The results are discussed in view of the large sequence similarity between both lipases.     

In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC

DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the biochemical role of E. coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state. In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1. However, DsbA is a more effective oxidant than DsbC of protein dithiols. In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media. On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced. DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results. A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds. Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively.     

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants

Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.     

Correlation of in vitro resistance of Staphylococcus aureus to tetracycline, doxycycline, and minocycline with in vivo use

A study was carried out to investigate the errors involved in obtaining volumes by means of ultrasound echograms and a small computer. The work was done on live pigs' kidneys (130-200 cm3). The errors include those associated with the scanner itself, those invloved in the interpretation of the contour boundaries, and those relating to the algorithms used to determine the volumes from the contours. An average deviation of 25.4 cm3 from actual volumes was found, while 58% of the measurements were within +/-10%. Computer programs allowing for the display of contours and intercepts of corresponding orthogonal scan planes were used to reanalyze the data resulting in an average deviation of 14.7 cm3 with 78% of the data resulting inan average deviation of 14.7 cm3 with 78% of the measurements with "/-10%.     

Characterization of grlA, grlB, gyrA, and gyrB mutations in 116 unrelated isolates of Staphylococcus aureus and effects of mutations on ciprofloxacin MIC

One hundred sixteen unrelated clinical isolates of Staphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the presence of mutations in the grlA, grlB, gyrA, and gyrB gene loci. Two mutations within grlA (located at codons 80 and 84) and two mutations within gyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.     

Substrate specificity of Staphylococcus hyicus lipase and Staphylococcus aureus lipase as studied by in vivo chimeragenesis

Staphylococcus hyicus lipase (SHL) and Staphylococcus aureus lipase (SAL) are highly homologous enzymes, yet they show remarkable differences in their biochemical characteristics. SHL displays a high phospholipase activity, hydrolyses neutral lipids, and has no chain length preference, whereas SAL only degrades short-chain fatty acid esters. To identify the regions in the primary sequence of SHL responsible for phospholipase activity and chain length selectivity, a set of histidine-tagged SAL/SHL chimeras was generated by in vivo recombination in Escherichia coli. Several classes of chimeric enzymes were identified on the basis of restriction site analysis. All chimeras were well-expressed as active enzymes. They were characterized for their specific activities on both phospholipids and p-nitrophenyl esters of various chain lengths. Phospholipase activity appeared to be determined by three regions, all located in the C-terminal domain of SHL. Testing of the enzymatic activity of the chimeras toward p-nitrophenyl esters showed that chain length selectivity is defined by elements within the region of residues 180-253. Moreover, also residues along the stretch 275-358 contribute to the binding of acyl chains. Interestingly, several chimeras were even more active than the parent enzymes on long-chain p-nitrophenyl esters.     

The effects of environmental conditions on the in vitro activity of selected antimicrobial agents against Escherichia coli

Serotonin (5-hydroxytryptamine, 5-HT) may play an important role in the pathogenesis of schizophrenia. Previous studies suggested that the efficacy of atypical neuroleptic drugs (e.g., risperidone and clozapine) on negative symptoms may be related to the 5-HT2a receptor. Although association studies between MspI polymorphism (T102C) and the 5-HT2a receptor gene and schizophrenia have been reported, their results are still controversial. The aim of this study was to examine the association between T102C polymorphism of the 5-HT2a receptor gene and schizophrenia as well as the association between the polymorphism and negative symptoms in a Japanese population (106 patients with schizophrenia and 109 healthy controls). No significant positive associations were observed. Our results suggest that the 5-HT2a receptor gene is not involved in the pathogenesis of schizophrenia or negative symptoms.     

Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNA(Ser) in vivo and in vitro

After ovulation in salmonids, the eggs are held in the peritoneal cavity and bathed in coelomic fluid. Using a chromogenic peptide substrate, the anti-protease activity of brook trout coelomic fluid was measured. Trypsin, chymotrypsin, and pancreatic elastase activities were significantly inhibited by coelomic fluid containing 5.0, 10.0, and 25.0 microgram of total protein, respectively. Using subtractive cDNA cloning, we have previously characterized a set of ovarian proteins called TOPs (trout ovulatory proteins) that are secreted into the coelomic fluid after ovulation. TOPs are most homologous to mammalian antileukoprotease, a heat- and acid-stable serine protease inhibitor. On the basis of this homology, we hypothesized that the anti-trypsin activity observed in the coelomic fluid was related to the presence of TOPs. In the present study, this hypothesis was supported by the acid- and heat-stability of the anti-trypsin activity present in coelomic fluid. Coelomic fluid could be heated to 50 degrees C or treated at a pH less than 5.2 without a significant decrease in the inhibitory activity. Further, coelomic fluid from which TOPs were immunoprecipitated had significantly less anti-trypsin activity than nonimmunoprecipitated controls. We propose that TOP proteins are uniquely produced by the ovary and secreted into the coelomic fluid to act as protease inhibitors following ovulation.     

In vitro studies of pharmacodynamic properties of vancomycin against Staphylococcus aureus and Staphylococcus epidermidis

The bactericidal activities of vancomycin against two reference strains and two clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis were studied with five different concentrations ranging from 2x to 64x the MIC. The decrease in the numbers of CFU at 24 h was at least 3 log10 CFU/ml for all strains. No concentration-dependent killing was observed. The postantibiotic effect (PAE) was determined by obtaining viable counts for two of the reference strains, and the viable counts varied markedly: 1.2 h for S. aureus and 6.0 h for S. epidermidis. The determinations of the PAE, the postantibiotic sub-MIC effect (PA SME), and the sub-MIC effect (SME) for all strains were done with BioScreen C, a computerized incubator for bacteria. The PA SMEs were longer than the SMEs for all strains tested. A newly developed in vitro kinetic model was used to expose the bacteria to continuously decreasing concentrations of vancomycin. A filter prevented the loss of bacteria during the experiments. One reference strain each of S. aureus and S. epidermidis and two clinical isolates of S. aureus were exposed to an initial concentration of 10x the MIC of vancomycin with two different half-lives (t1/2s): 1 or 5 h. The post-MIC effect (PME) was calculated as the difference in time for the bacteria to grow 1 log10 CFU/ml from the numbers of CFU obtained at the time when the MIC was reached and the corresponding time for an unexposed control culture. The difference in PME between the strains was not as pronounced as that for the PAE. Furthermore, the PME was shorter when a t1/2 of 5 h (approximate terminal t1/2 in humans) was used. The PMEs at t1/2s of 1 and 5 h were 6.5 and 3.6 h, respectively, for S. aureus. The corresponding figures for S. epidermidis were 10.3 and less than 6 h. The shorter PMEs achieved with a t1/2 of 5 h and the lack of concentration-dependent killing indicate that the time above the MIC is the parameter most important for the efficacy of vancomycin.     

Staphylococcus aureus cap5O and cap5P genes functionally complement mutations affecting enterobacterial common-antigen biosynthesis in Escherichia coli

BACKGROUND: Excision of large right atrial masses requires bicaval cannulation and cardiopulmonary bypass. Safe venous cannulation can be accomplished only by knowing the exact intracavitary location and extension of the mass to avoid fragmentation. Transthoracic echocardiography and intraoperative transesophageal echocardiography, although helpful, cannot always define the exact intracavitary relationships of the tumor. METHODS: We have used both intraoperative transesophageal and epicardial echocardiography to guide venous cannulation in 4 patients with large right atrial masses. Both echo images are used by the surgeon to select the exact site and method of cannulation to avoid fragmentation of the mass. Epicardial echocardiography complemented the images obtained by transesophageal echocardiography. RESULTS: The technique of combined transesophageal and epicardial echocardiography allowed safe venous cannulation in all 4 patients. Each of the right atrial masses was safely excised using case-specific cannulation techniques guided by the echocardiographic images. CONCLUSIONS: We propose the routine use of both intraoperative transesophageal and epicardial echocardiography in guiding venous cannulation for safe excision of large right atrial masses.     

In vitro effects of a high-molecular-weight heat-labile enterotoxin from enteroaggregative Escherichia coli

The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.     

In vivo and in vitro studies on interactions between the components of the hemolysin (HlyA) secretion machinery of Escherichia coli

The glycopeptide antibiotic vancomycin blocks cell wall synthesis in Escherichia coli only when it can reach its target site in the periplasm. In vivo, sensitivity to vancomycin is enhanced in the presence of the hemolysin (hly) determinant of E. coli or its translocator portion hlyBD. Two different mutations in hlyD alter the cell's susceptibility to vancomycin: mutations in the tolC-homologous region of hlyD increase vancomycin resistance, whereas mutations at the 3'-terminus of hlyD lead to hypersensitivity to vancomycin and to the accumulation of large periplasmic and cytoplasmic pools of this antibiotic in E. coli. These effects are only observed in the presence of functional HlyB and TolC, the two other components of the hemolysin secretion machinery. A defect in TolC causes hyperresistance to vancomycin, even when present together with a mutant HlyD protein which in the presence of TolC renders E. coli hypersensitive to vancomycin. Lipid bilayer experiments in vitro revealed specific interactions between TolC and vancomycin or HlyD protein. Second-site suppressor mutations in hlyD and hlyB were obtained, which abolish the hypersensitive phenotype caused by the 3'-terminal mutations in hlyD. Our results are compatible with the idea that (a) TolC, together with the TolC-homologous part of HlyD, forms a pore in the outer membrane through which hemolysin is released and vancomycin taken up; and (b) the C-terminal sequence of HlyD interacts with periplasmic loop(s) of HlyB to form a closed channel spanning the periplasm.     

Influence of subinhibitory concentrations of ceftazidime, ciprofloxacin and azithromycin on the morphology and adherence of P-fimbriated escherichia coli

The influence of subinhibitory concentrations (1/2, 1/4, 1/8, 1/16 and 1/32 x MIC) of ceftazidime, ciprofloxacin and azithromycin on the morphology and adherence of 29 wild-type P-fimbriated strains of Escherichia coli was studied. Bacterial adherence to the Buffalo green monkey (BGM) cell line was tested before and after treatment with antibiotics and detected by means of an immunofluorescence staining. Significant dose dependent reduction of bacterial adherence was observed, which correlated with the alterations in bacterial cell morphology. After exposure of strains to sub-MICs of antibiotics, normal shapes, spherical forms and filaments were noted. The greatest filamentation and the greatest loss of adherence ability occurred at 1/2 x MIC of ceftazidime. Treatment with sub-MICs of ciprofloxacin resulted in shorter filaments, while filamentation did not occur after bacterial exposure to sub-MICs of azithromycin. Azithromycin was least damaging to the adherence ability of E. coli and at a concentration of 1/2 x MIC caused globoid cell formation.     

Pharmacokinetic and pharmacodynamic models of the antistaphylococcal effects of meropenem and cloxacillin in vitro and in experimental infection

The efficacies of meropenem (MPM) and cloxacillin (CLC) against two Staphylococcus aureus strains were established in vitro. A pharmacodynamic model equation, based on the concept that the killing rate depends on concentration and time, was fitted to the numbers of CFU. The parameters of the equation are maximum killing rate, time point of maximum killing, and 50% effective concentration (EC50). The EC50s for the two strains were 0.047 and 0.040 mg/liter, respectively, for MPM and 0.105 and 0.121 mg/liter, respectively, for CLC. Calculated values of the parameters were used to predict the numbers of CFU at exponentially decreasing concentrations in vitro as well as in an experimental infection model. The prediction for in vitro conditions gave a satisfactory fit (R2, between 0.862 and 0.894). In vivo the numbers were predicted with the assumption that killing rate in vivo is proportional to that in vitro (R2, between 0.731 and 0.973). The proportionality factor ranged between 0.23 and 0.42; this variation was due mainly to covariation with growth rates in control animals, without other significant differences between antibiotics or strains.     

Hydrogen peroxide effects in Escherichia coli cells

We analyzed DNA lesions produced by H2O2 under low iron conditions, the cross adaptive response and the synergistic lethal effect produced by iron chelator-o-phenanthroline, using different Escherichia coli mutants deficient in DNA repair mechanisms. At normal iron levels the lesions produced by H2O2 are repaired mainly by the exonuclease III protein. Under low iron conditions we observed that the Fpg and UvrA proteins as well as SOS and OxyR systems participate in the repair of these lesions. The lethal effect of H2O2 is strengthened by o-phenanthroline if both compounds are added simultaneously to the culture medium. This phenomenon was observed in the wild type cells and in the xthA mutant (hypersensitive to H2O2). E. coli cells treated with low concentrations of H2O2 (micromolar) acquire resistance to different DNA damaging agents. Our results indicate also that pretreatment with high (millimolar) H2O2 concentrations protects cells against killing, by UV and this phenomenon is independent of the SOS system, but dependent on RecA and UvrA proteins. H2O2 induces protection against lethal and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). H2O2 also protects the cells against killing by cumene hydroperoxide, possibly with the participation of Ahp protein.     

In vitro activities of the oxazolidinone antibiotics U-100592 and U-100766 against Staphylococcus aureus and coagulase-negative Staphylococcus species

U-100592 and U-100766 are closely related antibiotics of the oxazolidinone class. Their in vitro activities were determined against 100 isolates of Staphylococcus aureus and 100 isolates of coagulase-negative Staphylococcus species by broth and agar dilution test methods. The MICs of both compounds by either test method at which 50 and 90% of isolates are inhibited were 2 and 4 micrograms/ml, respectively, for S. aureus and 1 to 2 micrograms/ml for coagulase-negative staphylococci. Time-kill assay with selected strains indicated a primarily bacteriostatic effect against staphylococci.