Escherichia coli and Lactobacillus plantarum responses to osmotic stress

Artificial insemination using cryopreserved semen is a common management tool of the contemporary livestock producer. However, cryopreservation is detrimental to sperm function and fertility, killing some 50% of the spermatozoa during the process. Prediction of cryopreservation damage from prefreeze samples remains elusive. Computer-automated sperm head morphometry was used in this study to determine the effects of cryopreservation on bovine sperm head morphometry. Semen was collected from 18 bulls and was divided. One portion was extended to 200 x 10(6) sperm/ml and a microscope slide was prepared, while the remaining portion was cryopreserved in a Triscitrate-yolk extender. After thawing, the cryopreserved samples were prepared on microscope slides. All slides were air dried and were stained with hematoxylin and rose bengal. The morphometric dimensions for length, width, width/length, area, and perimeter for a minimum of 200 sperm heads were analyzed from each slide by computer-aided sperm head morphometry analysis, and the mean measurements were recorded. Bull sperm heads were significantly (P < 0.01) smaller in cryopreserved spermatozoa than in the companion extended samples for length (8.56+/-0.07 vs. 8.63+/-0.08 microm), width (4.39+/-0.05 vs. 4.48+/-0.05 microm), area (28.42+/-0.07 vs. 29.14+/-0.08 microm), and perimeter (23.33+/-0.21 vs. 23.70+/-0.23 microm) for all bulls. Width/length was also different (0.513 vs. 0.519). In addition, differences (P < 0.05) were found within 14 of 18 bulls for at least four of the morphometric parameters. The percent change in measures after cryopreservation were correlated (P < or = 0.05) to the variability of the extended sample. Variations in sperm head measurements were lower (P < or = 0.05) in extended samples of the four bulls in which no changes occurred than in extended samples of the remaining 14 bulls. These data suggest that the variability in sperm head measurements of individual bulls, or ejaculates, may be an indicator of sperm cryosurvivability.     

Protein expression in response to folate stress in Escherichia coli

Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli. E. coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins. The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.     

Variation in toe-web response of turkey poults to phytohemagglutinin-P and their resistance to Escherichia coli challenge

PURPOSE: To evaluate the efficacy and safety of loteprednol etabonate 0.5% as prophylactic treatment for the ocular signs and symptoms of seasonal allergic conjunctivitis. METHODS: In this randomized, double-masked, placebo-controlled, parallel study, 293 adults with history of seasonal allergic conjunctivitis were treated with either loteprednol etabonate or vehicle (placebo) four times daily, beginning before the onset of the allergy season and continuing for 6 weeks. The primary efficacy measure was a primary composite score (sum of itching and bulbar conjunctival injection scores). Supportive efficacy measures were the investigator global assessment and a secondary composite score (sum of tearing, erythema, chemosis, and discomfort scores), all calculated during the 21-day peak pollen season. RESULTS: The proportion of patients who never developed moderate or severe signs and symptoms of allergy during the peak pollen season in the loteprednol etabonate treatment group was greater than that in the placebo group. For the primary composite score, this efficacy criterion was reached by 94% of patients (136/145) in the loteprednol etabonate group and 78% of patients (111/143) in the placebo group (P = .001). The magnitude of effect was similar for the investigator global assessment (86% [118/138] vs 64% [87/137]; P < .001) and, although not statistically significant, the secondary composite score (77% [112/145] vs 68% [97/143]; P = .092). None of the loteprednol etabonate-treated patients had an intraocular pressure increase of 10 mm Hg or more, whereas two placebo patients did. CONCLUSIONS: Loteprednol etabonate is generally effective in prophylaxis of seasonal allergic conjunctivitis and has an acceptable safety profile.     

Atypical Escherichia coli strains and their association with poult enteritis and mortality syndrome

To date, no definitive etiology has been described for Poult Enteritis and Mortality Syndrome (PEMS). However, two atypical Escherichia coli colony types are isolated consistently from moribund and dead poults afflicted with PEMS. To test the infectivity of these E. coli strains, poults were placed into floor pens in three isolation treatment rooms: 1) Control: no bacterial challenge, 2) E. coli colony Types 1 or 2 posthatch oral challenge: 10(8) cfu/per poult at 1 d, and 3) E. coli colony Types 1 or 2 posthatch oral challenge: 10(8) cfu/per poult at 6 d. Daily intramuscular injections of cyclophosphamide (100 micrograms per poult) from 1 to 5 d posthatch were given to half of the poults in each treatment. Atypical E. coli challenge caused BW depression, and cyclophosphamide treatment exacerbated the response. All E. coli-challenged poults developed diarrhea similar to PEMS. Mortality was increased by both atypical E. coli colony types, but at 21 d E. coli colony Type 2 caused greater mortality than colony Type 1. With cyclophosphamide treatment, mortality was exacerbated with both colony types, but colony Type 2 at 1 d caused the greatest mortality. Ultrastructural damage to ileum epithelium cell microvilli and subcellular organelles indicated that part of the BW depression could be attributed to malabsorption of nutrients. It was concluded that the atypical E. coli colony Types 1 and 2 play a significant role in the PEMS disease.     

Mucosal immunogenicity of genetically detoxified derivatives of heat labile toxin from Escherichia coli

Using a fixed dose of antigen, the immune response to detoxified mutants of LT-WT following intranasal (i.n.), subcutaneous (s.c.) and oral (i.g.) immunisation has been studied. When given i.n., both LT-WT and mutant toxin, K63, generated significant levels of toxin-specific IgG in the serum, and the levels of IgA in nasal and lung lavages were greater than those induced by rLT-B. In comparison, i.g. immunisation of mice with a similar quantity of either LT-WT or K63 toxin induced barely detectable levels of IgG in the sera. However, if the amount of protein used for i.g. immunisation was increased tenfold, relatively good levels of toxin-specific IgG were induced in the sera by both LT-WT or K63. Low levels of toxin-specific IgA were also observed in intestinal washes from these mice. Western blotting of the sera, using the native toxin as an antigen, demonstrated the presence of both anti-A and anti-B subunit antibodies. Most significantly, toxin-neutralising antibodies were induced in the serum, with the strongest activity being induced by the LT-WT, an intermediate activity induced by mutant K63 and a lower response by rLT-B. Together, these data show that ADP-ribosyltransferase is not necessary for mucosal immunogenicity of these proteins, and that the i.n. route of immunisation is more effective than the i.g. route of immunisation for the generation of both systemic (IgG) and mucosal (IgA) immune responses.     

Expression of the Streptomyces griseus alpha-amylase gene in Escherichia coli

The amy gene of Streptomyces griseus was not expressed in Escherichia coli cells due to the lack of recognition of the amy promoter by the E. coli RNA polymerase, as confirmed by using promoter-probe vectors. The expression of the amy gene in E. coli was detected only when the promoter-less gene was placed under the control of the lacZ promoter and was dependent on the level of IPTG added to the medium. The extracellular alpha-amylase detected in the culture broth seems to be released by cellular lysis. When the amy gene lacking both leader peptide and promoter was transcribed from the lacZ promoter, no alpha-amylase activity was detected but larger E. coli cells and inclusion bodies were observed.     

The dependence of the osmotic properties of Escherichia coli bacteria on temperature

The influence of water solutions of NaCl on the osmoresistance and reproduction of E. coli B/r and E. coli Bs-1 at different temperatures was studied. The increase in cell resistance was studied. The increase in cell resistance to NaCl hypertonia was noticed after the temperature rise from 20 to 37 degrees C. In this case the concentration of NaCl in the medium, which made this medium isotonic, was seen to increase too. On the contrary, when the temperature decreased to 0 degrees, the cell resistance to high concentrations of NaCl was found suppressed. In this case the concentration of NaCl in the medium, which made this medium isotonic, was seen to decrease. The salt tolerance of bacteria reproduction was found to depend on the temperature: the tolerance increased with the temperature rise, and vice versa. It is concluded that the solution temperature was to be considered as one of the major factors governing the osmotic bacterial cell homeostasis.     

Induction of heat shock gene expression in colonic epithelial cells after incubation with Escherichia coli or endotoxin

OBJECTIVE: The universal cellular response to stress is the expression of a family of genes known as heat shock or stress proteins. We investigated whether bacteria or bacterial products (endotoxin) can induce heat shock protein expression in human enterocytes. DESIGN: Controlled, in vitro study. SETTING: Cell culture laboratory. SUBJECTS: Human Caco-2 enterocyte cell line. MEASUREMENTS AND MAIN RESULTS: Incubation of confluent monolayers of Caco-2 cells with Escherichia coli C25 (1 x 10(9) bacteria/mL) for 1 hr at 37 degrees C was found to induce the expression of the 72-kilodalton molecular weight heat shock protein gene (heat shock protein-72), the major inducible form of the 70-kilodalton molecular weight heat shock protein family of stress proteins, as detected by Western blot analysis. The level of heat shock protein-72 induction after incubation with E. coli was similar to the response of Caco-2 cells to heat shock at 43 degrees C for 1 hr. The induction of heat shock protein-72 gene expression by E. coli was not purely due to the process of phagocytosis, since incubation of Caco-2 cells with latex beads (1 micron) failed to induce heat shock gene expression. To elucidate the possible mechanism of heat shock protein-72 induction mediated by bacteria, Caco-2 cells were incubated with E. coli endotoxin (200 micrograms/mL) for 1 hr at 37 degrees C. Such treatment was also found to induce the synthesis of heat shock protein-72. CONCLUSIONS: These results demonstrate that bacteria and/or bacterial products induce the heat shock gene expression in Caco-2 cells. Since intestinal epithelial cells are constantly in contact with bacteria and bacterial products, we speculate that the heat shock gene expression may be part of the natural mechanism of protection for these cells in the potentially harmful environment that may be present in the intestinal tract.     

Serum response of Escherichia coli strains causing dyspepsia and urinary tract infection: relation to alpha-hemolysin production and O type

A total of 109 alpha-hemolytic and 104 nonhemolytic Escherichia coli isolates from children with dyspepsia and urinary tract infections were investigated for resistance to the bactericidal activity of human serum. A significantly higher proportion of serum resistance was found in alpha-hemolytic E. coli isolates than in nonhemolytic isolates (P < 0.01). An association between the titer of alpha-hemolysin produced and serum resistance was found.     

Recombinant polyketide synthesis in Streptomyces: engineering of improved host strains

OBJECTIVE: Among the high risk groups for complications from influenza and pneumococcal disease, individuals aged 65 and older hospitalized within the previous year represent the group at highest risk. Studies have demonstrated that targeting hospitalized patients aged 65 and older for immunization before hospital discharge can be successful. This study addressed the efficacy of such a program within a managed care organization to immunize this highest risk group. DESIGN: A cross-sectional study. SETTING: Oxford Health Plans, a major managed care organization in New York serving a large Medicare population. PARTICIPANTS: A total of 106 Primary Care Physicians caring for 153 patients aged 65 and older, who were hospitalized in one of 10 high volume hospitals during October and November of 1996. Nine of these facilities were located in New York and one was in New Jersey. INTERVENTION: Patients aged 65 and older admitted to any of the 10 hospitals were identified daily. A fax was sent to each patient's primary care physician explaining the program and requesting that he/she administer influenza and/or pneumococcal vaccine to his/her patient before hospital discharge. Literature references citing past successful programs were included in the fax. MEASUREMENTS: Measurements included medical record documentation of influenza and pneumococcal immunization, both ordered and given, for the individual member before discharge; patient age; sex; and primary and secondary diagnoses. Physicians were sent follow-up questionnaires to determine reasons for not vaccinating. RESULTS: A total of 206 patients were admitted during the eligible time period. One hundred fifty-three hospitalized patients (average age = 74 years) participated. The median length of stay among this study population was 5 days (range, 1-63 days). The distribution of the median length of stay for the 25th and 75th percentiles was 3 and 9 days. The rate for influenza and pneumococcal immunization, both ordered and given, before hospital discharge was 1.96% for the influenza vaccine (n = 3) and .65% for the pneumococcal vaccine (n = 1), respectively. Results of a follow-up survey mailed to all physicians (n = 106) with eligible members in the study indicated that the most frequent reasons for not vaccinating included: patients were vaccinated before admission, patients were not stable enough to be vaccinated before discharge, and the acute care setting is not appropriate for vaccination. Response rate of 58% (n = 61) was achieved with an initial mailing and one follow-up telephone call to all previous nonresponders. Some physician survey responses do not correlate with data obtained from retrospective patients' claims analysis. CONCLUSION: Well-coordinated and timely attempts to encourage primary care physicians to immunize patients 65 years and older before hospital discharge were unsuccessful in our study. Rather than working with physicians, it may be that managed care organizations should work directly with hospitals to implement influenza and pneumococcal immunization programs.     

Gram-scale synthesis of recombinant chitooligosaccharides in Escherichia coli

Basilar membrane (BM) noise, measured as a velocity signal under the quiet acoustic condition, was investigated in the guinea pig. The cochleas of anesthetized young healthy guinea pigs were surgically exposed and a hole was made on the lateral wall of the scala tympani of the first cochlear turn for visualization of the BM and measurement of the BM velocity with a laser interferometer. The amplitude and frequency of the BM velocity noise were analyzed by a spectrum analyzer under different conditions. The spectrum of the BM velocity noise was a band limited function with a peak velocity at the topographic best frequency of the measured location on the BM. The peak velocity ranged to about 8 microm/s and depended on the physiological condition of the cochlea. Saline blockage of the external auditory canal or the middle ear did not change the BM noise. BM noise was much smaller, or was not evident, when the cochlear sensitivity decreased. The suppression tuning curve of the BM velocity noise indicates that the maximum suppression caused by an acoustic pure tone occurred at the best frequency location. A low sound level wide band acoustic noise given to the external ear canal produced a spectrum function having the same frequency and amplitude response as the BM noise. Electrical stimulation of the crossed olivocochlear bundle significantly depresses the BM velocity noise. These data demonstrate that the BM noise is a representation of internal rather than external noise. The amplitude and frequency of the BM noise reflect the usual cochlear sensitivity and frequency selectivity. Since the organ of Corti in the sensitive cochlea is a highly sensitive and tuned mechanical system, the internal (to the animal) noise responsible for the BM noise may originate from mechanical vibrations remote from the cochlea and propagated to the ear, or may be caused by Brownian motion of cellular structures in the cochlea.     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli

By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts. The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme. The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed. However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems. Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.     

Escherichia coli strains involved in diarrhea in France: high prevalence and heterogeneity of diffusely adhering strains

Two hundred sixty-two strains of Escherichia coli isolated from diarrheal stool specimens from infants, children, and adults hospitalized in Clermont-Ferrand, France, were studied to classify them in the previously described pathogenic groups of E. coli involved in diarrheal diseases. A total of 1.5% of them belonged to the enterotoxigenic E. coli pathotype, but none belonged to the enteroinvasive E. coli, enterohemorrhagic E. coli, or enteropathogenic E. coli pathotypes. Seventeen strains (6.5%) exhibited an aggregative pattern of adhesion to HEp-2 cells (EAggEC pathotype), but of these, three (17.6%) did not hybridize with the EAggEC DNA probe. Most of the strains involved in diarrhea belonged to the diffusely adhering E. coli group; 100 strains (38.2%) exhibited a diffuse adhesion (DA) to HEp-2 cells. Only eight strains (8.9%) from controls diffusely adhered to HEp-2 cells. The highly significant difference (P < 0.0001) between DA strains from patients and from controls suggests that the diffusely adhering E. coli strains should be considered pathogens. Only 33 of them (33%) hybridized with the previously described DA DNA probe, and only 2 (2%) hybridized with the AIDA DNA probe. Four different major proteins were observed in the bacterial surface extracts of the 33 strains positive with the DA DNA probe. In addition, 16 strains that diffusely adhered to HEp-2 cells induced a cytotoxic effect on HEp-2 cells that was characterized by pyknosis and lysis of the cytoplasmic membrane. This cytotoxic effect was correlated with the synthesis of a hemolysin. The genes involved in diffuse adhesion to HEp-2 cells were located on conjugative R plasmids in strains that did not hybridize with the DA or AIDA DNA probes.     

Characterization of Escherichia coli strains with gapA and gapB genes deleted

We obtained a series of Escherichia coli strains in which gapA, gapB, or both had been deleted. Delta gapA strains do not revert on glucose, while delta gapB strains grow on glycerol or glucose. We showed that gapB-encoded protein is expressed but at a very low level. Together, these results confirm the essential role for gapA in glycolysis and show that gapB is dispensable for both glycolysis and the pyridoxal biosynthesis pathway.     

Osmotic stress in viable Escherichia coli as the basis for the antibiotic response by polymyxin B

Cationic antimicrobial peptides, such as polymyxin B (PxB), below growth inhibitory concentration induce expression of osmY gene in viable E. coli without leakage of solutes and protons. osmY expression is also a locus of hyperosmotic stress response induced by common food preservatives, such as hypertonic NaCl or sucrose. High selectivity of PxB against Gram-negative organisms and the basis for the hyperosmotic stress response at sublethal PxB concentrations is attributed to PxB-induced mixing of anionic phospholipid between the outer layer of the cytoplasmic membrane with phospholipids in the inner layer of the outer membrane. This explanation is supported by PxB-mediated rapid and direct exchange of anionic phospholipid between vesicles. This mechanism is consistent with the observation that genetically stable resistance against PxB could not be induced by mutagenesis.     

Effect of high hydrostatic pressure on Escherichia coli and Pseudomonas fluorescens strains in ovine milk

Ovine milk that had been standardized to 6% fat was inoculated with Escherichia coli 405 CECT and Pseudomonas fluorescens 378 CECT at a rate of 10(6) and 10(7) cfu/ml, respectively, and treated with high hydrostatic pressure. Treatments consisted of combinations of pressure (300, 400, 450, and 500 MPa), temperature (2, 10, 25, and 50 degrees C), and time (5, 10, and 15 min). Inactivation (> 6 log cfu/ml) of both strains was observed at 50 degrees C for all pressures and treatment times. A similar level of inactivation occurred at > or = 450 MPa and 25 degrees C for E. coli and at > or = 400 MPa and 10 degrees C for P. fluorescens. Destruction was lowest at 10 degrees C for E. coli and at 25 degrees C for P. fluorescens. The test strain of E. coli was more baroresistance than was the P. fluorescens strain.