Cloning, expression and purification of extracellular serine protease Esp, a biofilm-degrading enzyme, from Staphylococcus epidermidis.

AIMS: Staphylococcus epidermidis Esp, an extracellular serine protease, inhibits Staphylococcus aureus biofilm formation and nasal colonization. To further expand the biotechnological applications of Esp, we developed a highly efficient and economic method for the purification of recombinant Esp based on a Brevibacillus choshinensis expression-secretion system. METHODS AND RESULTS: The esp gene was fused with the N-terminal Sec-dependent signal sequence of the B. choshinensis cell wall protein and a C-terminal hexa-histidine-tag gene. The recombinant Esp was expressed and secreted into the optimized medium as an immature form and subsequently activated by thermolysin. The mature Esp was easily purified by a single purification step using nickel affinity chromatography and showed proteolytic activity as well as Staph. aureus biofilm destruction activity. CONCLUSIONS: The purification yield of the developed extracellular production system was 5 mg recombinant mature Esp per 20-ml culture, which was much higher than that of an intracellular production system in Escherichia coli (3 mg recombinant Esp per 1-l culture). SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings will be a powerful tool for the production and purification of recombinant Esp and also applicable to a large variety of recombinant proteins used for basic researches and biotechnological applications.

How Vibrio cholerae survive during starvation.

Vibrio cholerae, a Gram-negative, motile, aquatic bacterium, is the causal agent of the diarrheal disease cholera. Cholera is a serious epidemic disease that has killed millions of people and continues to be a major health problem world-wide. The hypothesis that V. cholerae occupies an ecological niche in the estuarine environment requires that this organism is able to survive the dynamics of physiochemical stresses, including nutrient starvation. As a result of these stresses, bacteria in nature often exist in non-growth or very slow growth states with a low metabolic activity. Because microorganisms have little ability to control their environment, environmental changes have led to changes in cell function and structure. Such cellular responses can originate in one of two ways: by changes in genetic constitution or by phenotypic adaptation. In this review, we will focus on the phenotypic responses of V. cholerae of a given genotype to starvation stress.

Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation.

Vibrio parahaemolyticus is known to exist in a viable but nonculturable state when incubated at low temperature under starvation. It has long been debated whether the culturable cells which appear after temperature upshift are the result of true resuscitation or regrowth of a few residual culturable cells. Starved V. parahaemolyticus cells at 4 degrees C reached the nonculturable stage in about 12 days. The true resuscitation of nonculturable cells of V. parahaemolyticus occurred after spreading them onto an agar medium supplemented with H(2)O(2)-degrading compounds such as catalase or sodium pyruvate. The proposed method may be applicable to detecting the enteropathogen from environmental samples.

Ofloxacin and fleroxacin enhance superoxide production in human polymorphonuclear leukocytes by increasing phosphorylation in the signal transduction pathway.

Many antimicrobial agents including new quinolones (NQs) influence the cellular defense mechanisms such as polymorphonuclear leukocytes (PMNs), macrophages and lymphocytes. We examined the effects of NQs on superoxide (SO) production of PMNs following stimulation of phorbol myristate acetate (PMA). Ofloxacin (OFLX) and fleroxacin (FLRX) significantly augmented SO production of PMNs compared to lomefloxacin, sparfloxacin. Staurosporin and H-7, specific inhibitors of protein kinase C of SO production pathway in PMNs, inhibited augmented SO production by OFLX and FLRX in the concentration-dependent manner. NADPH oxidase activity was not influenced by OFLX in cell lysate assay system. These results suggest that OFLX and FLRX augmented PMN function through enhancing protein kinase activity, but not through direct enhancement of NADPH oxidase.

Chemiluminescence response of whole blood in patients undergoing urological operations.

Polymorphonuclear leukocytes (PMNs) are one of the most important components of the defence mechanisms against bacterial infection. The functions of PMNs are believed to be impaired in patients during the perioperative period. Bactericidal function of PMNs was investigated together with the luminol-dependent chemiluminescence (CL) reaction of whole blood in 23 patients, 12 undergoing open surgery and 11 undergoing endoscopic surgery. Blood samples were collected one day before surgery (day -1) and 2 hours (day 0), 24 hours (day 1) and 7 days (day 7) after surgery. Counts of whole white blood cells (WBCs), PMNs and lymphocytes were not different between the two surgery groups. CL responses in the open surgery group were increased on days 0, 1 and 7. In the endoscopic surgery group, CL response was increased on day 1, but not on day 0 or day 7. These results suggest that the PMN function during the perioperative period was not impaired, but increased just after surgery, mainly due to an increasing number of WBC caused by the surgical intervention.

[Comparative double-blind study of cefroxadine and cephalexin in the treatment of complicated urinary tract infection].

To evaluate the efficacy, safety, and utility of cefroxadine (CXD) for the treatment of complicated urinary tract infections, a double blind study comparing CXD with cephalexin (CEX) was carried out. Patient received either 1,500 mg/day of CXD 3 times a day, or 2,000 mg/day of CEX 4 times a day for 5 days by oral route, and the following results were obtained. Of the 305 patients, clinical efficacies were evaluated in 220 cases (CXD 105 cases, CEX 115 cases) except that excluded or dropped out. Side effect was evaluated in 301 cases (CXD 150 cases, CEX 151 cases). There was no statistically significant difference in the back ground characteristics between the 2 groups. Overall clinical assessment by the committee according to the "Criteria for Evaluation of Clinical Efficacy of Antimicrobial Agents on Urinary Tract Infection" patients evaluated as better than "good" were 64 of 105 (61.0%) for CXD and 75 of 115 (65.2%) for CEX. The difference between the 2 groups was not statistically significant. In effect on pyuria, patients evaluated as better than "decreased" were 58 of 105 (55.2%) for CXD and 69 of 115 (60.0%) for CEX. The difference between the 2 groups was not statistically significant. In effect of bacteriuria, patients evaluated as better than "decreased" were 57 of 105 (54.3%) for CXD and 69 of 115 (60.0%) for CEX. The difference between the 2 groups was not statistically significant. Analyses were stratified according to classification by the type of infection, diagnosis, degree of pyuria before treatment, and bacterial count before treatment. There were no statistically significant differences between the 2 treatment groups as to any item. In evaluation by attending physician, patients evaluated as better than "good" were 81 of 140 (57.9%) for CXD, and 85 of 141 (60.3%) for CEX. Statistically significant difference was not observed between the 2 groups. In drug usefulness by attending physician, patients evaluated as better than "usefulness" were 106 of 140 (75.7%) for CXD, and 109 of 141 (77.3%) for CEX. The difference between the 2 groups was not statistically significant. In evaluation of the infections with sensitive species to both CXD and CEX by the committee according to "Criteria for Evaluation of Clinical Efficacy of Antimicrobial Agents on Urinary Tract Infections, overall clinical efficacies were evaluated in 102 (CXD 48 cases, CEX 54 cases) which were infected with sensitive species. There was no statistically significant difference in the back ground characteristics between the 2 treatment groups.(ABSTRACT TRUNCATED AT 400 WORDS)

[Murine "sympathetic orchitis" induced by unilateral testicular injury and autoimmune response].

BACKGROUND: Experimental autoimmune orchitis (EAO) has been studied as an animal model for human male immunological infertility. Most EAO models have been induced by immunization with testis antigens in artificial adjuvants. In this paper, we report a more clinical EAO model. METHODS: Ten to 20 needle punctures were made to the unilateral testis of mice and it was crushed by a needle-holder. RESULTS: Contralateral EAO (so-called "sympathetic orchitis") was gradually induced starting on Day 28. Delayed type hypersensitivity (DTH), one of the cell-mediated immunities, to autologous testicular cells (TC) as well as anti-TC antibodies, humoral immunity, were both detected in those mice. Repeated crush(es) of the ipsilateral testis two weeks later (and four weeks later) as a booster did not enhance the contralateral lesion or autoimmune responses. CONCLUSION: Our present injury model mimics clinical testicular trauma; therefore, this testicular injury model can be very useful in studying the immunological mechanism of EAO and of human immunological male infertility.

Suitability of colchicine and superoxide dismutase for the suppression of renal scarring following an infection with bacteria showing mannose-sensitive pili.

Two new strains of Serratia marcescens were constructed by the gene manipulation method from the clinical isolate US 46, which has two kinds of pili--mannose-sensitive (MS) and mannose-resistant (MR) ones--on the cell surface. After cloning the genes of the MS and MR pili, either the MS or the MR gene was transferred to the nonpiliated Escherichia coli, and MS- or MR-piliated strains were obtained. In the experimental pyelonephritis model of rats, MS- or MR-piliated bacteria were inoculated directly to the renal parenchyma, and the following results were obtained. MS-piliated rather than MR-piliated strains stimulated severe scarring of the kidney, and this scarring was suppressed by treatment with colchicine or superoxide dismutase (SOD) during an early stage of the infection. These findings suggest that MS-piliated bacteria stimulated polymorphonuclear leukocytes, which released large amounts of superoxide resulting in renal scarring. SOD was hoped to be a drug capable of preventing renal scarring, and such a result was successfully obtained.

A cryptic fimbrial gene in Serratia marcescens.

The gene coding for the mannose-sensitive hemagglutinating fimbriae in Serratia marcescens US5 was cloned into Escherichia coli K4 with a cosmid vector system. One of the transformants, US5-1, expressed two morphologically distinct fimbriae, one that was 5-nm wide and one that was 3-nm wide. The latter fimbria was morphologically and serologically indistinguishable from that of strain US5. Genetic analysis of transformant US5-1 showed that the gene responsible for the 5-nm-wide fimbriae was located more than 10 kilobases away from the gene responsible for the 3-nm-wide fimbriae. The molecular sizes of the subunits of these two fimbriae, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 19 kilodaltons for the 3-nm-wide fimbriae and 20 kilodaltons for the 5-nm-wide fimbriae. Serologically, the 5-nm-wide fimbriae did not cross-react with monoclonal or polyclonal antibodies raised against the mannose-sensitive hemagglutinating fimbriae of strain US5. Strain EL101, which expressed only the 5-nm-wide fimbriae, did not agglutinate chicken or human erythrocytes. These experimental results suggest that the gene for the 5-nm-wide fimbriae is cryptic in strain US5 and is expressed in E. coli K4 only after it is moved by transformation.

Type 1 fimbriation and its phase switching in diarrheagenic Escherichia coli strains.

Type 1 fimbriae can be expressed by most Escherichia coli strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells. However, the role of type 1 fimbriae in enteric pathogenesis has been unclear. Expression of type 1 fimbriae in E. coli is phase variable and is associated with the inversion of a short DNA element (fim switch). Forty-six strains of diarrheagenic E. coli were examined for the expression of type 1 fimbriae. Only four of these strains were originally type 1 fimbriated. Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the fim switch, after serial passages in static culture. The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain E. coli K-12. None of the 16 strains of serovar O157:H7 or O157:H(-) expressed type 1 fimbriae after serial passages in static culture. The nucleotide sequence analysis of the fim switch region revealed that all of the O157:H7 and O157:H(-) strains had a 16-bp deletion in the invertible element, and the fim switch was locked in the "off" orientation. The results suggest that expression of type 1 fimbriae may be regulated differently in different E. coli pathogens causing enteric infections.

A comparison of solid and liquid media for resuscitation of starvation- and low-temperature-induced nonculturable cells of Aeromonas hydrophila.

Like many other gram-negative bacteria, starved cells of Aeromonas hydrophila can be induced into a viable but nonculturable (VBNC) state by incubation at low temperature, as shown here by using various bacterial enumeration methods. Starved A. hydrophila strain HR7 cells at 4 degrees C reached the nonculturable stage in about 45 days. The cells were resuscitated by either a solid medium resuscitation method, using solid agar amended with H2O2-degrading agents, catalase or sodium pyruvate, or a liquid medium resuscitation method, by incubating nonculturable cells in liquid media containing these compounds before spreading onto plates. The liquid medium resuscitation method using catalase resulted in nearly complete recovery of nonculturable cells.

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds.

Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 degrees C. Plate counts declined from 3 x 10(6) to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or alpha-ketoglutaric acid, plate counts increased to 10(4)-10(5) CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H2O2, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells.

Cloning and sequence of the gene encoding the major structural component of mannose-resistant fimbriae of Serratia marcescens.

Serratia marcescens US46, a human urinary tract isolate, exhibits mannose-resistant hemagglutination and agglutinates yeast cells, thereby indicating that it has two types of adhesins. We constructed a cosmid library for the DNA of this organism and isolated DNA clones carrying genes for mannose-sensitive (MS) and mannose-resistant (MR) fimbriae. On introduction of the cloned genes into Escherichia coli K-12, MS and MR fimbriae were formed. These fimbriae were functionally and morphologically indistinguishable from those of S. marcescens. Subcloning of these gene clusters revealed that the genes encoding MS fimbriae reside on a 9-kilobase (kb) DNA fragment, while those encoding MR fimbriae are present on a 12-kb fragment. Transposon insertion and maxicell analyses revealed that formation of MR fimbriae is controlled by several genes which reside on the 9-kb fragment. The nucleotide sequence of smfA, the gene encoding the major structural component of MR fimbriae, revealed that this gene encodes a 174-amino-acid polypeptide with a typical procaryotic signal peptide. The primary structure of the smfA product showed significant homology with the primary structure of the E. coli fimbrial subunit.

Direct inactivation of human polymorphonuclear leukocyte by hyperosmotic urea comparable to the renal medulla.

Hyperosmolarity in the renal medulla inhibits host defenses against bacterial pyelonephritis. Urea and NaCl contribute most to high osmolarity in the renal medulla. We therefore examined the inhibitory mechanism of urea on superoxide generation by human polymorphonuclear leukocytes. Superoxide production was inhibited by high concentration of urea. This inhibition was found to be direct and immediate. In addition, direct inactivation of NADPH oxidase, the key enzyme complex of superoxide generation, was shown by an NADPH oxidase activity assay using cell lysates of polymorphonuclear leukocytes stimulated by phorbol myristate acetate. The inhibitory effect of urea on NADPH oxidase was reversed by washing urea out of the assay system of cell lysates. Kinetic analysis of the inhibition of NADPH oxidase activity by urea showed decreased Vmax and Km, suggesting uncompetitive inhibition. These findings suggested that urea inactivated polymorphonuclear leukocyte superoxide production through a direct and uncompetitive inhibition of NADPH oxidase.

Renal scarring is enhanced by phorbol myristate acetate following infection with bacteria with mannose-sensitive pili.

Renal scarring is considered to develop in the later stages of chronic pyelonephritis. In our experimental model of pyelonephritis, bacteria with mannose-sensitive (MS) pili on their surface promoted renal scarring following inoculation into the renal parenchyma. The administration of cyclophosphamide to induce leukopenia and of superoxide dismutase to inactivate superoxide released from polymorphonuclear leukocytes (PMN) at the infection site suppressed any renal scarring following the infection. Conversely, the administration of phorbol myristate acetate at an early stage of infection significantly enhanced the renal scarring. These findings suggest that the PMNs which infiltrate the infection site and the superoxide they release play an important role in any renal scarring following infection with MS-piliated bacteria.

Increased renal scarring by bacteria with mannose-sensitive pili.

Renal scars are thought to be the end stage of chronic pyelonephritis and one of the most important causes of renal insufficiency and renal hypertension. The role of bacterial pili was examined in scar formation after an infection of newly constructed bacterial strains using the recombinant DNA technique, which possessed either mannose resistant (MR) or mannose sensitive (MS) pili of Serratia marcescens. Strains that differed in only a single virulence factor, namely, MR or MS pili, were used in a rat model of chronic pyelonephritis. In this model, MS-piliated bacteria stimulated renal scarring more severely than non-piliated or MR-piliated bacteria.

Significance of urinary endotoxin concentration in patients with urinary tract infection.

Endotoxin is a component of the outer membrane of gram-negative rods (GNR). Since GNR are responsible for the majority of urinary tract infection (UTI), we measured the concentration of endotoxin in urine using chromogenic endotoxin-specific assay and examined its diagnostic utility in patients with suspected UTI. In all 18 urine samples with an endotoxin concentration exceeding 350 pg/ml and 2 samples with 10-350 pg/ml of endotoxin concentration, GNR were detected at a count of 10(4) cfu/ml. Negative for endotoxin were 3 samples of culture positive for gram-positive cocci (GPC), 2 samples containing various bacterial contaminants and all 37 samples with no growth on culture. Two urine samples collected 5 h after antibiotic dosage showed negative culture for GNR but a significant concentration of endotoxin. In an in vitro experiment, a residual concentration of antibiotic in urine inhibited bacterial growth, leading to a false-negative culture. These results suggest that chromogenic endotoxin assay is a reliable method for diagnosing UTI caused by GNR and detecting false-negative culture of GNR.

Role of superoxide in renal scarring following infection by mannose-sensitive piliated bacteria.

The role of superoxide in scar formation following renal infection caused by mannose-sensitive (MS) piliated strains of bacteria was studied in the experimental pyelonephritis model using female Sprague-Dawley rats. The MS piliated strain stimulated renal scarring to a significantly greater extent than either the non-piliated or MR-piliated strain. Modulation of leukocytes by administering cyclophosphamide to induce neutropenia and colchicine to inhibit leukocyte migration was effective in preventing renal scarring. Treatment with superoxide dismutase during the early stage of infection was also effective in preventing scar formation. Finally, the production of superoxide by rat leukocytes was significantly larger following stimulation by MS piliated than either the non-piliated or MR piliated strains. These observations suggest that superoxide released from leukocytes plays a critical role in the development of renal scarring following a bacterial infection, especially by MS piliated strains.

Identification and nucleotide sequence of the gene determining the adhesion capacity of Serratia marcescens.

Three open reading frames, designated smfE, smfF, and smfG, within the mannose-resistant fimbria gene cluster of Serratia marcescens were identified. smfG, which is responsible for determining the receptor binding of S. marcescens, encodes a 280-amino-acid polypeptide with a typical prokaryotic signal sequence.