Biomedical subjects
Publications and source records attributed to S Gatermann.
Endobronchial actinomycosis was found to be the cause of right-sided atelectasis and haemoptysis in a 57 year old man without predisposing conditions. Fibreoptic bronchoscopy revealed occlusion of the intermediate bronchus by yellow-white masses. The diagnosis was confirmed histologically and by positive Actinomyces culture from bioptic material. Prolonged antibiotic treatment resulted in complete recovery, without need for surgical resection.
Staphylococcus saprophyticus, a well known cause of urinary tract infections, possesses several properties, such as hemagglutination of sheep erythrocytes, adherence to various cell types and production of urease, which may be virulence factors. In this contribution, we summarise the present knowledge about recently discovered surface proteins of Staphylococcus saprophyticus, a 95 kDa surface-associated protein (Ssp) and the hemagglutinin, a 160 kDa surface polypeptide. We describe culture conditions conducive to production of these surface proteins and discuss the molecular and clinical implications of our findings.
The 14C-urea breath test has been shown to be a reliable non-invasive method to detect the presence or absence of H. pylori infection. Alternatively, a number of techniques have been devised to detect circulating antibodies against H. pylori in serum, the most commonly used being enzyme-linked immunosorbent assays (ELISA). In the present study we compared the value of two ELISA antigen preparations, an acid glycine extract and a urease preparation, in relation to the results achieved in a 14C-urea breath test. Seventy-five gastroenterology outpatients were screened for the presence of H. pylori infection using the urea breath test. At the same time serum specimens were obtained. Thirty-seven patients had a positive breath test, i.e. they expired more than 2% of the oral 14C test dose within 60 min. Using the breath test as reference, sensitivity and specificity for the acid extract were 89.2% and 84.2% respectively, and for the urease ELISA 81.1% and 89.5%. Agreement between the two ELISAs was found in 82.7%, overall agreement between all three tests was observed in 77.3%. All three tests were found to be useful for monitoring therapy directed against H. pylori.
The role of the major secretory protein of Legionella pneumophila, a zinc protease, in Legionella infection is not known. Since an important step of the host reaction in Legionnaires' disease is the production of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages, we studied the interaction of Legionella protease and U-937 cells with respect to TNF-alpha. The Legionella protease was purified by fractionated precipitation, gel filtration and hydrophobic interaction chromatography. The purified enzyme was added to U-937 cells, a promyelocytic cell line. In the supernatants of PMA-treated U-937 cells we found low concentrations of TNF-alpha after incubation with protease. Therefore we pursued the hypothesis of direct enzymatic degradation of TNF-alpha by Legionella protease. Enzymatic cleavage of TNF-alpha was proven by SDS-PAGE, ELISA and TNF-alpha bioassay with L-929 cells. The degradation of TNF-alpha by the Legionella protease was shown in all three systems. Enzymatic degradation of TNF-alpha might be important for the pathogenesis of Legionnaires' disease.
The urease gene sequences of Staphylococcus epidermidis CNS23 were cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned sequences revealed four polypeptides (60, 17, 11 and 7.5 kDa) that were associated with enzyme activity. Southern hybridisation experiments showed high homologies with the urease genes of Staphylococcus saprophyticus.
An elastase of Staphylococcus epidermidis was purified by ion exchange chromatography on CM-Sepharose and characterised. Its M(r) is c. 21 kDa, its optimal temperature for activity is 42 degrees C and the pH optimum is 6.8. The enzyme is activated by cysteine and other SH-donators and inhibited by L-trans-epoxy-succinylleucylamido-(4-guanidino)butane (E64), an inhibitor of cysteine proteases, but not by 3,4-dichloroisocoumarin (3,4-DCI), an inhibitor of serine proteases. This finding suggests that the elastase of S. epidermidis is a cysteine protease. Because S. epidermidis elastase degrades human sIgA, IgM, serum albumin, fibrinogen, and fibronectin, this enzyme may be regarded as a virulence factor.
Many strains of Staphylococcus saprophyticus cause direct hemagglutination of sheep erythrocytes. For a high proportion of clinical isolates, a surface protein (Ssp) that is apparently not involved in this property has been described. In this study, S. saprophyticus CCM883, a hemagglutinating but Ssp-negative strain, was used for the identification, purification, and characterization of a 160-kDa surface polypeptide that appears to be the major component of the hemagglutinin. Expression of the protein required the addition to the growth medium of EDTA in micromolar quantities, suggesting an inhibitory role for some unidentified metal ion. The protein was purified by means of Sephacryl S-300 chromatography, and antisera were raised in rabbits. Antibody against this protein inhibited the hemagglutination of two other, unrelated strains and was used to demonstrate, by electron microscopy, the presence of the protein on the surface of the cells. In a confirmatory experiment, the purified antigen was incubated with erythrocytes and binding was detected by the Western immunoblot technique with the antibody to the 160-kDa polypeptide. These experiments indicate that this surface protein is the hemagglutinin of S. saprophyticus.
A 95-kDa protein was isolated from Staphylococcus saprophyticus 7108 grown on dialysis membranes placed on the surface of brain heart infusion agar. Strain CCM883 did not produce this protein. Ultrathin sections revealed the presence of very thin, tuftlike, 50- to 75-nm-long structures on the surface of strain 7108, whereas strain CCM883 was comparably smooth. The surface material could be removed by digestion with proteinase K, suggesting that the surface structures contain protein. High-resolution scanning electron microscopy showed a thick layer of surface material on strain 7108, whereas strain CCM883 appeared smooth. The 95-kDa protein was purified by Sephacryl S-300 chromatography, and an antiserum was raised in rabbits. This antiserum was used in immunogold labeling experiments, which showed that the protein is associated with the surface structures. Our experiments thus demonstrate the presence of a fibrillar protein on the surface of S. saprophyticus (Ssp for S. saprophyticus surface-associated protein).
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S. saprophyticus CCM883 and 9325 were found to adhere to the tubular cell line LLC-PK1. An ELISA technique was used to determine adherence of bacteria and inhibition of adherence by various carbohydrates. Only N-acetyl-galactosamine was found to significantly inhibit adhesion (p less than 0.001), which suggests that the surface component mediating adhesion recognizes structures on the target cell that contain this carbohydrate.
We studied the biochemical properties of the urease of Staphylococcus saprophyticus and the possible role of the urease in experimental urinary tract infections. For this purpose, the nonhemagglutinating and nonadherent strain 9325, which was isolated from a case of symptomatic urinary tract infection, was used. The urease was shown to have a Km of 6.64 mM urea and a Vmax of 4.59 mumol NH3.min-1.mg-1. The enzyme was inhibited by acetohydroxamic acid in a noncompetitive manner. By means of Sephacryl S-300 column chromatography, we determined a mean molecular weight (+/- standard error of the mean) of 420,000 +/- 16,000. To assess the contribution of S. saprophyticus urease to uropathogenicity, a urease-negative mutant was constructed by nitrosoguanidine mutagenesis. In the rat model of ascending unobstructed urinary tract infection, higher numbers of CFU.gram of tissue-1 and more-severe lesions were detected with the parent strain. Moreover, bladder stones were found in animals infected with the urease-positive strain only. Interestingly, the difference in mean bacterial counts of the bladders was found to be significant by the Wilcoxon two-sample test (P less than 0.05), whereas that between the kidney bacterial counts was not. Immunoblot studies revealed a faint antibody response in rats infected with the mutant strain, although bacteria could still be detected in the kidneys after 7 days. Sera of animals challenged with the parent strain reacted strongly with many antigens of S. saprophyticus. Our data indicate that urease is a major factor for invasiveness of S. saprophyticus, especially in the tissue of the bladder, whereas persistence in the urinary tract and nephropathogenicity of this organism are governed by factors other than urease.
The urease gene of Staphylococcus saprophyticus CCM883 was cloned and expressed in Staphylococcus carnosus TM300. In vitro translation of the cloned DNA sequences revealed six polypeptides (of 70, 47, 29, 27, 20, and 17 kilodaltons) that were associated with enzyme activity. Introduction of the cloned genes into a urease-negative mutant of S. saprophyticus restored the virulence of this strain, confirming our previous suggestion (S. Gatermann, J. John, and R. Marre, Infect. Immun. 57:110-116, 1989) that this enzyme is a major virulence factor of the organism and contributes mainly to cystopathogenicity.
We studied hemagglutinating and adherence properties in Staphylococcus saprophyticus isolates originating from symptomatic urinary tract infections. 12/13 (92%) of strains adhered to Hep cells and 11/13 (85%) were able to agglutinate sheep erythrocytes. Adherence properties differed markedly between strains (P less than 0.0001). Two strains, one hemagglutinating and adherent and one negative for both properties were chosen for experimental urinary tract infections. Results indicate that presence of the hemagglutinin favours colonization of kidney tissue.
Systemic immune response to Campylobacter pylori was detected by the immunoblot technique in serum samples from 200 patients, 129 blood donors, and 96 children. The results of the IgG immunoblot test showed excellent correlation with the detection of C pylori by culture and also with histopathological examination of the antrum, as well as with peptic ulcer disease. An IgA response also occurred and gave results comparable with those of the IgG immunoblot test, although on a quantitatively lower scale. The IgM immunoblots were of no help in the serodiagnosis of C pylori infection. The protein bands that seemed to be the most specific for C pylori and which were consistently observed in patients positive for C pylori were a 110 kilodalton and a 63 kilodalton band on the IgG immunoblot and an 89 kilodalton band on the IgA immunoblot. A 94 kilodalton and a 28 kilodalton band were also included in the evaluation. While immunoblot analysis may be used effectively for the serodiagnosis of C pylori infection and can distinguish between patients with normal antrum mucosa and those with gastritis, the test does not help to distinguish between those patients with antrum gastritis who subsequently develop peptic ulcers and those who do not.
A recombinant vaccine against hepatitis B derived from yeast cells (Gen-HB-Vax, Co. MSD/Behring) has been evaluated in 59 healthy young volunteers (37 men, 22 women) with an average age of 24.4 years. The seroconversion rate was 100%, and no major side effects were observed. During a follow-up period of 24 months concentrations of antibodies against HBsAg were shown to decline to one tenth within 16 to 17 months. Triple vaccination led to protective antibody titres for about 27 months on average. Based on these findings we suggest the following recommendations concerning revaccination: an anti-HBs titre of more than 10,000 mIU/ml four weeks after third vaccination should be reassessed 3-5 years later, and titres between 1000 and 10,000 mIU/ml after 2-3 years. A control of the antibody titre should be performed after 1-2 years if the titre is 200-1000 mIU/ml after the third vaccination, and after 6-12 months if it is 100-200 mIU/ml. Antibody titres between 10 and 100 mIU/ml should already be reassessed about 3-6 months later. We recommend an immediate revaccination for persons with anti-HBs titres below 10 mIU/ml. This corresponds to the course of antibody concentrations which could be seen in former studies with the conventional serum-derived vaccine. Maximum anti-HBs titres are slightly below those observed with the serum-derived vaccine.
Methicillin resistance is usually not detected by routine laboratory methods. With the aid of a method that facilitates in vitro detection of methicillin resistance 8.6% of our Staph. aureus strains and 42% of the coagulase-negative staphylococci proved to be resistant towards this antibiotic.
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97% of the vaccinees developed anti-HBs independently of the applied vaccine (experimental vaccine or H-B-Vax). With the experimental vaccine the mean antibody titre was 1095 IMU/ml four weeks after third inoculation. Follow up revealed that during a period of 18 month individual antibody titres declined continuously to approximately one tenth. Therefore the duration of protection depends on the titre of anti-HBs which was measured after the third immunization. A control of the antibody titres should be performed after about 3-5 years, when the antibody titres are greater than 1000 IMU/ml 4 weeks after vaccination. But a control should be made already after about 1 1/2-3 years if the antibody titres are 200-1000 IMU/ml. Antibody titres between 100 and 200 IMU/ml should be estimated about 6-18 months later and titres between 10 and 100 IMU/ml already about 3-6 months later. We recommend an immediate revaccination for persons with anti-HBs titres below 10 IMU/ml. Serological findings in hospital staff of the University in Hamburg revealed the presence of protective antibodies in 11,6% which is due to a previous hepatitis B infection. In this cases vaccination was unnecessary.