Rea Krausse

Antiviral effects of Glycyrrhiza species

Historical sources for the use of Glycyrrhiza species include ancient manuscripts from China, India and Greece. They all mention its use for symptoms of viral respiratory tract infections and hepatitis. Randomized controlled trials confirmed that the Glycyrrhiza glabra derived compound glycyrrhizin and its derivatives reduced hepatocellular damage in chronic hepatitis B and C. In hepatitis C virus‐induced cirrhosis the risk of hepatocellular carcinoma was reduced. Animal studies demonstrated a reduction of mortality and viral activity in herpes simplex virus encephalitis and influenza A virus pneumonia. In vitro studies revealed antiviral activity against HIV‐1, SARS related coronavirus, respiratory syncytial virus, arboviruses, vaccinia virus and vesicular stomatitis virus.

Molecular differentiation of bacteria by PCR amplification of the 16S–23S rRNA spacer

In general, operons for prokaryotic rRNA genes contain a transcribed spacer between 16S and 23S rRNA genes. The length and sequence of this spacer are expected to be highly variable. Polymerase chain reaction (PCR) permits the direct amplification of spacer DNA from small amounts of genomic DNA. Data for a wide range of microorganisms were accumulated, and the spacer lengths varied between 280 and 1300 bp. A further differentiation was achieved by a high-resolution gel electrophoresis method, single-strand conformation polymorphism (SSCP). It was possible to differentiate 15 Mycoplasma species, to analyze mixed samples and to distinguish Streptococcus strains and Clostridium botulinum serotypes. Spacer analysis is a promising method for the differentiation of diverse and closely related species, and also useful in analysis of mixed samples.

In Vitro Activities of New Fluoroquinolones against Campylobacter jejuni and Campylobacter coli Isolates Obtained from Humans in 1980 to 1982 and 1997 to 2001

ABSTRACT The antibacterial activities of three newly developed fluoroquinolones (gatifloxacin, levofloxacin, and moxifloxacin) against a total of 307 gastrointestinal human isolates of Campylobacter jejuni and Campylobacter coli collected during 1980 to 1982 and 1997 to 2001 were examined and compared to those of ciprofloxacin and the unrelated antibacterial agents, clarithromycin, erythromycin, and tetracycline by using the agar plate dilution method. All of the fluoroquinolones exhibited a good activity against Campylobacter, and some of them were more active than ciprofloxacin, the macrolides, and tetracycline. Among the fluoroquinolones, gatifloxacin and moxifloxacin showed the highest anticampylobacter activity, with MICs at which 50% of the isolates tested are inhibited (MIC50s) and MIC90s of 0.125 and 4 μg/ml, respectively; the MIC50 for both levofloxacin and ciprofloxacin was 0.25, and the MIC90s were 16 and 32 μg/ml, respectively. About 30% of the strains were found to be resistant to at least one fluoroquinolone. Resistance to gatifloxacin occurred in 9.8% of the isolates tested, and resistance to the other fluoroquinolones occurred in 19.9 to 27.4% of the isolates tested; the frequency of cross-resistance was 35.7 to 100%. An increase in fluoroquinolone resistance from 0% in 1980 to 1982 to 11.8 to 29% in 1997 and 1998, 8.2 to 31.8% in 1999 and 2000, and 12.1 to 30.3% in 2001 was found. A total of 61.4 to 73.2% of the C. jenuni strains resistant to erythromycin, clarithromycin, and/or tetracycline were susceptible to fluoroquinolones; gatifloxacin showed the highest percentage of inhibition. These results show that the newer fluoroquinolones with their potent activity could be used to treat infections with C. jejuni and C. coli. However, when these drugs are used, one must consider the increase in resistance and the high cross-resistance to these antimicrobial agents.

Clinical Relevance of CagA-specific Antibodies Related to CagA Status of Heliobacter pylori Isolates Using Immunofluorescence Test and PCR

AbstractBackground: The cagA (cytotoxin-associated gene A) protein is found in about 50% of Helicobacter pylori strains; its clinical relevance in gastroduodenal disease is uncertain. Patients and Methods: The frequency of IgG antibodies to cagA was studied by using a commercial Western blot assay in sera of 189 patients with endoscopically and histologically confirmed gastroduodenal disease. In addition, 38 H. pylori strains isolated from biopsies were analyzed by immunofluorescence test (IFT) and PCR for detection of cagA protein and cagA gene sequences, respectively. Results: 54.3–60.0% of all patients with gastrointestinal diseases (chronic gastritis, gastric or duodenal ulcer and chronic duodenitis) and 28.6% with a normal mucosa were found to be positive for anti-cagA IgG antibodies. There was no significant difference in anti-cagA IgG seroprevalence between the different clinical entities. CagA-positive (cagA+) H. pylori strains were detected in 44.7% and 50% of the 38 isolates by PCR and IFT, respectively. 22 of 23 patients infected with cagA+ strains had anti-cagA antibodies. Using PCR as a gold standard, the sensitivity and specificity of the cagA IgG Western blot were 100.0% and 35.0%, respectively; the sensitivity and specificity of the cagA IFT were 76.5% and 71.4%, respectively. The incidence of the cagA+H. pylori strains detected either by PCR or IFT was significantly higher (p < 0.05 and p < 0.01, respectively) in patients with chronic duodenitis, gastric or duodenal ulcer compared to patients with chronic gastritis (66.7%, 80% and 30.4%, respectively). Conclusion: In this study the cagA-specific serological status in H. pylori infections as diagnosed by IgG Western blot was of no predictive value for severity of disease. In contrast, the cagA status of H. pylori isolates, diagnosed by IFT or PCR, was a predictive marker for severe disease and, therefore, also of clinical relevance in the assessment of the virulence of the infecting strain.

The reaction of campylobacter species on the chemiluminescence, chemotaxis and hemagglutination

The influence of Campylobacter species (sp.) on the luminol-dependent chemiluminescence (CL) during phagocytosis, the chemotaxis and the agglutination of different erythrocyte species was investigated. CL was measured directly with undiluted whole blood samples and isolated polymorphonuclear neutrophil granulocytes (PMNG). The chemotactic response of PMNG to various Campylobacter sp. was performed using an agarose technique. All the Campylobacter sp. investigated showed a different CL response pattern. When opsonized Campylobacter strains were used, the CL response of PMNG was greater than in the presence of nonopsonized strains. No correlation was found between CL response and bacterial killing. For the investigation of the chemotaxis the filtered supernatants of Campylobacter cultures were used as cytotoxins and compared with the chemotactic peptide, formyl-methionyl-leucine-phenylalanine (FMLP). All the supernatants of cultures of Campylobacter sp. were chemotactic for PMNG. A strong mannose-resistant hemagglutination (MRHA) with glycine-hydrochloride extract was observed with C. fetus ssp. fetus and a weak reaction, with C. coli. There was no correlation between chemiluminescence, chemotaxis and hemagglutination. The weak stimulation of PMNG by C. fetus ssp. fetus and most of the C. jejuni/coli isolates suggest that this behavior could be a cause of pathogenicity.

Pharmacokinetics of imipenem in patients undergoing major colon surgery

SummaryTen patients about to undergo a colorectal operation lasting an average of three hours received 500 mg each of imipenem and cilastatin i.v. preoperatively. During the operation blood and tissue samples were taken in order to determine the serum kinetics of the substances as well as the levels of imipenem in the cutis, subcutis, fascia, muscle, parietal peritoneum and colon. The imipenem concentrations were measured by HPLC. The mean peak serum concentration was 26 mg/l, the mean half-life 55 min and the AUC 40 mg/l/h−1. The serum pharmacokinetics of imipenem was subject to substantially larger fluctuations in this patient group than in subjects or patients without surgery. Imipenem rapidly penetrates into tissue, with peak concentrations being reached after 10–25 min. The highest imipenem concentrations were found in the colon, the lowest in the cutis and subcutis. After 1 h a level of 8 mg/kg imipenem was still found in the colon. The concentrations were > 1 mg/kg in all tissues for more than 3 h p. a. and thus within this period exceeded the MICs ofEscherichia coli andBacteriodes fragilis, the indicator organisms of intraabdominal infections.ZusammenfassungZehn Patienten, die sich einer kolorektalen Operation von durchschnittlich drei Stunden Länge unterziehen mußten, erhielten präoperativ je 500 mg Imipenem und Cilastatin i.v. Während der Operation wurden Blut und Gewebeproben für eine Serumpharmakokinetik sowie zur Bestimmung der Konzentrationen von Imipenem in der Kutis, Subkutis, Faszie, im Muskel, im Peritoneum parietale und Dickdarm entnommen. Die Imipenem-Konzentrationen wurden mit der HPLC-Methodik bestimmt. Die durchschnittliche Serumspitzenkonzentration lag bei 26 mg/l, die mittlere Halbwertszeit bei 55 min und die AUC bei 40 mg/l/h−1. Die Serumpharmakokinetik von Imipenem unterlag bei dieser Patientengruppe wesentlich größeren Schwankungen als bei Probanden oder Patienten ohne Operation. Imipenem penetriert schnell in das Gewebe. Nach 10–25 min wurden die höchsten Konzentrationen gemessen. Der Dickdarm weist die höchsten, Kutis und Subkutis die niedrigsten Imipenem-Konzentrationen auf. Im Dickdarm sind nach einer Stunde noch 8 mg/kg Imipenem nachzuweisen. In allen Geweben lagen die Konzentrationen mehr als drei Stunden p. a.=1 mg/kg und damit über diesen Zeitraum höher als die MHK-Werte vonEscherichia coli undBacteroides fragilis, den Leitkeimen bei intraabdominellen Infektionen.

Identification of anaerobic bacteria using high-performance liquid chromatography.

20 species of the genera Clostridium, Bacteroides, Fusobacterium and the type strains of Peptostreptococcus anaerobius, Peptococcus asaccharolyticus, Veillonella parvula and Propionibacterium acnes were examined for the production of volatile (VFA) and nonvolatile (NVFA) short-chain fatty acids using high-performance liquid chromatography (HPLC) with a column for organic acids (Aminex HPX-87H). 10 min are needed for sample preparation and the VFA and NVFA were detected simultaneously in a single chromatographic run. The total time required to run each chromatogram was approximately 60 min. With regard to the production of short-chain fatty acids in culture media it was possible to identify the species of the genera rapidly and clearly. The results illustrate the role of HPLC in determinating short-chain fatty acid products as an additional means for rapid differentiation between closely related anaerobic bacterial species. The aim of the method developed is to establish a complete automatization for the identification of anaerobic bacteria using an automatic sampling system and a microprocessor-controlled chromatography unit.

Studies on the identification of Campylobacter species using biochemical tests and high-performance liquid chromatography.

A total of 56 strains of Campylobacter jejuni (C. jejuni) isolated from diarrhoeal patients were characterized by biochemical tests. The following reactions were performed: hydrolysis of hippurate, reduction of nitrate and nitrite, activity of deoxyribonuclease, hydrolysis of Tween(R) 40, 60 and 80. Including the hydrolysis of the different Tweens(R), 14 biotypes could be distinguished. 19 out of the 56 strains of C. jejuni and the nomenclatural type strains of C. jejuni, C. coli, C. laridis, C. fetus subsp. fetus, subsp. venerealis, C. faecalis, C. sputorum subsp. sputorum, subsp. mucosalis, subsp. bubulus were examined for the production of volatile (VFA) and non-volatile (NVFA) short-chain fatty acids using high performance liquid chromatography (HPLC) with a column for organic acids (Aminex HPX-87 H). A standard mixture of 21 short-chain fatty acids was taken as reference. By this method 5 biotypes of C. jejuni could be characterized. The most frequent biotype was type 1 (78.9%). All the biotypes produced succinic, acetic and butyric acids. Differences existed in the production of pyruvic, malonic, formic and isobutyric acids. C. jejuni could rapidly and clearly be distinguished from C. coli, C. fetus subspp. and catalase-negative Campylobacter species. No qualitative differences were found between the subspecies of C. fetus, C. sputorum subsp. sputorum and subsp. bubulus, C. sputorum subsp. mucosalis and C. faecalis were characterized by presence of fumaric and malonic acid, respectively.

Chlamydia pneumoniae Infection and Restenosis in Patients with Coronary Heart Disease

Abstract.Background: The aim of this study was to establish whether Chlamydia pneumoniae is implicated in the development of restenosis in patients with coronary heart disease (CHD) after percutaneous transluminal coronary angioplasty (PTCA). Patients and Methods: 67 patients were selected for study after they underwent control angiography after PTCA. Sera were tested for anti-chlamydial antibodies with a genusspecific ELISA and a species-specific microimmunofluorescence test (MIFT). Oropharyngeal specimens were examined for the presence of antigen with a Chlamydia immunofluorescence test (IFT), C. pneumoniae IFT and semi-nested PCR. In addition, anamnestic findings were also included. To determine the general level of antibodies, an age- and sexmatched control group of 180 persons was also examined for Chlamydia and C. pneumoniae serology. Results: Coronary angiography revealed that 31 of the 67 patients had developed a restenosis. There was no significant correlation between serological and angiographic findings. However, the MIFT showed a higher positive rate, especially in IgA, in the restenosis group. C. pneumoniae was detected in the oropharynx by PCR and/or IFT in 20.8% and 16.0% of the cases in patients with and without a restenosis. PCR found more C. pneumoniae-positive cases in the restenosis patients than IFT. No association was found between the detection of Chlamydia antigen and serology. The women with restenosis were more frequently smokers (p = 0.012). Men with restenosis were significantly older (p = 0.015). C. pneumoniae serology based on the rELISA or the MIFT did not show any correlation with restenosis. Conclusion: No evidence was found to suggest that positive C. pneumoniae serology is a risk factor for the development of restenosis. However, whether the species-specific serological test, especially for IgA-antibodies, and the detection of C. pneumoniae in oropharyngeal specimens by PCR might be reliable diagnostic markers in these cases remains to be determined.

A Modified Procedure for the Identification of Anaerobic Bacteria by High Performance Liquid Chromatography — Quantitative Analysis of Short-Chain Fatty Acids

We have developed a new rapid method for analysing volatile and non-volatile short-chain fatty acids using high-performance liquid chromatography. Within 50 min, 22 fatty acids in a standard mixture could be detected in a single chromatographic run. The fatty acids released by anaerobic bacteria in the culture media were ether-extracted and analysed with an Aminex HPX-87H column. Using a microprocessor-controlled chromatography unit, a quantitative analysis of the fatty acids produced in bacterial cultures was possible. Resolution, rapidity and sensitivity were improved as compared to previous methods by using an eluent of 5% acetonitrile in 0.01 N H2SO4 and changing the column temperature to 35 degrees C.