Purification and characterisation of two GST's forms from Rhizobium leguminosarum with a high affinity to herbicides.

Cytosolic glutathione transferases are a family of multifunctional proteins that catalyse the conjugation of GSH to a large variety of endogenous and exogenous compounds. These enzymes have been widely studied in mammals and, to a lesser extent, in plants. In plants, GSTs can detoxify herbicides; they are also induced by pathogenic infection and are likely to be involved in defence responses. GSTs are found in pathogenic and not pathogenic prokaryotes but the functional role played by these enzymes in the cell still remains to be clarified. Here we report the purification and characterisation of two GST forms from Rhizobium leguminosarum that play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes. These bacterial GSTs from R. leguminosarum have immunological characteristics that are different among them and they are characterised both by a high affinity to herbicides.

Morphological and biochemical variations of Haemophilus influenzae type b induced by pH and temperature changes.

Haemophilus influenzae type b ATCC 10211 was cultured at different temperatures (25 degrees C-49 degrees C) and pH values (5.7-8.7) either in liquid or semisolid medium. Morphological variations of individual cells were noted by optical microscopy depending upon the conditions of growth. At higher temperatures filaments were produced whereby the length of individual cells increased compared to cultures grown at 37 degrees C. Filaments were also observed at lower pH values. Culture conditions also affected colonial morphology. At low pH values colonies had an enhanced lobulated contour and were more wrinkly and rougher than at higher pH. The changes in cellular and colonial morphology were correlated with distinct outer membrane protein profiles. The changes in temperature and pH did not affect identification of the microorganism by the API system.

Evidence for an oral-faecal transmission of Helicobacter pylori infection in an experimental murine model.

An experimental murine model was used to evaluate the possible animal-to-animal transmission of Helicobacter pylori and the mechanism involved. Twenty-four Balb/C mice were infected with H. pylori type I strain culture and kept with 24 noninoculated mice to evaluate the possible transmission of the microorganism. Twelve inoculated mice were housed with 12 noninoculated mice in a grated cage (supporting an oral-oral transmission); the remaining inoculated and noninoculated mice were housed in another cage without grating on the floor (supporting a faecal-oral transmission). The bacterial colonization was assessed by culture and immunohistochemistry. The systemic antibody response to H. pylori and the histopathological changes were evaluated; controls were examined at 2, 4, 8, 12 weeks after the start of the experiment. Faecal samples were also collected from each mouse on the day before sacrifice, to assess the presence of H. pylori by culture and by immunohistochemistry. In the gastric mucosa of inoculated mice, histopathological changes were recorded at each control time and H. pylori was detected both by immunohistochemistry and by a systemic antibody response; the microorganism was also cultured at 2, 4, 8 weeks postinoculation. H. pylori was detected in noninoculated mice, housed in the cage without grating, using an immunoperoxidase technique at 2, 4, 8 weeks after starting the experiment, and these positive values were supported by histopathological changes, and, in one case, at 8 weeks, also by the serum immune response. No colonies of H. pylori were detected by culturing faecal samples from either noninoculated or inoculated mice. The results obtained in this study seem to support an oral-faecal route as the mode of transmission of H. pylori infection in this animal model.

The effect of oxygen on the growth and cell morphology of Helicobacter pylori.

The in vitro effect of progressive oxygen decrease on the growth and morphology of Helicobacter pylori was studied. H. pylori ATCC 43,504 was used for the experiments. The strain inoculated in Brucella broth plus fetal calf serum was incubated under a controlled atmosphere with oxygen concentration from 5 to 0%. CFU ml-1 and bacterial morphology were detected at the time of spreading and at 24 h, 72 h, 7 days and 14 days. A detailed ultrastructural investigation of the bacterial cells, grown in different experimental conditions, was performed by scanning electron microscopy. Oxygen deprivation produced a rapid reduction of CFU ml-1. In particular, a significant reduction of viable bacteria was recorded at 72 h of incubation in the presence of 1% oxygen and anaerobiosis, and 0 CFU ml-1 was found after 7 days of incubation at the above mentioned oxygen concentrations. The coccoid phenotype was already prevalent after 24 h of incubation with a progressive tendency to aggregate in clusters. These clusters were progressively larger, depending on the reduction of oxygen concentration, since the aggregation phenomenon can be the expression of a hypothesized mechanism of protection among bacterial cells.

Recovery of Helicobacter pylori ATCC43504 from a viable but not culturable state: regrowth or resuscitation?

Studies were conducted following the formation and characterization of the coccoid morphology of Helicobacter pylori. H. pylori ATCC43504 was incubated in brucella broth plus 2% fetal calf serum at three different temperatures: 37 degrees C, room temperature and 4 degrees C in a microaerophilic environment, and readings were taken at 2, 7, 15, 30 and 45 days. At control times, the total and the viable count, viability tests with tetrazolium salts, and ultrastructural studies were carried out. On solid media, H. pylori became nonculturable after 7 days of incubation at room temperature and 4 degrees C, and after 15 days of incubation at 37 degrees C. At these times of incubation, after subculturing in liquid medium under the same conditions, the growth of H. pylori was detected until the 15th day from cultures incubated at 4 degrees C and until the 30th day from cultures stored at 37 degrees C, and at room temperature. Ultrastructural studies showed a gradual reduction of integrity of bacterial cells that remained stable at 30 and 45 days of incubation: 30% of whole cells of bacteria incubated at 37 degrees C and room temperature and 50% in bacteria incubated at 4 degrees C. The viability of the VNC (viable nonculturable) state was assessed by studying the reduction of tetrazolium salts INT (p-iodonitrophenyl tetrazolium violet) and CTC (cyanoditolyl tetrazolium chloride) to their respective formazans and this was linked to the cellular respiration. At 45 days of incubation, when bacterial regrowth was not observed in solid or in liquid medium, different resuscitation methods were applied to evaluate a possible resuscitation of VNC H. pylori. No significant growth on solid medium was observed.

Helicobacter pylori isolated from stomach corpus and antrum: comparison of DNA patterns.

The genomic DNA of Helicobacter pylori was studied in strains isolated from two different sites of the stomach: the corpus and the antrum. 70 strains of H. pylori were found in 36 patients; 34 out of the 36 patients harboured the strain in both districts analysed. Restriction endonuclease analysis with Hae III and Hind III was used to compare the DNA patterns of strains isolated from the anatomical sites studied. Two pairs of DNA samples were not digested by these enzymes. 27 of the 32 pairs of the digested DNA appeared similar to each other. The analysis of chromosomal DNA in the remaining five pairs showed different electrophoretic patterns. These results indicate that the gastric mucosa can be colonized, at the same time, by strains of H. pylori with different genomic patterns, and this aspect can be important for epidemiological studies.

Comparative in vitro activity of cefodizime and other antibiotics against pathogens recently isolated in Italy.

In the present study we tested the susceptibility to cefodizime on 1,985 selected nosocomial pathogens isolated in five laboratories. Moreover, we evaluated the epidemiology of the resistance of the tested strains to cefodizime and to other antibiotics clinically available in Italy. The susceptibility to cefodizime was determined with both MIC (microdilution method) and the agar diffusion method (Kirby-Bauer). The Kirby-Bauer method was used to compare the antibiotics. Cefodizime was equivalent in activity to ceftazidime and ceftriaxone and was more active than piperacillin and amoxicillin + clavulanic acid. The activity of gentamicin (where tested) was generally comparable to that of cefodizime; ciprofloxacin had lower percentages of resistance against some species of Enterobacteriaceae and staphylococci.

Failure to detect Helicobacter pylori in nasal mucus in H pylori positive dyspeptic patients.

Stomach biopsies and samples of nasal mucus were cultured in patients with dyspeptic symptoms who underwent endoscopy to evaluate the possible route of transmission of Helicobacter pylori (H pylori). 42 patients were examined. For each patient two biopsies from the stomach corpus and antrum were taken and, before endoscopy, one nasal swab was obtained. Biopsy samples were tested for urease test, microbiological culture, and histological examination. The nasal swab was processed for microbiological examination. H pylori was not found in the nasal mucus of any of the patients, including the 36 who had H pylori in gastric biopsies.

Microbiological evidence of Helicobacter pylori from dental plaque in dyspeptic patients.

To evaluate the possible route of transmission of Helicobacter pylori, stomach biopsies and dental plaques were cultured from patients with dyspeptic symptoms who underwent endoscopy. A total of 31 patients were examined. Twenty patients out of thirty one (64%) were H. pylori positive in gastric biopsy. Among the microorganisms isolated in dental plaque only one sample (corresponding to a patients with duodenal ulcer H. pylori positive) showed colonies morphologically and biochemically compatible with H. pylori. Proteic patterns of whole cells and restriction endonuclease analysis with Hind III and Hae III endonucleases of DNA extracted from the strain subcultured from a stomach biopsy and from dental plaque of the same patient indicated that both sites were infected with the same strain of H. pylori.

Differentiation of Clostridium difficile, Clostridium bifermentans, Clostridium sordellii, and Clostridium perfringens from diarrheal stool by API ZYM and API LRA oxidase test.

A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed. It consists of a combination of API ZYM and API LRA Oxidase tests. The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar). Fifty-five strains of Clostridium difficile, C. bifermentans, C. sordellii, and C. perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed. The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.

Incidence of lower respiratory tract infections caused by Mycoplasma, Chlamydia and Legionella: an Italian Multicenter Survey.

A collaborative retrospective study based on serologic diagnosis was conducted to assess the etiological role sustained by privileged pathogens in Italy. The results obtained indicate the Mycoplasma, Chlamydia and Legionella are important etiologic agents of lower respiratory tract infections in Italy since they account for about 31% of the cases taken into consideration in this survey. We found a high incidence of M. pneumoniae (12.3%), C. pneumoniae (10.5%) and L. pneumophila (8.3%). These results are in line with similar figures reported in the recent literature. While the data gathered in our survey do not allow us to clarify the nature of the agents involved in the etiology of the majority (70%) of the respiratory infections occurring in Italy, it seems safe to assume that after Streptococcus pneumoniae and Haemophilus influenzae, the privileged pathogens represent the most common cause of lower respiratory tract infections.

[Microbiological aspects of antibiotics with immunomodulating action]. / Aspetti microbiologici degli antibiotici ad azione immunomodulante.

Through the introduction of a 7-mercapto-1,3-thiazole chain at position 3' of the dihydrothiazine ring, cefodizime, which is structurally similar to cefotaxime, has acquired a number of remarkable immunomodulatory properties while retaining a potent antimicrobial spectrum of activity. Cefodizime penetrates in fact readily through the bacterial cell wall and interacts with its molecular targets in such a way that at high concentrations cell death and lysis are rapidly induced. Its spectrum of action encompasses the Enterobacteria, Neisseriae, Haemophilus, Moraxella catarrhalis, methicillin-susceptible staphylococci and streptococci, with pneumococci included. Cefodizime is devoid of useful potency against Pseudomonas, Acinetobacter and enterococci. Given the wide occurrence of strains synthesizing beta-lactamases in several primary pathogens of community-acquired and nosocomial infections, the complete stability of cefodizime towards the most prevalent of these hydrolytic enzymes (TEM-1, TEM-2, SHV-1, BRO-1 and the staphylococcal penicillinases) seems reassuring. Only a few chromosomally-coded and extended spectrum beta-lactamases produced by gram-negative microorganisms inactivate the new cephalosporin. Since the distribution of pathogens carrying these enzymes depends on the local trends of antibacterial consumption and cannot be easily predicted, a large multicenter study in Italy has recently assessed the antibacterial potency of cefodizime, in comparison with suitable drugs, on 1985 selected nosocomial strains. In this survey cefodizime was more effective in vitro than amoxicillin-clavulanate, gentamicin and piperacillin while being substantially similar in the rates of eradication of gram-negative and gram-positive organisms to other third generation cephalosporins like ceftazidime and ceftriaxone.(ABSTRACT TRUNCATED AT 250 WORDS)

Helicobacter pylori: a fickle germ.

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.

Coccoid Helicobacter pylori not culturable in vitro reverts in mice.

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the "dormant" forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD550 = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5 x 10(9) CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.

Twelve aberrant strains of Staphylococcus aureus subsp. aureus from clinical specimens.

A new biovar of Staphylococcus aureus subsp. aureus was isolated from human clinical specimens and described on the basis of studies of 12 isolates that were compared with 11 standard reference strains. Both DNA hybridization experiments and numerical taxonomy analysis demonstrated that these strains were strictly related to S. aureus subsp. aureus; however, they were significantly different from the latter. The atypical strains belonging to the new biovar can be distinguished from typical S. aureus subsp. aureus strains by their alpha-chymotrypsin, alpha-glucosidase, beta-N-acetylglucosaminidase, lipase (C-14), and leucine arylamidase enzymatic activities and novobiocin resistance. Thus, the combination of alpha-glucosidase and beta-N-acetyl-glucosaminidase is more useful for distinguishing these S. aureus strains from the other, typical ones.

Enterococcus flavescens sp. nov., a new species of enterococci of clinical origin.

Four yellow-pigmented group D enterococci of uncertain taxonomic position were isolated from several humans with severe infections. The results of DNA composition, DNA-DNA hybridization, fatty acid content, and biochemical property studies demonstrated that these organisms were slightly related to other previously described yellow-pigmented enterococcal species and constitute a new species, for which we propose the name Enterococcus flavescens. The type strain of E. flavescens is strain CCM 4239 [corrected].

New plate medium for growth and detection of urease activity of Helicobacter pylori.

A new medium for detection of urease activity and isolation of Helicobacter pylori is proposed. This medium, containing Columbia Agar Base, was supplemented with IsoVitaleX, hemin, urea, and phenol red (nonselective medium [NSM]). Both bacterial growth and color change were evaluated and compared with growth in the same medium supplemented with cefsulodin, vancomycin, polymyxin B sulfate, and amphotericin B (selective medium [SM]). Twenty-five recent clinical isolates and antral biopsy specimens from 33 patients who underwent endoscopy were examined. The isolates showed a rapid color change and good growth at 5 days of incubation with NSM and SM. H. pylori-positive biopsies revealed a color change within 36 h, and bacterial growth was better appreciated in NSM, but with more contaminating flora than in SM.

Double sugar-tyrosine medium improves O-1 phage Salmonella screening.

A modification of the procedure for O-1 phage Salmonella screening is presented. The novel method is based on the use of two media, i.e., a new medium (double sugar-tyrosine [DST]), which permits the combination of adonitol and sucrose fermentation and tyrosine clearing tests, and the previously described o-nitrophenyl-beta-D-galactopyranoside urease indole medium. In comparative trials, the new procedure and the conventional one were used to screen for Salmonella isolates from 553 lactose-negative strains of members of the family Enterobacteriaceae. The O-1 phage test, performed on DST medium, recognized the same number of phage-susceptible Salmonella strains as did the standardized method; however, it permitted the correct identification of a greater number of phage-resistant strains for discard (95.6 versus 85.3%). In particular, DST medium presented a higher efficacy than triple sugar iron agar (which is the corresponding medium in the reference procedure) in correctly identifying phage-negative cultures for discard (69.1 versus 28.5%).

Enhanced clearing of Helicobacter pylori after omeprazole plus roxithromycin treatment.

The in vitro antibacterial activity of omeprazole against eight strains of Helicobacter pylori was evaluated. Minimum inhibitory concentration (MIC) values were 32 micrograms/ml and 64 micrograms/ml (MIC50 and MIC90 respectively). We performed a randomized single blind study comparing the efficacy of omeprazole alone (for 4 weeks) or combined with roxithromycin (for 2 weeks) in the treatment of duodenal ulcer and chronic active gastritis associated with H. pylori infection, H. pylori was eradicated in 75% of patients treated with omeprazole alone whereas the patients treated with the combination of these drugs were completely free from H. pylori at the end of the therapy.

T-mod pathway, a reduced sequence for identification of gram-negative urinary tract pathogens.

In this paper, we describe a reduced sequence of identification that includes T-mod medium, a selective and differential isolation medium which allows accurate presumptive identification of the most common gram-negative bacteria encountered in urine samples. The present study, performed on bacteria isolated from 1,762 independent urine samples, has shown that a few selected tests (lysine and ornithine decarboxylase, urease and trehalose fermentation tests) improve the identification accuracy of T-mod, making it possible both to identify the less frequent species and to prevent some misidentifications of Klebsiella pneumoniae and Proteus mirabilis. The proposed work flow agreed with conventional identification protocols to a 99.3% extent and allowed identification of 87.4% of the isolates directly from the primary plate, 11.4% after 1 to 3 additional tests, and 1.2% after an identification gallery.