Antifungal activity of oils macerates of North Sardinia plants against Candida species isolated from clinical patients with candidiasis.

Antibiotic resistance is one of the bigger problem of public health: the rise of multi-drug resistant organisms causes a challenge in the treatment of infective diseases. Anti-Candida resistance to conventional antifungal agents has increased in the last period. Our research was intended to evaluate antimicrobial activity of oil macerate (OM) of Helichrysum microphyllum Cambess. subsp. tyrrhenicum Bacch., Brullo & Giusso and OM of Hypericum perforatum subsp. angustifolium against several clinical strains. The study included 30 patients with candidiasis who had not received any antifungal treatment before they were enrolled. A collection of 30 clinical isolates belonging to 5 different species of Candida spp. was selected for this study. The data obtained showed an interesting activity of both Oil Macerate especially against C. krusei and C. parapsilosis. On the contrary, H. microphyllum Oil Macerate has had a better activity than the H.perforatum especially in relation to C. glabrata and C. krusei.

Antimicrobial activity of Austroeupatorium inulaefolium (H.B.K.) against intracellular and extracellular organisms.

The antimicrobial activity of Austroeupatorium inulaefolium (H.B.K.) essential oil was studied in different pathogens species and its cytotoxicity activity was determinated on different cellular lines. Despite the good antibacterial activity of A. inulaefolium, it has been cytotoxic at low concentrations. Consequently it might be interesting to determine the antimicrobial activity and cytotoxicity of the major compounds of this essential oil.

Change in Caco-2 cells following treatment with various lavender essential oils.

Lavender is an aromatic evergreen shrub diffused in the Mediterranean basin appreciated since antiquity. The genus Lavandula is part of Lamiaceae family and includes more than 20 species, among which true lavender (L. vera D.C. or L. angustifolia Miller.) and spike lavender (L. latifolia Medikus); there are also numerous hybrids known as lavandins (L. hybrida Rev.). L. vera, spike lavender and several hybrids are the most intensely used breeding species for the production of essential oils. Lavender and lavandin essential oils have been applied in food, pharmaceutical and other agro industries as biological products. In their chemical composition, terpenes linalool and linalyl acetate along with terpenoids such as 1,8-cineole are mostly responsible for biological and therapeutic activities. This study evaluates cytotoxic activity of essential oils derived from four lavender species on human epithelial colorectal adenocarcinoma cells. Analysis of pre- and post-treatment cell morphology has been performed using scanning electron microscope.

Interferon-γ release assay in people infected with immunodeficiency virus.

The present study aimed to use QuantiFERON TB Gold in tube (Cellestis Limited, Carnegie, Victoria, Australia) as a tool for the screening for tubercular infection in HIV-positive patients. Seventy-three HIV-positive subjects were tested. For each individual, QuantiFERON TB in tube was performed. The immunoassay was negative in 53 subjects, positive in eight and indeterminate in 12. The data obtained indicate that factors such as the CD4 cell count and their percentage, as well as the stage of the disease, could affect the performance of the interferon-γ release assay in populations at risk such as HIV-positive subjects.

A molecular method for the recovery and identification of enteric virus in shellfish.

In this paper we report the results of an investigation into the presence of enteric viruses in shellfish from the waters around Sardinia. Twenty two samples of shellfish were examined using a rapid and sensitive technique to concentrate and detect viral RNA in shellfish tissues. After recovery of viral particles, RNA was extracted, transcribed into cDNA and amplified using "nested PCR". Testing with enterovirus-specific RT-PCR produced positive results in over 13% of specimens. The virus detection procedure appears to be effective. In some circumstances it could be a better test of water quality than conventional monitoring techniques.

Strain variation in Mediterranean and Red Sea Mycobacterium marinum isolates.

Four different PCR fingerprinting techniques were tested to distinguish possible strain variations in fourteen Mycobacterium marinum isolates, thirteen from Mediterranean and Red Sea fishes and one from a patient in Sardinia, Italy. PCR ribotyping and ERIC (enterobacterial repetitive consensus sequences)-PCR were found to be non-discriminative, whereas IS (insertion sequences)-PCR and GTG (GTG sequences repeats)-PCR could distinguish the clinical isolate from the piscine isolates, two Italian piscine isolates from all other isolates, but not the Greek isolates from the Israeli isolates. Our results indicate that GTG-PCR and IS-PCR have superior discriminative properties and are thus useful molecular tools for epidemiological studies of M. marinum.

Virulence genes in halophilic Vibrio spp. isolated in common mussels.

Twenty-five Vibrio strains belonging to nine different species, isolated in common mussels, were examined for the presence of different virulence genes: ctxA, tcpA, toxR, toxS, ace, zot and vpi previously found in pathogenic Vibrio cholerae strains. Our results suggest that there is a wide dissemination of Vibrio cholerae virulence genes among the various Vibrio species tested. This finding raises the question of whether a different approach should be taken to study "environmental" Vibrio strains.

Rapid identification of cutaneous infections by nontubercular mycobacteria by polymerase chain reaction-restriction analysis length polymorphism of the hsp65 gene.

BACKGROUND: Nontubercular mycobacteria (NTM) may cause cutaneous infections which are difficult to interpret due to the variability of the clinical manifestations. This study involved eight patients (four men and four women) with primary cutaneous infections caused by NTM; the skin lesions included dermo-hypodermal abscesses, suppurative granulomas, and papulonodules localized on the legs, arms, hands, and face. The general condition of the patients was relatively good and they were not immunosuppressed. METHODS: All samples were processed with standard methods and the isolates were identified by pattern restriction analysis after polymerase chain reaction (PCR-PCA) amplification of the heat shock protein of 65 kDa. RESULTS: In this way, we were able to identify three Mycobacterium chelonae strains, two Mycobacterium marinum, two Mycobacterium fortuitum, and one Mycobacterium avium. The lesions disappeared in 3 or 4 weeks after treatment with two or more antimicrobials. CONCLUSIONS: For a correct diagnosis of cutaneous infection by NTM, demonstrating the presence of mycobacteria is essential; routinely available techniques lack sensitivity and are extremely tedious; often mycobacteria are not seen after acid-fast stain. We used PCR-PCA to identify mycobacteria grown in liquid media; the time of identification of mycobacteria was shortened relative to conventional methods.

Isolation and identification of Mycobacterium neoaurum from a patient with urinary infection.

Mycobacterium neoaurum is a novel species of Mycobacteria, until now only isolated from catheters in immunosuppressed patients. This report describes the isolation and identification of M. neoaurum from urine obtained from a hospitalized patient.

Molecular basis of rifampin and isoniazid resistance in Mycobacterium bovis strains isolated in Sardinia, Italy.

Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoB gene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.

Antibacterial activity of ozonized sunflower oil (Oleozon).

AIMS: To evaluate the antimicrobial effect of the ozonized sunflower oil (Oleozon) on different bacterial species isolated from different sites. METHODS AND RESULTS: The effect of Oleozon on Mycobacteria, staphylococci, streptococci, enterococci, Pseudomonas and Escherichia coli was tested. The sunflower oil was ozonized at the Centro de Investigaciones del Ozone (CENIC, Havana, Cuba) by an ozone generator. MICs were determined by the agar dilution method. For Mycobacteria, the MIC of Oleozon was determined on solid medium by a microdrop agar proportion test. Oleozon showed antimicrobial activity against all strains analysed, with an MIC ranging from 1.18 to 9.5 mg ml-1. CONCLUSION: Oleozon showed a valuable antimicrobial activity against all micro-organisms tested. Results suggest that Mycobacteria are more susceptible to Oleozon than the other bacteria tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The wide availability of sunflower oil makes Oleozon a competitive antimicrobial agent. These results should prompt the setting up of some clinical trials to compare Oleozon with other antimicrobial agents.

Virulence factors in Vibrio alginolyticus strains isolated from aquatic environments.

In a microbiological monitoring carried out in various aquatic environment of Sardinia Island (Italy) Vibrio alginolyticus with different virulence phenotypes appeared widely spread. Hemolysis, hemoagglutination and protease production might be together particularly in strains isolated from polluted environments. Adherence capacities to two epithelial cells (Hep-2 and Caco-2) available in laboratory were widely spread in the examined bacterial strains. The adhesion degree was influenced by the utilized cellular clone. The lack of a correspondence between adhesion capacity and more traditional virulence tests do not permit its replacement at screening level.

Different strategies for molecular differentiation of Mycobacterium bovis strains isolated in Sardinia, Italy.

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed.

Microbiological diagnosis of tuberculosis: a comparison of old and new methods.

The aim of our study was to evaluate the diagnostic value of various methods widely used in microbiological diagnosis of tuberculosis: direct smear examination for acid-fast bacilli, cultural identification in Lowestein-Jensen (L-J) medium, the radiometric BACTEC 460 system, and Polymerase Chain Reaction (PCR). Three hundred and ninety-three clinical samples of sputum (375), gastric aspirate (3), pleural fluid (12) and urine (3) were taken from 125 patients hospitalized at our Institute for suspected pulmonary tuberculosis, between January 1995 and June 1997. On completion of diagnosis, 35 were found to be affected by active tuberculosis (30 pulmonary, 4 pleural and 1 urinary) and 90 by other non-tubercular diseases (pneumonia, lung cancer, non-tubercular pleural effusion, etc.). In our study, direct smear examination for acid-smear bacilli gave diagnostic value results of 88% and positive predictive value of 91.67%. Cultural identification in L-J and BACTEC 460 TB radiometric system media resulted in diagnostic values of 96.80% and 94.40%, respectively, and positive predictive values of 100% for both of them. Finally, One-Tube Nested-PCR, a variant which uses specific primers for the IS6110 insertion sequence specific for Mycobacterium tuberculosis, gave us 88.80% (91.43% sensitivity and 87.78% specificity) diagnostic value results, and 74.42% (11 false-positives) positive predictive value. On the basis of our results, we can affirm that PCR is a good method for microbiological diagnosis of tuberculosis, given its high sensitivity and specificity and unparalleled rapidity. However, the high number of false-positives that we found suggests that results obtained should be confirmed with BACTEC, which considerably reduces the time required for identification, and makes it possible to carry out an antibiotic assay rapidly.

Evaluation of a nonradiometric system (BACTEC 9000 MB) for detection of mycobacteria in human clinical samples.

This study was carried out to evaluate the rate of recovery and time required for detection of mycobacteria from pulmonary and extrapulmonary human clinical samples, by using a fluorescence-quenching-based oxygen sensor (BACTEC 9000 MB; Becton Dickinson Microbiology Systems, Sparks, Md.). The results were compared with those obtained by microscopy, conventional culture in Lowenstein-Jensen (LJ) medium, and a BACTEC radiometric system (BACTEC 460 TB; Becton Dickinson). Of the 779 clinical samples processed, 364 from pulmonary sites and 415 from extrapulmonary sites, 62 (7.9%) were positive for mycobacterial isolates; of the positive samples, 59 (95.1%) were detected with the fluorescent BACTEC 9000 MB system, 57 (91.9%) were detected with the radiometric system (BACTEC 460 TB), and 43 (69.3%) were detected with LJ conventional culture. The mean times to detection of all mycobacteria with BACTEC 9000 MB and BACTEC 460 TB were similar (10.3 and 10.0 days, respectively). The results obtained indicate that the nonradiometric BACTEC (BACTEC 9000 MB) system is as efficient as Bactec 460 TB and significantly more efficient than LJ for the rapid recovery of mycobacteria from both pulmonary and extrapulmonary clinical specimens. Though the BACTEC 9000 MB system is recommended for respiratory specimens, we demonstrated that it can be successfully used also for recovery of mycobacteria from clinical specimens from various extrapulmonary sites.