Distribution of a Specific 500-Base-Pair Fragment in Mycobacterium bovis Isolates from Sardinian Cattle

Amplification of a specific, 500-bp fragment from Mycobacterium bovis isolates and use of the fragment to differentiate between Mycobacterium tuberculosis and M. bovis was previously reported (J. G. Rodriguez, G. A. Meja, P. Del Portillo, M. E. Patarroyo, and L. A. Murillo, Microbiology 141:2131–2138, 1995). In the present study, 30 M. bovis isolates from Sardinian cattle were examined for the presence of this 500-bp fragment; 4 of the 30 isolates lacked the fragment. This result indicates that identification of M. bovis strains by amplification of the 500-bp sequence may lead to false-negative results

Simple and rapid identification of different species of Mycobacteria by PCR

A simple polymerase chain reaction (PCR) assay for rapid identification of different species of mycobacteria was developed. This PCR is based on the use of conserved sequences to amplify the genome of several mycobacterial species. The amplification patterns obtained were specific and reproducible for the species tested. In particular, we could identify Mycobacterium tuberculosis and Mycobacterium bovis (both produced the same pattern), Mycobacterium avium, Mycobacterium kansasii, Mycobacterium xenopi, Mycobacterium chelonae, Mycobacterium peregrinum, Mycobacterium fortuitum, Mycobacterium gordonae and Mycobacterium smegmatis. Moreover, due to the numerous copies of the target sequences present in the genome, the PCR showed a very high level of sensitivity

Distribution of <i>Vibrio cholerae</i> virulence genes among different <i>Vibrio</i> species isolated in Sardinia, Italy

The members of the genus Vibrio include harmless aquatic strains as well as strains capable of causing epidemics of cholera. Diarrhoea caused by Vibrio cholerae is attributed to cholerae enterotoxin (CT) codified by the ctx operon and regulated by a number of virulence genes such as toxT, toxR and toxS. Fifty-two Vibrio strains were isolated from different aquatic environments in and around Sardinia and searched by PCR for the presence of ctxA, zot, ace, toxR, toxS, toxT, tcpA and vpi virulence genes in the genomes of the isolates. The toxR operon was found in 27 Vibrio alginolyticus strains out of 42 analysed, in three out of four V. cholerae non-O1 strains and in three Vibrio parahaemolyticus isolates. A positive amplification for the virulence pathogenic island (vpi) was produced by five V. alginolyticus strains. Finally, the ace expected amplification fragment was found in two V. alginolyticus isolates whereas the amplification with zot primers produced the expected fragment in one V. alginolyticus isolate. Differentiation of these strains with a PCR fingerprinting technique revealed no association between the presence of virulence genes and a particular fingerprinting pattern. Although most Vibrio species are considered non-pathogenic or only potentially harmful to humans, the finding of V. cholerae virulence genes in other members of the genus Vibrio, and the recent reports of the creation and evolution of pandemic strains of V. cholerae, may give a new perspective to the significance of these results

Incidence of virulence determinants in clinical <i>Enterococcus faecium</i> and <i>Enterococcus faecalis</i> isolates collected in Sardinia (Italy)

Enterococci are widely distributed in the environment; within the human body, they are normal commensals of the oral cavity, gastrointestinal tract and vagina. In recent years, enterococci have become one of the most frequent causes of acquired nosocomial infections worldwide. The molecular mechanism of virulence of these bacteria is still not completely understood. The aims of this work were to characterize phenotypically 47 isolates of Enterococcus faecalis and Enterococcus faecium collected in Sardinia (Italy) by their abilities to adhere to different epithelial cell lines (Vero and Caco-2 cells) and to associate their phenotypes with the presence of known virulence genes detected within their genomes by PCR. The following genes were amplified: AS (aggregation substance), esp (surface protein gene), ace (accessory colonization factor), efaA (E. faecalis endocarditis antigen) and gelE (gelatinase). The virulence genes were detected in E. faecalis isolates only, with the exception of esp, which was found in both species. The phenotypic and genotypic results were also compared with the susceptibility of isolates to various antibiotics

Virulence factors of <i>Enterococcus faecium</i> and <i>Enterococcus faecalis</i>

Enterococcus spp. are widely diffuse in the environment, within the human body they are normal commensal of the oral cavity, gastrointestinal tract and vagina, such enviroments are complex, feeding-rich and oxigen-poor. Enterococci became one of the most frequent cause of acquired nosocomial infections world-wide (they are responsible about 16% of nosocomial infections). The molecular mechanism of virulence of these bacteria is still not completely understood. The aim of this work was to characterise phenotypically different strains of E. faecalis and E. faecium isolated in Sardinia (Italy) by their property to adhere to different epithelial cell lines (Vero and Caco2 cells) and to associate their phenotype to the presence of known virulence genes which have been searched within their genomes by PCR. Amplification of the following genes was perforrned: The aggregation substance AS, the surface protein gene esp, the accessory colonisation factor ace, the E. faecalis endocarditis antigen efaA and gelatinase gel-E

Strain variation in Mediterranean and Red Sea <i>Mycobacterium marinum</i> 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

Differentiation of <i>Vibrio alginolyticus</i> strains isolated from Sardinian waters by ribotyping and a new rapid PCR fingerprinting method

We investigated the usefulness of a novel PCR fingerprinting technique, based on the specific amplification of genomic regions, to differentiate 30 Vibrio alginolyticus strains isolated in Sardinian waters. The different profiles obtained were scanned and analyzed by a computer program in order to determine genetic relationships. The results were then compared with the patterns obtained by ribotyping with HindIII, KpnI, and XbaI restriction enzymes. PCR fingerprinting could differentiate the strains analyzed into 12 different patterns, whereas ribotyping with XbaI, which produced the highest number of patterns, generated only 7 different profiles. This study revealed the superior discriminative power of the proposed technique for the differentiation of related V. alginolyticus strains and the potential use of PCR fingerprinting in epidemiological studies

Genotypic changes in DNA fingerprinting patterns of <i>Mycobacterium tuberculosis</i> strains from HIV-positive persons in Sardinia

Amongst infectious diseases, tuberculosis is still the major killer in the world. In some developing countries tuberculosis infection has reached epidemic proportions and is increasing worldwide. This rapid growth is thought to be correlated in part to the movement of people from developing countries, where tuberculosis largely occurs, to industrialized countries. It is also due in part to AIDS, as Mycobacterium tuberculosis infection appears to be one of the first indications of a loss of immune system function, and those infected are susceptible to a number of opportunistic infections, including infections by other mycobacteria, and finally because the expectation of life is increased. In the present study, we applied a computer-assisted method to analyse DNA fingerprints and construct dendrograms of M. tuberculosis strains isolated from HIV-positive patients

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

Characterization of new insertion-like sequences of <i>Enterococcus hirae</i> and their dissemination among clinical <i>Enterococcus faecium</i> isolates

Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci. We tested several enterococcal ATCC strains and found that only E. hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence. Moreover, we wanted to investigate the dissemination of this new IS among E. faecium strains. We analyzed 131 clinical E. faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates. The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species