Outbreak of mastitis in sheep caused by multi-drug resistant Enterococcus faecalis in Sardinia, Italy.

An outbreak of infective mastitis due to Enterococcus faecalis occurred in an intensive sheep farm in north Sardinia (Italy). E. faecalis, which is only rarely isolated from sheep milk, was unexpectedly found in 22·3% of positive samples at microbiological examination. Forty-five out of the 48 E. faecalis isolates showed the same multi-drug resistance pattern (cloxacillin, streptomycin, kanamycin, clindamycin, oxytetracycline). E. faecalis isolates were analysed by pulsed-field gel electrophoresis, and all 45 multi-drug resistant strains showed an indistinguishable macrorestiction profile, indicating their clonal origin. To our knowledge, this is the first report of an outbreak of mastitis in sheep caused by E. faecalis.

Trichomonas vaginalis degrades nitric oxide and expresses a flavorubredoxin-like protein: a new pathogenic mechanism?

Besides possessing many physiological roles, nitric oxide (NO) produced by the immune system in infectious diseases has antimicrobial effects. Trichomoniasis, the most widespread non-viral sexually transmitted disease caused by the microaerophilic protist Trichomonas vaginalis, often evolves into a chronic infection, with the parasite able to survive in the microaerobic, NO-enriched vaginal environment. We relate this property to the finding that T. vaginalis degrades NO under anaerobic conditions, as assessed amperometrically. This activity, which is maximal (133 +/- 41 nmol NO/10(8) cells per minute at 20 degrees C) at low NO concentrations (< or = 1.2 microM), was found to be: (i) NADH dependent, (ii) cyanide insensitive and (iii) inhibited by O(2). These features are consistent with those of the Escherichia coli A-type flavoprotein (ATF), recently discovered to be endowed with NO reductase activity. Using antibodies against the ATF from E. coli, a protein band was immunodetected in the parasite grown in a standard medium. If confirmed, the expression of an ATF in eukaryotes suggests that the genes coding for ATFs were transferred during evolution from anaerobic Prokarya to pathogenic protists, to increase their fitness for the microaerobic, parasitic life style. Thus the demonstration of an ATF in T. vaginalis would appear relevant to both pathology and evolutionary biology. Interestingly, genomic analysis has recently demonstrated that Giardia intestinalis and other pathogenic protists have genes coding for ATFs.

Molecular identification of pathogenicity genes and ERIC types in Vibrio cholerae O1 epidemic strains from Mozambique.

The phenotypic and genotypic profiles of the V. cholerae strains causing the Mozambican 1997-8 epidemic were characterized to provide a reference for comparison with other epidemic strains. A total of 75 strains of V. cholerae O1 isolated in different provinces, were analysed. Strains were characterized by PCR for detecting toxin genes (ctxA, zot and ace), virulence associated genes (tcpA. nanH, hlyA and torR) and ERIC sequences. All V. cholerae strains were serotype O1, Ogawa, biotype El Tor. MIC testing showed a high proportion of strains multi-resistant to drugs (100% to cotrimoxazole and 52% to tetracycline) and susceptibility to ciprofloxacin. The isolates contained two intact copies of the CTX genetic element and all other genes tested. PCR of restricted DNA revealed two ERIC types: the first in provincial isolates, also predominant in other African epidemic strains, and the second in Maputo isolates (the national capital).

Mycoplasma hominis and Trichomonas vaginalis symbiosis: multiplicity of infection and transmissibility of M. hominis to human cells.

We recently reported that most Trichomonas vaginalis isolates cultured in vitro are infected by Mycoplasma hominis. In this work, we have characterized some aspects of the relationships between the two microorganisms. PCR, cultivation, and immunological methods revealed that the number of M. hominis organisms carried by T. vaginalis in culture varied from isolate to isolate, suggesting a specific multiplicity of infection. Moreover, infected T. vaginalis isolates were able to pass bacteria not only to M. hominis-free protozoa, but also to human-derived epithelial cells. The in vitro transmission of the bacterium from T. vaginalis to both uninfected parasite isolates and human epithelial cells suggests a role for T. vaginalis as a carrier of the M. hominis infection in vivo.

Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli.

Diarrheagenic E. coli comprise a diverse group of microorganisms responsible for gastrointestinal diseases in humans. On the basis of their virulence traits they are distinguished from the non-pathogenic E. coli and classified in several categories. Molecular methods represent the most reliable techniques for distinguishing pathogenic from non-pathogenic E. coli and characterising their pathogenic features. In this paper we report the development of a set of three multiplex PCR assays for the simultaneous and rapid identification of diarrheagenic E. coli belonging to ETEC, EPEC, EHEC and EIEC groups. Assay 1 utilizes primer pairs specific for genes coding for ST and LT toxins of ETEC, and for the E. coli beta-glucuronidase (uidA); assay 2 detects the presence of the eae and bfpA genes of EPEC, and assay 3 recognizes stx1 and stx2 of EHEC, and ial of EIEC. This technique has been validated on 190 E. coli isolated in Angola, Italy and Mozambique from feces of children with diarrhea. Results obtained with the set of multiplex PCR demonstrated 100% accordance with those obtained for the same isolates by PCR on single target genes. The proposed set of multiplex PCRs is the first reported assay that allows the simultaneous characterization of the four categories of diarrheagenic E. coli.

A pre-existing infection by Mycobacterium avium subsp. avium modulates anti-Cryptococcus neoformans and anti-Candida albicans activities in human macrophages.

Mycobacterium avium is a facultative intracellular microorganism, able to survive and multiply within mammalian macrophages by circumventing antimicrobial mechanisms. In this study we hypothesize that pre-existing M. avium infection could result in macrophage superinfections by other microorganisms. We found that 24 h after ingestion of M. avium at a low multiplicity of infection, macrophages are unable to efficiently produce superoxide anions when over-stimulated with phorbol esters, and that the generation of oxidative burst is only partially restored 72 h after bacteria ingestion. We also demonstrate that intracellular killing of Cryptococcus neoformans is markedly impaired in human macrophages that have previously ingested M. avium (but not other bacteria such as Escherichia coli). This inhibitory effect is observed with live mycobacteria, but not when heat-inactivated bacteria are ingested. In contrast, when Candida albicans is given to macrophages instead of C. neoformans, an enhancement of intracellular killing is observed, suggesting that cytocidal mechanisms other than respiratory burst are involved in the anti- Candidacidal activity of macrophages.

Host and tissue specificity of Trichomonas vaginalis is not mediated by its known adhesion proteins.

Adhesion of Trichomonas vaginalis is believed to be dependent on four adhesion proteins, which are thought to bind to vaginal epithelial cells in a specific manner with a ligand-receptor type of interaction. However, the specific receptors on the host cell have not yet been identified. In this work, the ability of the T. vaginalis adhesins to bind to cells of different histologic derivations and from different species has been studied. HeLa, CHO, and Vero cell lines; erythrocytes from different species; and a prokaryote without a cell wall, Mycoplasma hominis, were employed in order to investigate the cell specificity of the T. vaginalis adhesins. We observed that the T. vaginalis adhesins are able to bind to the different cell types to the same extent, suggesting that the host and tissue specificity of T. vaginalis adhesion should not be due to specificity of the parasite adhesins. Our results suggest that the data published to date on the subject are probably artifactual and that the experiments reported in the literature are not appropriate for identification of protozoan adhesins.

Brucella abortus infection acquired in microbiology laboratories.

We report an outbreak of laboratory-acquired Brucella abortus infection originating in the accidental breakage of a centrifuge tube. A total of 12 laboratory workers were infected (attack rate of 31%), with an incubation time ranging from 6 weeks to 5 months. Antibody titers were evaluated weekly in all personnel exposed, allowing the diagnosis of the infection in most cases before the onset of clinical symptoms, so that specific therapy could be administrated.

The flagellated parasite Trichomonas vaginalis: new insights into cytopathogenicity mechanisms.

Our knowledge concerning cytopathogenicity of Trichomonas vaginalis has been enriched in the past by numerous findings. In this paper, we review the latest advances in the field and discuss the different mechanisms and molecules responsible for the parasite's virulence.

Identification of Trichomonas vaginalis alpha-actinin as the most common immunogen recognized by sera of women exposed to the parasite.

A study on presence of antibodies to Trichomonis vaginalis in serum was done on a group of 500 pregnant, asymptomatic Angolan women. A serologic screening, done by ELISA, revealed that 41% of the women had IgG and IgM against the parasite. Analysis of sera by immunoblotting revealed that 94.4% of sera with anti-T. vaginalis IgG class antibodies were reactive against a common immunogenic protein of 115 kDa. The common immunogen was identified as the protozoan alpha-actinin. All sera recognizing the 115-kDa antigen were reactive against both native and recombinant T. vaginalis alpha-actinin and nonreactive against human alpha-actinin. The findings presented in this work offer a new tool for epidemiologic studies and open new perspectives for vaccination.

Development of a nested PCR for detection of Cryptococcus neoformans in cerebrospinal fluid.

We report the development of a nested-PCR-based assay for the detection of Cryptococcus neoformans in cerebrospinal fluid. The specificity and sensitivity of the test were assessed. The technique was then applied to 40 cerebrospinal fluid samples. We obtained positive reactions for all 21 clinical samples from patients who had been previously diagnosed as having cryptococcal meningitis by conventional techniques and negative reactions for all 19 negative controls. Nested PCR is here compared with other diagnostic methods currently used in patients' follow-up exams during anticryptococcal therapy.

Cloning and molecular characterization of a cDNA clone coding for Trichomonas vaginalis alpha-actinin and intracellular localization of the protein.

We have identified and sequenced a cDNA clone coding for Trichomonas vaginalis alpha-actinin. Analysis of the obtained sequence revealed that the 2,857-nucleotide-long cDNA contained an open reading frame encoding 849 amino acids which showed consistent homology with alpha-actinins of different species. Such homology was particularly significant in regions which have been reported to represent the actin-binding and Ca2+-binding domains in other alpha-actinins. The deduced protein was also characterized by the presence of a divergent central region thought to play a role in its high immunogenicity. A study of protein localization performed by immunofluorescence revealed that the protein is diffusely distributed throughout the T. vaginalis cytoplasm when the cell is pear shaped. When parasites adhere and transform into the amoeboid morphology, the protein is located only in areas close to the cytoplasmic membrane and colocalizes with actin. Concomitantly with transformation into the amoeboid morphology, alpha-actinin mRNA expression is upregulated.

Properties of recombinant bacteriophage T4 tail sheath protein and its deletion fragments.

A vector for expression of recombinant bacteriophage T4 tail sheath protein (gp18) under control of phage T7 promoter in Escherichia coli cells has been constructed. The entire length recombinant gp18 (659 amino acids) polymerizes in vivo into extended polysheaths. To study gp18 folding mechanisms, six vectors for expression of deletion mutants have been constructed. Three proteins--1N, 2N, and 3N--contain, respectively, 268, 316, and 372 amino acids of the gp18 N-tail region. The other three fragments--1C, 2C, and 3C--contain, respectively, 455, 356, and 288 amino acids of the gp18 C-tail. The fragments 1N, 2N, 1C, 2C, and 3C form insoluble aggregates during expression. However, fragment 3N accumulates in soluble form in the cellular cytoplasm and does not form polymeric structures; this has allowed an effective purification method to be developed for it. The interaction of monoclonal antibodies against recombinant gp18 with protein fragments and with phage sheath before and after contraction has been studied. The fragment 3N seems to be a stable domain of native phage sheath gp18.

Contact-dependent disruption of the host cell membrane skeleton induced by Trichomonas vaginalis.

This report presents evidence showing that the pathogenetic process of the protozoan parasite Trichomonas vaginalis involves degradation of the target cell membrane skeleton; spectrin, the most representative protein within this structure, has been identified as the main molecular target. Degradation of the target cell spectrin is accomplished only upon contact with the parasite, and immunochemical and immunofluorescence studies performed with the erythrocyte as a model demonstrate that degradation of the protein takes place before target cell lysis. A preliminary characterization of the effectors involved has led to the identification of a nonsecreted 30-kDa proteinase which is characterized by a high specificity for spectrin. This molecule is suggested as the main effector responsible for cytoskeletal disruption.

Tracking of clinical and environmental Vibrio cholerae O1 strains by combined analysis of the presence of toxin cassette, plasmid content and ERIC PCR.

Clinical and environmental Vibrio cholerae O1 strains associated with the cholera epidemic in the Luanda province of Angola from 1991 to 1994 were tracked by toxin distribution, plasmid content and chromosomal polymorphism of the enterobacterial repetitive intergenic consensus (ERIC) sequences by PCR fingerprinting. To follow the distribution of ace, zot and ctxA toxin genes, 6 specific PCR tests were applied to 100 Vibrio strains, after preliminary hybridization experiments. Clinical isolates of Vibrio cholerae O1 were characterized by high stability of the toxigenic cassette and the presence of a large conjugative multi-resistant plasmid of incompatibility class C. Such characteristics were present in all isolates during the four years of the epidemic. Environmental strains, isolated from the river supplying water to the Luanda population showed three different genetic profiles: the presence of both cassette and plasmid, the presence of cassette only or absence of both. To assess the clonal relationship between the clinical isolates and the three groups of environmental strains, the strains were analyzed by PCR ERIC polymorphism. This analysis, supported by the toxin and plasmid content, suggested the stability of the epidemic strain in clinical cases during the epidemic and led to the finding that there was a strict genetic relationship of the epidemic strain with the environmental ones as characterized by the presence of the toxin cassette. The role of the water supply from Bengo River as a reservoir of the Vibrio epidemic strain is discussed.

Extracellular release by Trichomonas vaginalis of a NADP+ dependent malic enzyme involved in pathogenicity.

This report presents evidence showing that Trichomonas vaginalis releases in the extracellular environment a functional form of NADP(+)-dependent malic enzyme. The protein which is likely responsible for the oxidative decarboxilase activity had already been identified in previous studies as P65, one of the five adhesive proteins of the protozoan. The same protein had also been described as AP65 by other authors, which identified it as one of the four surface proteins specifically responsible for binding of the parasite to the target cell in a ligand-receptor fashion. Gene characterization studies performed on P65 by different authors revealed that the nucleotide sequences of the genes coding for P65 display a striking homology with the ones coding for the trichomonad malic enzyme. The experiments performed in this work demonstrate that P65 is secreted and retains its adhesive properties in the extracellular environment, being able to bind both erythrocytes and HeLa cells. Therefore, an oxidative decarboxylase activity assay was performed on T. vaginalis cell-free filtrates, in order to assess if the released P65 displays cathalitic properties. The assay revealed that parasite-free supernatants exhibit an oxidative decarboxylase activity which is NADP(+)-dependent. On the basis of the most recent findings on T. vaginalis pathogenetic mechanism, which involves pH-dependent perforins, a role for the secreted enzyme as part of the system is proposed.

Trichomonas vaginalis haemolysis: pH regulates a contact-independent mechanism based on pore-forming proteins.

There is a controversy in literature about involvement of secreted factors in the pathogenetic mechanisms of Trichomonas vaginalis, described mostly as contact-dependent. We found that the protozoan, under triggering conditions, is able to release molecules that lead to lysis without direct contact between parasite and target cells as a prerequisite. In this paper we characterize contact-independent cytotoxicity using the red blood cell as a cellular model. Contact-independent haemolysis is a phenomenon were pH exerts a key role, triggering the secretion of a lytic molecule and regulating its activity. A partial physicochemical characterization of the haemolytic factor suggests that a protein of M(r) > 30 kDa could be the effector responsible for damage. Furthermore, the parasite-induced membrane permeabilization, detected by measuring potassium escape from the target cell, and an effective osmotic protection by carbohydrates allowed us to relate the previously described pore-forming mechanism involved in contact-dependent cytotoxicity with the contact-independent lysis.