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Development of a multiplex PCR assay for the identification of pathogenic genes of Escherichia coli in milk and milk products.

A multiplex PCR for the simultaneous detection of some pathogenic genes of enteropathogenic, enterotoxigenic and verocytotoxin-producing Escherichia coli was developed. In this study primers found in literature as well as primers to the purpose designed were used. In this way, it was possible to generate specific fragments of 96, 170, 229, 285, 348, 414 and 510 bp for Hlya, St, EaeA, Lt, Vt1, UidA and Vt2 genes, respectively. When applied to bacterial strains experimentally inoculated in milk and milk products, the proposed PCR showed a detection limit of 5 x 10(4)CFU/ml for Hyla, St, Eaea, Vt1 primers, while for Lt and Vt2 primers the limit resulted of 10(6)CFU/ml.

Animals↗

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).

Cholera↗

Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations.

Knowledge about the world phylogeny of human mitochondrial DNA (mtDNA) is essential not only for evaluating the pathogenic role of specific mtDNA mutations but also for performing reliable association studies between mtDNA haplogroups and complex disorders. In the past few years, the main features of the East Asian portion of the mtDNA phylogeny have been determined on the basis of complete sequencing efforts, but representatives of several basal lineages were still lacking. Moreover, some recently published complete mtDNA sequences did apparently not fit into the known phylogenetic tree and conflicted with the established nomenclature. To refine the East Asian mtDNA tree and resolve data conflicts, we first completely sequenced 20 carefully selected mtDNAs--likely representatives of novel sub-haplogroups--and then, in order to distinguish diagnostic mutations of novel haplogroups from private variants, we applied a 'motif-search' procedure to a large sample collection. The novel information was incorporated into an updated East Asian mtDNA tree encompassing more than 1000 (near-) complete mtDNA sequences. A reassessment of the mtDNA data from a series of disease studies testified to the usefulness of such a refined mtDNA tree in evaluating the pathogenicity of mtDNA mutations. In particular, the claimed pathogenic role of mutations G3316A, T3394C, A4833G and G15497A appears to be most questionable as those initial claims were derived from anecdotal findings rather than e.g. appropriate association studies. Following a guideline based on the phylogenetic knowledge as proposed here could help avoiding similar problems in the future.

Asian People↗

Identification of pathogenic neisseriae by genetic transformation.

The detection of pathogenic neisseriae by genetic transformation of a naturally occurring proline auxotroph of Neisseria gonorrhoeae strain F62 is described. Of 169 clinical isolates of N. gonorrhoeae, approximately 90% gave a positive transformation assay. Twelve clinical isolates of N. meningitidis and stock cultures of the various meningococcal serogroups also gave a positive result. However, the sensitivity of the assay was found to be approximately 1000-fold lower with N. meningitidis as test organism. Eleven other members of the family Neisseriaceae failed to transform the recipient organism. Although proline requirement did not appear to limit the value of the assay greatly, it probably was the main reason for negative results. The sensitivity of the assay and its ability to detect non-viable gonococci suggests that this method merits further investigation as a possible aid to diagnosis of gonococcal infection in special circumstances.

Bacteriological Techniques↗

The identification of pathogenic yeast strains by electrophoretic analysis of their chromosomes.

Epidemiological studies require characterisation of pathogenic yeasts at and below the species level. The chromosomes of 130 strains of four pathogenic species of the genus Candida, isolated from clinical material, were separated by pulsed field electrophoresis with the clamped homogeneous electric field (CHEF) technique. Each species was characterised by a distinct electrophoretic karyotype (EK). Furthermore, smaller variations of the EK amongst strains belonging to the same species appeared to offer a useful means of strain differentiation. A karyotyping system is proposed for C. albicans. The EKs were assigned to a code of four numbers which designated the number of bands that could be resolved in each of four sets of chromosomes. Morphotypes of the colonies of C. albicans on malt agar plates, which did not correlate with the EK, could provide a complementary means of strain characterisation in epidemiological studies.

Candida↗

Laboratory identification of pathogenic Neisseria with special regard to atypical strains: an evaluation of sugar degradation, immunofluorescence and co-agglutination tests.

Sugar degradation tests (SDT) were compared with immunofluorescence (IFL) and co-agglutination (COA) tests for the diagnosis of Neisseria gonorrhoeae (GC) and Neisseria meningitidis (MC). Somewhat more than 5% of the GC strains and 8% of the MC strains were misinterpreted by SDT. On most occasions the disagreement between SDT and serological tests was due to the inability of the MC strains (less so for GC strains) to degrade sugars correctly. Because of this, three out of 15 strains (20%) from pharyngeal specimens were primarily considered to be GC by SDT but were identified as MC by COA tests. Deficiencies in sugar degradations were also found in a group of clinical problem strains. Many of them were unable or had a decreased ability to degrade glucose or maltose but were diagnosed distinctly as MC by the COA test. There were no false positives with the IFL or COA tests, but 2% of the GC strains and 26% of the MC carrier strains (non-groupable) were not identified by COA. Both IFL and COA tests are good adjuncts to SDT for the diagnosis of GC and clinically significant MC, since the results are reliable and the tests rapid and simple to perform.

Agglutination Tests↗

Isolation and identification of pathogenic Naegleria from Florida lakes.

Five cases of primary amoebic meningoencephalitis associated with swimming in freshwater lakes have been recorded in Florida over the past 14 years. The present study demonstrated that pathogenic Naegleria, the causative agent, is relatively widespread. Twelve of 26 lakes sampled only once yielded the amoeba. Populations in three of five lakes sampled routinely reached levels of one amoeba per 25 ml of water tested during the hot summer months. Overwintering in freshwater lake bottom sediments was demonstrated, showing that thermal-discharge pollution of waters plays a miniscule, if any, role in the maintenance of pathogenic Naegleria in nature in this semitropical area.

Animals↗

Isolation and identification of pathogenic microorganisms at wastewater-irrigated fields: ratios in air and wastewater.

Samples of air and corresponding wastewater samples were taken at wastewater spray-irrigated fields. The concentrations of salmonellae and enteroviruses present in these samples were determined and compared with those of coliforms, and the ratios between them were calculated. The most common Salmonella serotype in the air was Salmonella ohio, whereas in the wastewater, Salmonella anatum was the most common. Enteroviruses isolated and identified were poliovirus, echovirus, and coxsackievirus type B. From the ratios of salmonellas to coliforms and enteroviruses to coliforms in the air, as compared to these ratios in the wastewater, it was concluded that the suitability of coliforms as an indication of airborne contamination caused by spray irrigation is questionable.

Aerosols↗

Isolation and identification of pathogenic Naegleria australiensis (Amoebida, Vahlkampfiidae) from a spa in northern Italy.

Samples from therapeutic swimming pools and mud basins were cultured for free-living amoebae. Seven strains of pathogenic Naegleria species were isolated. Although some of the strains were as virulent as Naegleria fowleri, the etiological agent of primary amoebic meningoencephalitis, they were identified as Naegleria australiensis with the indirect fluorescent-antibody technique. The virulence of the isolates for mice corresponded with the cytopathic effect for Vero cells. The N. australiensis strains were isolated from swimming pools with water temperatures ranging from 32 to 35 degrees C and from mud with temperatures from 25 to 43 degrees C. The presence of pathogenic N. australiensis in the swimming pools did not correlate with bacterial indicators.

Amoeba↗

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.

Base Sequence↗