Comparison of different NAT assays for the detection of microorganisms belonging to the class Mollicutes.

BACKGROUND: Mollicutes detection can be cumbersome due to their slow growth in vitro. For this reason, the use of DNA based on generic molecular tests represents an alternative for rapid, sensitive and specific detection of these microorganism. For this reason, six previously described nucleic acid testing assays were compared to evaluate their ability to detect microorganisms belonging to the class Mollicutes. METHODS: A panel of 61 mollicutes, including representatives from the Mycoplasma, Acholeplasma, Mesoplasma, Spiroplasma and Ureaplasma genus, were selected to evaluate the sensitivity and specificity of these assays. A total of 21 non-mollicutes, including closely related non-mollicutes species, were used to evaluate specificity. Limits of detection were calculated to determine the analytical sensitivity of the assays. The two best performing assays were subsequently adapted into real-time PCR format, followed by melting curve analysis. RESULTS: Both assays performed satisfactorily, with a 100% specificity described for both assays. The detection limits were found to be between 10-4 and 10-5 dilutions, equivalent to 15 to 150 genome copies approximately. Based on our work, both van Kuppeveld and Botes real-time PCR assays were found to be the best performing tests in terms of sensitivity and specificity. Furthermore, Botes real-time PCR assay could detect phytoplasmas as well. CONCLUSIONS: These assays can be very useful for the rapid, specific and sensitive screening cell line contaminants, clinical samples as well as detecting non-culturable, unknown species of mollicutes or mollicutes whose growth is slow or difficult.

Comparative genomic analysis reveals novel facts about Leptospirillum spp. cytochromes.

Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb3 oxidases. Genes encoding cbb3 oxidase are clustered, with predicted genes involved in cbb3 maturation proteins. Duplication of cbb3 encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc1 and b6f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen.

Vigilancia epidemiológica de enfermedades transmitidas por alimentos en la Provincia de Río Negro, Argentina, 1993-2001 / Surveillance of foodborne diseases in the province of Rio Negro, Argentina, 1993-2001

A total of 39 outbreaks of foodborne diseases affecting 958 people in the province of Rio Negro, Argentina between 1993 and 2001 are described and evaluated. The main causal agents were identified involving food, sites of occurrence, risk factors and notification system used. Salmonella spp (38%), Trichinella spiralis (15%), Escherichia coli (13%) and Staphylococcus aureus (15%) were the most frequent agents present in outbreaks. Salmonella spp produced the largest number of cases (52%). Food involved were cooked meat (36%), cheese (10%), sandwiches (10%), deserts (10%) and ice cream (8%). Indeed, ice creams were involved in the largest number of cases and of people affected. In relation to the source of food, 41% of outbreaks were caused by homemade meals, 23% by catering or ice cream parlor, 13% in family parties, 8% in county fairs and 8% in hotel restaurants. In 28% of the outbreaks the etiological agent was identified exclusively by epidemiological analysis, in 64% isolation of the agent was carried out, and in 8% of the cases, a final diagnosis could not be obtained. Validity of epidemiological studies in foodborne disease, the necessity of strengthening the notification system of outbreaks, and the importance of good practices in food handling are analyzed.

Se describen 39 brotes de enfermedades transmitidas por alimentos que afectaron a 958 personasen la provincia de Río Negro, Argentina, en el período 1993- 2001. Se identifican los agentes causales, los alimentos involucrados, los sitios de ocurrencia, los factores de riesgo involucrados y los mecanismosde notificación empleados. Salmonella spp (38%), Trichinella spiralis (15%), Escherichia coli (13%) y Staphylococcus aureus (15%) resultaron los agentes más frecuentes en los brotes. Salmonella spp. tambiénprodujo el mayor número de casos (52%). Los principales alimentos involucrados resultaron cárneos (36%),quesos (10%), fiambres y sándwiches (10%), postres (10%) y helados (8%). El mayor número de casos, por suparte, fue causado por la ingestión de helados (37%). Con relación al origen de los alimentos, 41% de los brotesfueron causados por comidas elaboradas en los domicilios, 23% en establecimientos comerciales, 13% enfiestas familiares, 8% en fiestas comunitarias y 8% en restaurantes de hoteles. En el 28% de los brotes fueidentificado el agente etiológico por análisis epidemiológico exclusivamente, en el 64% se logró el aislamientodel agente, mientras que en el 8% de los casos no se logró el diagnóstico definitivo. Se analiza el valor de laencuesta epidemiológica en los estudios de enfermedades transmitidas por alimentos, la necesidad de fortalecerel sistema de notificación médica de casos y brotes y la importancia de las buenas prácticas en la manipulaciónde alimentos.

Vigilancia epidemiológica de enfermedades transmitidas por alimentos en la Provincia de Río Negro, Argentina, 1993-2001 / Surveillance of foodborne diseases in the province of Rio Negro, Argentina, 1993-2001

A total of 39 outbreaks of foodborne diseases affecting 958 people in the province of Rio Negro, Argentina between 1993 and 2001 are described and evaluated. The main causal agents were identified involving food, sites of occurrence, risk factors and notification system used. Salmonella spp (38%), Trichinella spiralis (15%), Escherichia coli (13%) and Staphylococcus aureus (15%) were the most frequent agents present in outbreaks. Salmonella spp produced the largest number of cases (52%). Food involved were cooked meat (36%), cheese (10%), sandwiches (10%), deserts (10%) and ice cream (8%). Indeed, ice creams were involved in the largest number of cases and of people affected. In relation to the source of food, 41% of outbreaks were caused by homemade meals, 23% by catering or ice cream parlor, 13% in family parties, 8% in county fairs and 8% in hotel restaurants. In 28% of the outbreaks the etiological agent was identified exclusively by epidemiological analysis, in 64% isolation of the agent was carried out, and in 8% of the cases, a final diagnosis could not be obtained. Validity of epidemiological studies in foodborne disease, the necessity of strengthening the notification system of outbreaks, and the importance of good practices in food handling are analyzed.(AU)

Se describen 39 brotes de enfermedades transmitidas por alimentos que afectaron a 958 personasen la provincia de Río Negro, Argentina, en el período 1993- 2001. Se identifican los agentes causales, los alimentos involucrados, los sitios de ocurrencia, los factores de riesgo involucrados y los mecanismosde notificación empleados. Salmonella spp (38%), Trichinella spiralis (15%), Escherichia coli (13%) y Staphylococcus aureus (15%) resultaron los agentes más frecuentes en los brotes. Salmonella spp. tambiénprodujo el mayor número de casos (52%). Los principales alimentos involucrados resultaron cárneos (36%),quesos (10%), fiambres y sándwiches (10%), postres (10%) y helados (8%). El mayor número de casos, por suparte, fue causado por la ingestión de helados (37%). Con relación al origen de los alimentos, 41% de los brotesfueron causados por comidas elaboradas en los domicilios, 23% en establecimientos comerciales, 13% enfiestas familiares, 8% en fiestas comunitarias y 8% en restaurantes de hoteles. En el 28% de los brotes fueidentificado el agente etiológico por análisis epidemiológico exclusivamente, en el 64% se logró el aislamientodel agente, mientras que en el 8% de los casos no se logró el diagnóstico definitivo. Se analiza el valor de laencuesta epidemiológica en los estudios de enfermedades transmitidas por alimentos, la necesidad de fortalecerel sistema de notificación médica de casos y brotes y la importancia de las buenas prácticas en la manipulaciónde alimentos.(AU)

A dual-specific Glu-tRNA(Gln) and Asp-tRNA(Asn) amidotransferase is involved in decoding glutamine and asparagine codons in Acidithiobacillus ferrooxidans.

The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.

Conserved amino acids near the carboxy terminus of bacterial tyrosyl-tRNA synthetase are involved in tRNA and Tyr-AMP binding.

Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.

tRNA recognition and evolution of determinants in seryl-tRNA synthesis.

We have analyzed the evolution of recognition of tRNAsSerby seryl-tRNA synthetases, and compared it to other type 2 tRNAs, which contain a long extra arm. In Eubacteria and chloroplasts this type of tRNA is restricted to three families: tRNALeu, tRNASer and tRNATyr. tRNALeuand tRNASer also carry a long extra arm in Archaea, Eukarya and all organelles with the exception of animal mitochondria. In contrast, the long extra arm of tRNATyr is far less conserved: it was drastically shortened after the separation of Archaea and Eukarya from Eubacteria, and it is also truncated in animal mitochondria. The high degree of phylo-genetic divergence in the length of tRNA variable arms, which are recognized by both class I and class II aminoacyl-tRNA synthetases, makes type 2 tRNA recognition an ideal system with which to study how tRNA discrimination may have evolved in tandem with the evolution of other components of the translation machinery.

Bacterial populations in samples of bioleached copper ore as revealed by analysis of DNA obtained before and after cultivation.

The composition of bacterial populations in copper bioleaching systems was investigated by analysis of DNA obtained either directly from ores or leaching solutions or after laboratory cultures. This analysis consisted of the characterization of the spacer regions between the 16 and 23S genes in the bacterial rRNA genetic loci after PCR amplification. The sizes of the spacer regions, amplified from DNAs obtained from samples, were compared with the sizes of those obtained from cultures of the main bacterial species isolated from bioleaching systems. This allowed a preliminary assessment of the bacterial species present in the samples. Identification of the bacteria was achieved by partial sequencing of the 16S rRNA genes adjacent to the spacer regions. The spacer regions observed in DNA from columns leached at different iron concentrations indicated the presence of a mixture of different bacteria. The spacer region corresponding to Thiobacillus ferrooxidans was the main product observed at high ferrous iron concentration. At low ferrous iron concentration, spacer regions of different lengths, corresponding to Thiobacillus thiooxidans and "Leptospirillum ferrooxidans" were observed. However, T. ferrooxidans appeared to predominate after culture of these samples in medium containing ferrous iron as energy source. Although some of these strains contained singular spacer regions, they belonged within previously described groups of T. ferrooxidans according to the nucleotide sequence of the neighbor 16S rRNA. These results illustrate the bacterial diversity in bioleaching systems and the selective pressure generated by different growth conditions.

CFTR mutations in Chilean cystic fibrosis patients.

An analysis of five of the most common cystic fibrosis (CF) mutations worldwide (delta F-508, R-553X, G-551D, N-1303K and G-542X) was performed in 36 Chilean patients. Polymerase chain reaction (PCR) amplification of the DNA followed by allele specific restriction enzyme analysis was used for detection. The overall frequencies of the mutations in the chromosomes analyzed were 29.2% for delta F-508 and 4.2% for R-553X (n = 72). The G-542X, G-551D and N-1303 K mutations were absent in the Chilean sample. Our data suggest however that delta F-508 is not the most common CF mutation in Chilean patients. delta F-508 and R-553X account for only 33.4% of the alleles; 66.6% of them do not respond to the probes used and still remain uncharacterized.

Thiobacillus ferrooxidans tyrosyl-tRNA synthetase functions in vivo in Escherichia coli.

The tyrosyl-tRNA synthetase gene (tyrZ) from Thiobacillus ferrooxidans, an acidophilic, autotrophic, gram-negative bacterium that participates in bioleaching of minerals, was cloned and sequenced. The encoded polypeptide (TyrRZ) is 407 amino acids in length (molecular mass; 38 kDa). The predicted protein sequence has an extensive overall identity (44%) to the sequence of the protein encoded by the Bacillus subtilus tyrZ gene, one of the two genes encoding tyrosyl-tRNA synthetases in this microorganism. Alignment with Escherichia coli TyrRS revealed limited overall identity (24%), except in the regions of the signature sequence for class I aminoacyl-tRNA synthetases. Complementation of an E. coli strain with a thermosensitive mutation in TyrRS showed that the protein encoded by the T. ferrooxidans tyrZ gene is functional and recognizes the E. coli tRNA(Tyr) as a substrate. TyrZ is a single-copy gene as revealed by Southern blot analysis. The gene was localized upstream from the putative promoters of the rrnT2 ribosomal RNA operon. Although no rho-independent transcription terminator was found between the two genes, a 1.3-kb RNA hybridized to a DNA probe derived from the tyrZ gene. The functional relationship between these two transcription units is discussed.

Transposition of IST2 in Thiobacillus ferrooxidans.

The genome of Thiobacillus ferrooxidans contains at least two different repetitive DNA elements. One of these elements, termed IST2 has been sequenced and shown to exhibit the characteristics of a typical prokaryotic insertion sequence. Furthermore, preliminary evidence has implicated IST2 in genomic rearrangements, although the mechanism of rearrangement, whether by transposition or recombination, has not been established. In this report we provide evidence from detailed restriction enzyme analyses and DNA sequencing data that support a model of transposition, consistent with the notion that IST2 is a mobile insertion sequence.

[Molecular genetic analysis of 2 Chilean cystic fibrosis patients and their families]. / Análisis genético molecular de la fibrosis quística en dos pacientes chilenos y sus familias.

As a contribution to establish the real incidence of cystic fibrosis (CF) and the prevalent mutations in the Chilean population a method for the detection of delta F-508 and R-553X, two of the most frequent mutations described worldwide, has been implemented. The method is based on the polymerase chain reaction (PCR) amplification of DNA followed by allele specific restriction enzymatic digestion. The application of this techniques allowed to confirm CF diagnosis in two patients and to detect asymptomatic carriers in both families. One of the patients showed normal sweat electrolyte concentration.

[Molecular genetic study of cystic fibrosis in the Chilean population. Relationship to its clinical expression]. / Estudio genético molecular de la fibrosis quística en la población Chilena. Relación con su expresión clínica.

Aiming to establish a genotype-phenotype relationship and to search a clinical expression in heterozygotes, 25 Chilean subjects with Cystic Fibrosis and 165 relatives were subjected to a clinical-molecular study. The most common mutations found worldwide were studied: delta F-508, G-542X, N-1303K, R-553X and G551D. Clinical and laboratory assessment comprised chest X-rays, spirometry, clinical evaluation, nutritional assessment, sweat test and carotenemia. Age at diagnosis was lower among homozygotes for the mutation delta F-508. In this group, Brasfield and Schawchman scores were better, probably due to an earlier initiation of treatment. No other differences were found among genotypic groups or relatives. Genetic markers indicated a higher european component of the sample, compared to the general Chilean population.

Isolation and nucleotide sequence of the Thiobacillus ferrooxidans genes for the small and large subunits of ribulose 1,5-bisphosphate carboxylase/oxygenase.

The genes encoding for the large (rbcL) and small (rbcS) subunits of ribulose-1,5-bisphosphate carboxylase (RuBisCO) were cloned from the obligate autotroph Thiobacillus ferrooxidans, a bacterium involved in the bioleaching of minerals. Nucleotide sequence analysis of the cloned DNA showed that the two coding regions are separated by a 30-bp intergenic region, the smallest described for the RuBisCO genes. The rbcL and rbcS genes encode polypeptides of 473 and 118 amino acids, respectively. Comparison of the nucleotide and amino acid sequences with those of the genes for rbcL and rbcS found in other species demonstrated that the T. ferrooxidans genes have the closest degree of identity with those of Chromatium vinosum and of Alvinoconcha hessleri endosymbiont. Both T. ferrooxidans enzyme subunits contain all the conserved amino acids that are known to participate in the catalytic process or in holoenzyme assembly.

Identification and structural analysis of a ribosomal RNA gene promoter from Thiobacillus ferrooxidans.

The 5'-terminus of a rRNA operon (rrnT2) from Thiobacillus ferrooxidans was characterized. The rRNA promoters from this microorganism were identified by means of a functional assay in Escherichia coli. DNA sequencing of the promoter region, upstream the 16 S rRNA gene, showed the presence of a consensus sequence for bacterial ribosomal promoters. Other features such as a 'discriminator' sequence, antiterminator elements and an upstream hexanucleotide common to several rRNA operons were also found. Two other putative transcription promoters were also identified.

Characterization of the two rRNA gene operons present in Thiobacillus ferrooxidans.

The organization of rRNA genes from the autotrophic, acidophilic bacterium Thiobacillus ferrooxidans has been examined. Two rRNA operons were found in this microorganism by means of genomic hybridization studies. Recombinant plasmids, pTR-3 and pTR-1 that carry a portion of 16/23 S rDNA from one operon and the 5'-flanking region of the second operon, respectively, were identified and characterized.