Comparative in silico design and validation of GPS™ CoVID-19 dtec-RT-qPCR test.

AIMS: Providing a ready-to-use reverse transcriptase qPCR (RT-qPCR) method fully validated to detect the SARS-CoV-2 with a higher exclusivity than this shown by early published RT-qPCR designs. METHODS AND RESULTS: The specificity of the GPS™ CoVID-19 dtec-RT-qPCR test by analysis of sequence alignments was approached and compared with other RT-qPCR designs. The GPS™ CoVID-19 dtec-RT-qPCR test was validated following criteria of UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012. Diagnostic validation was achieved by two independent reference laboratories, the Instituto de Salud Carlos III, (Madrid, Spain), the Public Health England (Colindale, London, UK), and received the label CE-IVD. The GPS design showed the highest exclusivity and passed all parameters of validation with strict acceptance criteria. Results from reference laboratories 100% correlated with these obtained by using reference methods and showed 100% of diagnostic sensitivity and specificity. CONCLUSIONS: The CE-IVD GPS™ CoVID-19 dtec-RT-qPCR test, available worldwide with full analytical and diagnostic validation, is the more exclusive for SARS-CoV-2 by far. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering the CoVID-19 pandemic status, the exclusivity of RT-qPCR tests is crucial to avoid false positives due to related coronaviruses. This work provides of a highly specific and validated RT-qPCR method for detection of SARS-CoV-2, which represents a case of efficient transfer of technology successfully used since the pandemic was declared.

Phylogenetic analyses of the genus Aeromonas based on housekeeping gene sequencing and its influence on systematics.

The phylogenies derived from housekeeping gene sequence alignments, although mere evolutionary hypotheses, have increased our knowledge about the Aeromonas genetic diversity, providing a robust species delineation framework invaluable for reliable, easy and fast species identification. Previous classifications of Aeromonas, have been fully surpassed by recently developed phylogenetic (natural) classification obtained from the analysis of so-called 'molecular chronometers'. Despite ribosomal RNAs cannot split all known Aeromonas species, the conserved nature of 16S rRNA offers reliable alignments containing mosaics of sequence signatures which may serve as targets of genus-specific oligonucleotides for subsequent identification/detection tests in samples without culturing. On the contrary, some housekeeping genes coding for proteins show a much better chronometric capacity to discriminate highly related strains. Although both, species and loci, do not all evolve at exactly the same rate, published Aeromonas phylogenies were congruent to each other, indicating that, phylogenetic markers are synchronized and a concatenated multigene phylogeny, may be 'the mirror' of the entire genomic relationships. Thanks to MLPA approaches, the discovery of new Aeromonas species and strains of rarely isolated species is today more frequent and, consequently, should be extensively promoted for isolate screening and species identification. Although, accumulated data still should be carefully catalogued to inherit a reliable database.

Draft Genome Sequence of Aeromonas lusitana sp. nov. Strain DSM 24905T, Isolated from a Hot Spring in Vila-Real, Portugal.

Aeromonas lusitana sp. nov. is an isolate derived from a study aimed at characterizing Aeromonas spp. from water sources used for recreation and agricultural purposes and assessing the implications these organisms have for human and animal health. We present here the 4.52-Mbp draft genome sequence of this novel species.

Draft Genome Sequence of Aeromonas cavernicola sp. nov. DSM 24474T, Isolated from a Cavern Brook in the Moravia Region of the Czech Republic.

Species of the Aeromonas genus can be found in numerous environmental milieus, including various water sources, and some species cause disease in animals. We present here the draft genome sequence for Aeromonas cavernicola DSM 24474T, a novel species isolated from a freshwater brook within a cavern in the Czech Republic.

Phylogenetic diversity of Aeromonas from "alheira," a traditional Portuguese meat product.

"Alheira" is a traditional smoked meat sausage produced in the north of Portugal, representing an important economic resource for the region. This meat product has been subjected to research studies with the aim of detecting the presence of common foodborne pathogens, but, to our knowledge, isolation of emerging foodborne Aeromonas from alheira has never been previously described. Present work attempts to evaluate the Aeromonas species diversity of 84 isolates of Aeromonas spp. collected from 32 alheira samples. All presumptive Aeromonas isolates were subjected to genotyping by enterobacterial repetitive intergenic consensus-polymerase chain reaction analysis. The isolates presenting a different pattern were subjected to gyrB gene sequencing for species classification, and the species A. hydrophila, A. salmonicida, A. caviae, A. media, and A. allosaccharophila were identified. The Aeromonas species diversity found has not been previously described in any other meat product evaluated in previous studies. It is also important to highlight the presence of A. hydrophila and A. caviae because they were previously associated with illness in humans, including gastroenteritis.

Phylogenetic identification of Aeromonas from pigs slaughtered for consumption in slaughterhouses at the North of Portugal.

In the present study, 710 isolates of Aeromonas spp. have been collected from pig carcasses, diaphragm muscle, faeces, dehairing equipment and water in slaughterhouses at the North of Portugal. The isolates were obtained from a total of 154 samples. All presumptive Aeromonas isolates were subjected to ERIC-PCR analysis and those which presented a different pattern were taken and the species classified by gyrB gene sequencing. We have found the species A. hydrophila, A. salmonicida, A. bestiarum, A. caviae, A. media, A. veronii, A. allosaccharophila, A. simiae and A. aquariorum. To our knowledge, this extent of Aeromonas species diversity has not been previously described from meat or from the slaughter environment, perhaps due to the unreliability of available identification methods. A noticeable level of isolate redundancy (strains with identical gyrB sequence) from different samples collected in different dates was also obtained, indicating that only a few predominant strains of these species persist at the slaughter system. It is also important to emphasise the presence of Aeromonas species previously associated with illness in man.

Aeromonas rivuli sp. nov., isolated from the upstream region of a karst water rivulet.

Two freshwater isolates (WB4.1-19(T) and WB4.4-101), sharing 99.9 % 16S rRNA gene sequence similarity, were highly related to Aeromonas sobria (99.7 % similarity; 6 bp differences). A phylogenetic tree derived from a multi-locus phylogenetic analysis (MLPA) of the concatenated sequences of five housekeeping genes (gyrB, rpoD, recA, dnaJ and gyrA; 3684 bp) revealed that both strains clustered as an independent phylogenetic line next to members of Aeromonas molluscorum and Aeromonas bivalvium. The DNA-DNA reassociation value between the two new isolates was 89.3 %. Strain WB4.1-19(T) had a DNA-DNA relatedness value of <70 % with the type strains of the other species tested. Phenotypic characterization differentiated the two novel strains from all other type strains of species of the genus Aeromonas. It is concluded that the two new strains represent a novel species of the genus Aeromonas, for which the name Aeromonas rivuli sp. nov. is proposed, with the type strain WB4.1-19(T) (=CECT 7518(T)=DSM 22539(T)=MDC 2511(T)).

Aeromonas aquariorum sp. nov., isolated from aquaria of ornamental fish.

During a survey to determine the prevalence of Aeromonas strains in water and skin of imported ornamental fish, 48 strains presumptively identified as Aeromonas were isolated but they could not be identified as members of any previously described Aeromonas species. These strains were subjected to a polyphasic approach including phylogenetic analysis derived from gyrB, rpoD and 16S rRNA gene sequencing, DNA-DNA hybridization, MALDI-TOF MS analysis, genotyping by RAPD and extensive biochemical and antibiotic susceptibility tests in order to determine their taxonomic position. Based on the results of the phylogenetic analyses and DNA-DNA hybridization data, we describe a novel species of the genus Aeromonas, for which the name Aeromonas aquariorum sp. nov. is proposed, with strain MDC47T (=DSM 18362T =CECT 7289T) as the type strain. This is the first Aeromonas species description based on isolations from ornamental fish.

Updated phylogeny of the genus Aeromonas.

Recent phylogenetic studies of the genus Aeromonas based on gyrB and rpoD gene sequences have improved the phylogeny based on 16S rRNA gene sequences first published in 1992, particularly in the ability to split closely related species. These studies did not include the recently described species Aeromonas simiae and Aeromonas molluscorum and only a single strain of Aeromonas culicicola was available for analysis at that time. In the present work, these Aeromonas species and newly isolated strains of A. culicicola were examined. Sequence analysis indicates that A. simiae and A. molluscorum belong to non-described phylogenetic lines of descent within this genus, which supports the original description of both species. The most closely related species are Aeromonas schubertii and Aeromonas encheleia, respectively, which is consistent with 16S rRNA gene sequencing results. However, while the five strains of A. molluscorum showed nucleotide differences in their gyrB and rpoD gene sequences, the only two known A. simiae strains exhibited identical gene sequences, suggesting that they are isolates of the same strain. On the basis of the rpoD gene sequence phylogeny, A. culicicola strains from the original description and new isolates from drinking water and ornamental fish clustered within the species Aeromonas veronii, suggesting inconsistencies with previous results. Other strains with previously controversial taxonomy and new isolates from other studies were included in this study in order to clarify their phylogenetic affiliation at the species level.

First record of the rare species Aeromonas culicicola from a drinking water supply.

We describe the recovery of the rare species Aeromonas culicicola, so far known only in mosquitoes in India, from a drinking water supply in Spain. Typing, using enterobacterial repetitive intergenic consensus-PCR, revealed that the 27 new isolates belonged to 3 very closely related strains. These strains were genetically identified by 16S rRNA gene sequencing. Spanish strains differed from the mosquito strains in three nucleotide positions. The AHCYTOEN gene was present in these water strains, which may have a public health significance.

Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.

The phylogenetic relationships of all known species of the genus Aeromonas, and especially Aeromonas bestiarum and Aeromonas salmonicida, were investigated on 70 strains using the rpoD sequence, which encodes the sigma70 factor. This analysis was complemented with the sequence of gyrB, which has already proven useful for determining the phylogenetic relationships in the genus. Nucleotide sequences of rpoD and gyrB showed that both genes had similar substitution rates (< 2 %) and a similar number of variable positions (34 % for rpoD versus 32 % for gyrB). Strain groupings by analysis of rpoD, gyrB and a combination of both genes were consistent with the taxonomic organization of all Aeromonas species described to date. However, the simultaneous analysis of both clocks improved the reliability and the power to differentiate, in particular, closely related taxa. At the inter-species level, gyrB showed a better resolution for differentiating Aeromonas sp. HG11/Aeromonas encheleia and Aeromonas veronii/Aeromonas culicicola/Aeromonas allosaccharophila, while rpoD more clearly differentiated A. salmonicida from A. bestiarum. The analysis of rpoD provided initial evidence for clear phylogenetic divergence between the latter two species.

Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences.

The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase. Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions. The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria. This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date. In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species. The present analysis was useful for the elucidation of reported discrepancies between different DNA-DNA hybridization sets. Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains. In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas.

Sequence microdiversity at the ribosomal RNA operons of Escherichia coli pyelonephritogenic strains.

OBJECTIVE: To determine whether Escherichia coli strains isolated from patients with uncomplicated acute pyelonephritis can be distinguished from those isolated from patients with complicated acute pyelonephritis on the basis of the genetic background. METHODS: In total, 103 E. coli strains isolated from patients with acute pyelonephritis (59 uncomplicated pyelonephritis (UAP) and 44 complicated pyelonephritis (CAP)) were characterized by RFLP of the intergenic spacer region 16S-23S rRNA, the presence of three alternative sequences found in the polymorphic V6 loop of the 16S rRNA gene, the presence of the pap gene, and antibiotic susceptibility. RESULTS: At similarity levels of 70%, four RFLP groups (alpha1, alpha2, beta1 and beta2) were discerned. Strains from UAP were statistically significant for alpha RFLP, with a strong association with the presence of the pap gene, V6-I sequence and antibiotic multisensitivity. Strains from CAP randomly belonged to the alpha or beta RFLP groups, with a very low presence of the pap gene, and random presence of V6 sequences, and were multiresistant to antibiotics. When the CAP strains were distributed according to underlying pathology, non-obstructive cases had RFLP and V6 polymorphisms similar to those of UAP cases, while obstructive cases were clearly distinct. CONCLUSIONS: UAP and non-obstructive CAP E. coli strains are sensitive to antimicrobials, show a high level of the pap gene and belong to the selective, homogeneous and highly protected molecular alpha2 group, where no recombinations, deletions or insertions are present. On the contrary, obstructive and vesicorenal reflux E. coli strains show significant antimicrobial resistance, high intercistronic heterogenicity (wide presence of block nucleotidic substitutions, deletions or insertions) and significantly lower virulence.

Características moleculares de cepas de escherichia coli causantes de pielonefritis agudas / Molecular characteristics of Escherichia coli that cause acute pielonefritis

A un total de 103 cepas de E. Boli aisladas de enfermos con pielonefritis aguda (59 cepas de pielonefritis no complicadas (PNAnoC) y 44 de pielonefritis complicadas (PNAC)) y 72 cepas de la colección ECOR se les ha determinado el tipo de secuencia V6 (posiciones 1000-1040) del gen 16S-rRNA, la posesión del gen pap y la secuencia espacio intergénico 16S-23S-rRNA mediante amplificación por PCR usando 7 cebadores específicos, digestión con endonucleasas, electroforesis y análisis de los polimorfismos de longitud de fragmentos de restricción (RFLP). Con niveles de similaridad del 70 por ciento se han formado 4 grupos denominados a-1, a-2, beta-1 y beta-2. El subgrupo a-2 se ha correlacionado con el B-2 de MLEE considerado de maxima virulencia.Las cepas aisladas de PNAnoC mostraron una asociación causal significativa (p <0,01) con el subgrupo molecular a-2 (74 por ciento) , secuencia V6-1(91,2 por ciento) y presencia del gen pap (85,3 por ciento). Por contra, en las cepas aisladas de PNAC no se observó relación de causalidad (p = n. s.) entre los grupos moleculares, la secuencia V6-I y la presencia del gen pap. La agrupación de las cepas de PNAC según el diagnóstico de la patología de base (no obstructiva: 13 casos, obstructiva: 20 casos y reflujo vesico-renal: 11 casos) demostró que para los casos no obstructivos se conservaba una estructura molecular estrechamente relacionada a los casos de PNAnoC, mientras que para el resto la pertenencia al grupo alfa/beta y tipo de secuencia era aleatorio con una presencia del gen pap muy baja (12,9 por ciento). La incidencia de polirresistencia a los antibióticos en el grupo a fue bastante menor que en el grupo beta (5,6 por ciento versus 25 por ciento respectivamente).En el grupo beta la frecuencia del gen pap en cepas polirresistentes disminuyó más de 4 veces respecto a las cepas sensibles o con monorresistencia de su propio grupo o más de 6 veces respecto a las del grupo a Se concluye que las cepas de PNAnoC poseen una elevada virulencia y homogeneidad y están altamente protegidas de recombinaciones o reordenaciones con patrón molecular de: subgrupo a-2 + presencia gen pap + secuencia V6-I + multisensibilidad a los antibióticos. Las cepas de PNAC (excepto los casos no obstructivos) tienen gran variabilidad intercistrónica (grupo alfa/beta, tipo de secuencia V6), poseen una baja virulencia y son mas polirresistentes a los antibióticos. (AU)

Typing of clinical and environmental Aeromonas veronii strains based on the 16S-23S rDNA spacers.

Genetic relationships of Aeromonas veronii strains isolated from human and environmental sources were investigated by restriction fragment length polymorphism (RFLP) of the polymerase chain reaction-amplified intergenic spacer region (ISR) flanked by the 16S and 23S rRNA genes. When using endonucleases AluI, HinfI and CfoI the 16S-23S rDNA-RFLP patterns showed considerable overall similarity, although most strains yielded specific profiles. Several intra-specific lines of descent comprised clinical strains linked to isolates from environmental sources. Strains having identical patterns may be individuals derived from highly similar, if not the same, microorganism. Results suggest that the ISR sequence-based method can be used to demonstrate colonization of a public water supply with a particular microorganism. In addition it could be very useful for tracing recurrent episodes of diarrhea and Aeromonas infection outbreaks.

Extended method for discrimination of Aeromonas spp. by 16S rDNA RFLP analysis.

A previously described molecular method, based on 16S rDNA RFLP analysis, for the identification of Aeromonas spp. was unable to separate the species Aeromonas salmonicida, Aeromonas bestiarum and the recently described Aeromonas popoffii. In this study, the method has been extended with endonucleases AIwNI and PstI for the identification of these species. A molecular frame for the identification of all known Aeromonas spp. is presented.