Antonio Martínez-Murcia

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.

Sequencing of Bacterial and Archaeal 16S rRNA Genes Directly Amplified from a Hypersaline Environment

Summary The new methods based on PCR amplification and sequencing of 16S rRNA genes from DNA samples extracted directly from the environment allow the description of microbial diversity in natural ecosystems without the need for cultivation. We have applied this technique to an extreme environment presumed to have very low diversity: the crystallizer ponds of a marine saltern (salinity over NaCl saturation). The molecular methodology has shown that indeed very low diversity can be found here. Bacteria (formerly eubacteria) are largely minoritary, and only a cluster of closely related sequences was found, all distant relatives of the α-Proteohacteria (82% to Rhodopseudoinonas marina). Halophilic Archaea were shown by hybridization to be, as expected, the largest component of biomass in this environment. All the archaeal clones sequenced again were highly similar to each other suggesting that they are probably members of the same genus. However, all the sequences diverged considerably from those of the described genera of halophilic Archaea. In fact the data are consistent with the idea that the 16S rDNA genes directly amplified from the saltern correspond to members of an undescribed genus which seems to be abundant in the sample. This is remarkable since many collection strains sequenced come specifically from this same saltern. Furthermore, 16S rDNA obtained from archaeal cultures isolated from the same sample had no homology to the sequences obtained by PCR amplification, instead they appear to be members of the well known genus Haloarcula. However, this is in agreement with the findings of other authors who by culture have obtained organisms different from those indicated by the sequences retrieved directly by PCR. Possible explanations are discussed.

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.

16s-23s rDNA intergenic sequences indicate that Leuconostoc oenos is phylogenetically homogeneous

The study of the intra-specific phylogenetic structure of Leuconostoc oenos is essential to understand the participation of several strains in malo-lactic fermentation (MLF). RFLP of the PCR-amplified 16S-23S rDNA intergenic spacer region (ISR) was performed in Leuc. oenos and other related species. The RFLP patterns with seven endonucleases were identical for the 37 Leuc. oenos strains, but differed from those obtained for all other species tested. This method could provide an invaluable insight for molecular identification of the wine leuconostocs. The RFLP relationships of members of the genera Leuconostoc and Weissella were highly similar to those previously reported by 16S and 23S rRNA sequencing studies. The 16S-23S rDNA ISR was sequenced in five strains of Leuc. oenos. A single tRNA(Ala) was detected. The ISR sequence seems to be identical in the two rRNA (rrn) operons found in Leuc. oenos and no significant sequence variation was observed between strains that revealed relative differences as previously shown by PFGE. Results from the present study demonstrated that Leuc. oenos is phylogenetically a very homogeneous species (according to DNA-DNA hybridization studies) and sustain that this species is different from the genus Leuconostoc. The extremely conserved ISR of these organisms suggests that Leus. oenos strains currently isolated and characterized must have spread with the transfer of viticulture rather than coming from indigenous populations.

Patterns of sequence variation in two regions of the 16S rRNA multigene family of Escherichia coli.

Sequence heterogeneities of variable positions located at regions V1 and V6 of 56 cloned 16S rRNA genes were determined from six Escherichia coli strains. These nucleotides were involved in secondary structure base-pairing of stem-loops. Compensatory and single mutations have occurred but secondary structure was conserved. Eight different sequences were found in the stem at region V1 indicating that in these sites mutation rates are higher than those of homogenizatin processes. Region V6 showed two different structures (V6-I and V6-II) although heterogeneities were determined in nine sites. Strains ECOR52 and ECOR56 only showed the V6-I sequence, ECOR35 showed V6-II, whereas clones from ECOR42 and ECOR49 showed both types of V6 structures. Results were confirmed by PCR using V6 sequence-specific probes. Stem V6-II was also found in 16S rRNA sequences deposited in the RDP (Ribosomal Database Project) belonging to distantly related taxa; ancestral sequence V6-II seems to be homogenized in all rrn operons of the multigene family of strain ECOR35 producing effects of distortion in the molecular clock, similar to those that homoplasies could produce. V6 sequence-specific probes were applied to the 72 ECOR strains: half showed both V6-I and V6-II, and the rest had one or another. Only strain ECOR24 did not yield products in the PCR test and sequencing of 12 cloned 16S rRNA genes revealed a third form, V6-III, also found in the RDP. Concerted evolution by homogenization of the rRNA family may induce chronometric distortions responsible for a loss of ultrametricity in phylogenetic trees, particularly, of very closely related micro-organisms.

Sequence Diversity in the 16S–23S Intergenic Spacer Region (ISR) of the rRNA Operons in Representatives of the Escherichia coli ECOR Collection

Abstract. The ribosomal RNA multigene family in Escherichia coli comprises seven rrn operons of similar, but not identical, sequence. Four operons (rrnC, B, G, and E) contain genes in the 16S–23S intergenic spacer region (ISR) for tRNAGlu-2 and three (rrnA, D, and H) contain genes for tRNAIle-1 and tRNAAla-1B. To increase our understanding of their molecular evolution, we have determined the ISR sequence of the seven operons in a set of 12 strains from the ECOR collection. Each operon was specifically amplified using polymerase chain reaction primers designed from genes or open reading frames located upstream of the 16S rRNA genes in E. coli K12. With a single exception (ECOR 40), ISRs containing one or two tRNA genes were found at the same respective loci as those of strain K12. Intercistronic heterogeneity already found in K12 was representative of most variation among the strains studied and the location of polymorphic sites was the same. Dispersed nucleotide substitutions were very few but 21 variable sites were found grouped in a stem-loop, although the secondary structure was conserved. Some regions were found in which a stretch of nucleotides was substituted in block by one alternative, apparently unrelated, sequence (as illustrated by the known putative insertion of rsl in K12). Except for substitutions of different sizes and insertions/deletions found in the ISR, the pattern of nucleotide variation is very similar to that found for the 16S rRNA gene in E. coli. Strains K12 and ECOR 40 showed the highest intercistronic heterogeneity. Most strains showed a strong tendency to homogenization. Concerted evolution could explain the notorious conservation of this region that is supposed to have low functional restrictions.

Description of prokaryotic biodiversity along the salinity gradient of a multipond solar saltern by direct PCR amplification of 16S rDNA

New methods based on PCR amplification of 16S rRNA genes from DNA samples extracted directly from the environment allow the description of microbial diversity in natural ecosystems without the need for cultivation. We have applied this technique to an extreme environment presumed to have very low diversity: the crystallizer ponds of a marine saltern with salinity over NaCl saturation. The molecular methodology has shown that indeed very low diversity can be found here. Prokaryotes belonging to the Bacteria domain are a minor component and only members of a closely related cluster of sequences were found, all relatives of the α-Proteobacteria (ca. 83% to Rhodopseudomonas marina). Halophilic Archaea were as expected the largest component of biomass in this environment. All the clones sequenced corresponded again to a highly homologous cluster (probably members of the same genus). However, all the sequences diverged considerably from the ones of the described genera of halophilic Archaea, in fact the data are consistent with the idea that the 16S rRNA genes directly amplified from the saltern correspond to members of an undescribed genus. This is remarkable since many collection strains sequenced come specifically from this saltern. Furthermore, 16S rDNA obtained from archaeal cultures isolated from the same sample had no homology to the sequences obtained by PCR amplification, instead they appear to be members of the well known genus Haloarcula. However, this concurs with the findings of other authors who obtained different organisms by culture from those detected by the sequences retrieved directly by PCR. A possible explanation is that culturability, in standard media, is the exception rather than the rule. To study the biodiversity gradient present along the salinity gradient found in a multi-pond solar saltern we have also applied a novel molecular strategy. This method is based on the restriction digestion of a population of 16S rDNA sequences directly amplified from an environmental sample. Digested fragments separated by polyacrylamide gel electrophoresis generate characteristic profile data for estimation of diversity and overall similarities between the organisms of different environments. The methodology has been applied to a set of five ponds covering the salinity gradient from about twice that of seawater (6.4%) to NaCI precipitation (30.8%). Bacterial (eubacterial) diversity estimated from the complexity of the banding pattern obtained by restriction of the amplicons from the different ponds decreased with increasing salinity while for Archaea (archaebacteria) the reverse was true i.e. the higher the salinity the higher the number of bands. The similarities in taxonomic composition of the prokaryotic populations present in those ponds were evaluated from the number of restriction bands shared by the different samples. The relationships found among the different environments were independent of the enzyme used for digestion and were consistent with previous descriptions obtained by the study of isolates from the different environments. This technique appears to be promising as a rapid method for microbial biodiversity fingerprinting useful to compare several environments and detect major shifts in species composition of the microbial population.

Reclassification of Aeromonas hydrophila subsp. dhakensis Huys et al. 2002 and Aeromonas aquariorum Martínez-Murcia et al. 2008 as Aeromonas dhakensis sp. nov. comb nov. and emendation of the species Aeromonas hydrophila.

Previous studies indicate that Aeromonas aquariorum and Aeromonas hydrophila subsp. dhakensis are the same taxon and suggest that they should be synonymized. Using a polyphasic approach, the phenotypic and phylogenetic relationship of A. aquariorum with the 3 defined A. hydrophila subspecies (i.e. dhakensis, hydrophila, ranae) was investigated. Phylogenetic trees derived from the 16S rRNA, rpoD or gyrB genes and a multilocus phylogenetic analysis (with the concatenated sequences of gyrB, rpoD, recA, dnaJ and gyrA) confirmed that both A. aquariorum and A. hydrophila subsp. dhakensis are a unique taxon, different from the other A. hydrophila subspecies, corroborating the phenotypic and DNA-DNA hybridization (DDH) results. A formal synonymization of A. aquariorum and A. hydrophila subsp. dhakensis and a reclassification of both as Aeromonas dhakensis sp. nov. comb nov. is therefore proposed.

Clinical Relevance of the Recently Described Species Aeromonas aquariorum

ABSTRACT Twenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized as Aeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided.

Phylogenetic positions of Aeromonas encheleia, Aeromonas popoffii, Aeromonas DNA hybridization group 11 and Aeromonas group 501.

The 16S rDNA sequences of the recently described Aeromonas encheleia and Aeromonas popoffii, were determined and compared with data from all known Aeromonas sp. Diagnostic 16S rDNA regions were also sequenced for some strains previously considered as an extension of A. encheleia and a strain of Aeromonas Group 501 (formerly Enteric Group 501). Results indicated that A. encheleia and A. popoffii are phylogenetically separated species as originally described. A conclusion about HG11 taxonomic status is not recommended until previous discrepancies are clarified by further DNA-DNA hybridization and sequencing studies.