Value of recN sequences for species identification and as a phylogenetic marker within the family “Leuconostocaceae”

The genera Leuconostoc, Oenococcus, and Weissella (family “Leuconostocaceae”) constitute a group of lactic acid bacteria of great interest in food microbiology. From the taxonomic point of view, they are considered phylogenetically coherent according to their 16S rRNA gene sequences and other macromolecules. These three genera were the focus of the present study; specifically, the resolution and discriminatory power of recN (encoding a DNA repair and genetic recombination protein) as a molecular marker at the species level were investigated. For this purpose, partial sequences (about 1200 nt) were obtained from 23 type strains and from several additional strains following direct amplification of recN and subsequent sequencing. Phylogeny was evaluated according to different treeing methods (neighbor joining, maximum likelihood, and maximum parsimony) and the inclusion of variability filters. The results showed that recN, used either alone or in combination with 16S rRNA data, can serve as a phylogenetic marker as well as a tool for species identification.[Int Microbiol 2008; 11(1): 33-39

Value of recN sequences for species identification and as a phylogenetic marker within the family “Leuconostocaceae”

The genera Leuconostoc, Oenococcus, and Weissella (family “Leuconostocaceae”) constitute a group of lactic acid bacteria of great interest in food microbiology. From the taxonomic point of view, they are considered phylogenetically coherent according to their 16S rRNA gene sequences and other macromolecules. These three genera were the focus of the present study; specifically, the resolution and discriminatory power of recN (encoding a DNA repair and genetic recombination protein) as a molecular marker at the species level were investigated. For this purpose, partial sequences (about 1200 nt) were obtained from 23 type strains and from several additional strains following direct amplification of recN and subsequent sequencing. Phylogeny was evaluated according to different treeing methods (neighbor joining, maximum likelihood, and maximum parsimony) and the inclusion of variability filters. The results showed that recN, used either alone or in combination with 16S rRNA data, can serve as a phylogenetic marker as well as a tool for species identification.Macian Rovira, M. Carmen, [email protected] ; Garay Auban, Esperanza, [email protected]

Aerobic and facultative anaerobic heterotrophic bacteria associated to Mediterranean oysters and seawater

A comparative study on the composition and seasonal fluctuations of the main heterotrophic bacterial groups and species isolated from Mediterranean oysters and their growing-seawater was carried out. For the study we used 574 strains isolated from Marine Agar (MA) and submitted to numerical analysis of phenotypic traits in previous studies, plus 323 isolates recovered on Thiosulphate Citrate Bile Sucrose (TCBS) agar from the same samples and identified in this study. Oyster samples were dominated by halophilic fermentative bacteria during most of the year with predominance of two Vibrio species, V. splendidus (at temperatures lower than 20°C), and V. harveyi (at higher temperatures). On the contrary, Vibrio spp. was not the predominant microbiota of seawater, where most isolates had remained unidentified but corresponded to α-Proteobacteria, as shown by rDNA hybridization with phylogenetic probes in this study. Among the strict aerobes that could be identified, none of them showed a clear dominance, and many different groups were represented in very low percentages, in contrast with the major species from oyster samples. Shannon-Weaver diversity index revealed significant differences between both types of samples. No apparent seasonality was found in the distribution of seawater species, in sharp contrast with oyster-associated [email protected]; [email protected]; [email protected]

Chryseobacterium potabilaquae sp. nov., Chryseobacterium aquaeductus sp. nov. and Chryseobacterium fistulae sp. nov., from drinking water systems

A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them revealed that strains CECT 9293T and CECT 9393T could perhaps be related at the species level as they shared 99.5 % similarity. However, whole genome sequencing was performed and the subsequent calculation of relatedness indices, average nucleotide identity and estimated DNA-DNA hybridization, ruled out that possibility and confirmed instead that each of the strains should be considered a separate species in the genus Chryseobacterium. Having clarified their status, we also performed phylogenomic analyses and searched for possible environmental or non-type material sequences that could be related to any of them at the species level. In parallel, the strains were characterized phenotypically and compared to their closest relatives to determine diagnostic traits to support their formal proposal. The proposed species are Chryseobacterium potabilaquae sp. nov. with the type strain CECT 9293T (=LMG 32084T), Chryseobacterium aquaeductus sp. nov. with the type strain CECT 9390T (=LMG 32085T) and Chryseobacterium fistulae sp. nov. with the type strain CECT 9393T (=LMG 32086T)

Rapid and improved identification of drinking water bacteria using the Drinking Water Library, a dedicated MALDI-TOF MS database

According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment facilities. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is potentially such a method, although most of the currently available mass spectral libraries have been developed in a clinical setting and have limited environmental applicability. In this work, a MALDITOF MS drinking water library (DWL) was defined and developed by targeting bacteria present in water intended for human consumption. This database, made up of 319 different bacterial strains, can contribute to the routine microbiological control of either treated drinking water or mineral bottled water carried out by water treatment and distribution operators, offering a faster identification rate compared to a clinical sample-based library. The DWL, made up of 96 bacterial genera, 44 of which are not represented in the MALDI-TOF MS bacterial Bruker Daltonics (BDAL) database, was found to significantly improve the identification of bacteria present in drinking water

Drinking Water Library: nuevo recurso para identificar cepas bacterianas en aguas de consumo

De acuerdo con el RD 140/2003, el agua apta para el consumo humano no debe mostrar presencia de los indicadores bacterianos Escherichia coli, Clostridium perfringens y Enterococcus sp. Pero, aunque no se detecten estos indicadores, el agua del grifo no es estéril y puede tener cierta cantidad de microrganismos que muchas veces puede generar colonias en las placas de cultivo. Lejos de representar un peligro para la salud humana, si se dispone de un método rápido, sencillo, robusto y fiable para la identificación de estas colonias, se puede avanzar mucho en el conocimiento de la microbiología del agua potable. El proyecto Drinking Water Library ha permitido desarrollar la primera base de datos para MALDI-TOF MS específica para bacterias presentes en aguas de consumo

Detection and Differentiation of Several Food-Spoilage Lactic Acid Bacteria by Multiplex Polymerase Chain Reaction, Capillary Gel Electrophoresis, and Laser-Induced Fluorescence

In this work, a complete analytical procedure is investigated to differentiate several food-spoilage lactic acid bacteria. To do that, a method involving multiplex Polymerase Chain Reaction (PCR), capillary gel electrophoresis (CGE), and laser-induced fluorescence (LIF) is developed. The PCR-CGE-LIF protocol allows the simultaneous detection and differentiation of the genera Leuconostoc and Carnobacterium, the nonmotile group of species within the genus Carnobacterium, and the three species of the group individually (C. divergens, C. gallinarum, and C. maltaromicum). The capability of this approach is clearly illustrated through the sensitive and efficient analysis of the two closest amplicons, with sizes equal to 397 and 412 bp, showing very different yields in all of the amplification reactions tested. These two fragments, which could not be resolved by agarose gel electrophoresis (AGE), are clearly distinguishable by CGE-LIF even when very different areas for both peaks are obtained. The PCR-CGE-LIF method also allows the sensitive detection of these bacteria, demonstrating both a significant resolution improvement compared with traditional AGE and the usefulness of this approach to solve real-life analytical challenges. Good reproducibility of the CGE-LIF procedure is shown for the analysis of multiplex PCR samples with percent relative standard deviation values for migration times and corrected peak areas as low as 0.80 and 6.50 for the same sample and three different days (n = 12), respectively.Peer reviewe