Lorén Jg

The role of pH in the ‘glucose effect’ on prodigiosin production by non‐proliferating cells of Serratia marcescens

The production of prodigiosin by non‐proliferating cells of Serratia marcescens is inhibited by addition of glucose or different carbon sources to the induction medium. The induction in acidic external pH, mimicking the effects produced by the carbon sources, reduced prodigiosin synthesis, and the prodigiosin production seems to be related to the length of the low pH period. Buffering at pH 7·5 increased pigment production in media with repressing carbon sources. This study reveals that the inhibitory effect of carbon sources on prodigiosin production may be due to a lowering of the pH of the medium.

Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov.

Although Anoxybacillus and Geobacillus, two genera of thermophilic bacteria close to the genus Bacillus, have only been described recently, the number of species in these genera has increased rapidly. Four thermophilic, lipolytic strains (DR01, DR02, DR03 and DR04) isolated from a hot spring in Veracruz (Mexico), which could not be identified phenotypically, were subjected to 16S rRNA gene sequence analysis. Three strains were identified as belonging to the genus Anoxybacillus, but strain DR03 was identified as Geobacillus pallidus. This result led us to perform a phylogenetic analysis of the genera Anoxybacillus and Geobacillus based on 16S rRNA gene sequences from all the type strains of these genera. Phylogenetic trees showed three major clusters, Anoxybacillus-Geobacillus tepidamans, Geobacillus sensu stricto and Geobacillus pallidus, while the 16S rRNA gene sequences of G. pallidus (DR03 and the type strain) showed low similarity to sequences of Anoxybacillus (92.5-95.1 %) and Geobacillus (92.8-94.5 %) species, as well as to Bacillus subtilis (92.2-92.4 %). In addition, G. pallidus could be differentiated from Anoxybacillus and Geobacillus on the basis of DNA G+C content and fatty acid and polar lipid profiles. From these results, it is proposed that Geobacillus pallidus should be classified in a novel genus, for which we propose the name Aeribacillus, as Aeribacillus pallidus gen. nov., comb. nov. The type strain of Aeribacillus pallidus is H12(T) (=ATCC 51176(T) =DSM 3670(T) =LMG 19006(T)).

Biochemical identification and numerical taxonomy of Aeromonas spp. isolated from environmental and clinical samples in Spain

Aims: To study the phenotypic characteristics of Aeromonas spp. from environmental and clinical samples in Spain and to cluster these strains by numerical taxonomy. 
Methods and Results: A collection of 202 Aeromonas strains isolated from bivalve molluscs, water and clinical samples was tested for 64 phenotypic properties; 91% of these isolates were identified at species level. Aeromonas caviae was predominant in bivalve molluscs and Aerom. bestiarum in freshwater samples. Cluster analyses revealed eight different phena: three containing more than one DNA‐DNA hybridization group but including strains that belong to the same phenospecies complex (Aerom. hydrophila, Aerom. sobria and Aerom. caviae), Aerom. encheleia, Aerom. trota and three containing unidentified Aeromonas strains isolated from bivalve molluscs. 
Conclusions:Aeromonas spp. are widely distributed in environmental and clinical sources. A selection of 16 of the phenotypical tests chosen allowed the identification of most isolates (91%), although some strains remain unidentified, mainly isolates from bivalve molluscs, suggesting the presence of new Aeromonas species. Numerical taxonomy was not in total concordance with the identification of the studied strains. 
Significance and Impact of the Study: Numerical taxonomy of Aeromonas strains isolated from different sources revealed the presence of potentially pathogenic Aeromonas spp., especially in bivalve molluscs, and phena with unidentified strains that suggest new Aeromonas species.

Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target

An analysis of the universal target (UT) sequence from the cpn60 gene was performed in order to evaluate its usefulness in phylogenetic and taxonomic studies and as an identification marker for the genus Aeromonas. Sequences of 555 bp, corresponding to the UT region, were obtained from a collection of 35 strains representing all of the species and subspecies of Aeromonas. From the analysis of these sequences, a range of divergence of 0-23.3% was obtained, with a mean of 11.2+/-0.9%. Comparative analyses between cpn60 and gyrB, rpoD and 16S rRNA gene sequences were carried out from the same Aeromonas strain collection. Sequences of the cpn60 UT region showed similar discriminatory power to gyrB and rpoD sequences. The phylogenetic relationships inferred from cpn60 sequence distances indicated an excellent correlation with the present affiliation of Aeromonas species with the exception of Aeromonas hydrophila subsp. dhakensis, which appeared in a separate phylogenetic line, and Aeromonas sharmana, which exhibited a very loose phylogenetic relationship to the genus Aeromonas. Sequencing of cpn60 from 33 additional Aeromonas strains also allowed us to establish intra- and interspecific threshold values. Intraspecific divergence rates were <or=3.5%, while interspecific divergence rates fell between 3.7 and 16.9%, excluding A. sharmana. In this study, cpn60 UT sequencing was shown to be a universal, useful, simple and rapid method for the identification and phylogenetic affiliation of Aeromonas strains.

The effect of pH on prodigiosin production by non‐proliferating cells of Serratia marcescens

The synthesis of prodigiosin by non‐proliferating cells of Serratia marcescens was examined at various pH values between 5.5 and 9.5. During incubation in unbuffered medium, pH changed and prodigiosin production was similar regardless of the initial pH. Variations in pigment production were noted when buffers were employed in cultures of non‐proliferating cells. The optimum pH for prodigiosin production was 8.0–8.5. Proline oxidase was also measured. The results suggest that the effect of pH may be related to the amount of proline which can be incorporated into prodigiosin.

Genetic relationships between clinical and environmental Vibrio cholerae isolates based on multilocus enzyme electrophoresis

A total of 107 isolates of Vibrio cholerae, including 29 strains belonging to serogroup O139, were studied using multilocus enzyme electrophoresis (MLEE) to determine allelic variation in 15 housekeeping enzyme loci. All loci were polymorphic and 99 electrophoretic types (ETs) were identified from the total sample. No significant clustering of isolates was detected in the dendrogram generated from a matrix of coefficients of distances with respect to serogroup, biotype or country of isolation. The mean genetic diversity of this V. cholerae population (H:=0.50) was higher than reported previously. Linkage disequilibrium analysis of the MLEE data showed a clonal structure for the entire population, but not in some of the population subgroups studied. This suggests an epidemic population structure. The results showed that the O139 strains were not clustered in a unique ET, in contrast to previous MLEE studies. This higher genetic variation of the O139 serogroup is concordant with ribotyping studies. The results also confirm that the O139 and O1 ElTor isolates are genetically more closely related to each other than to all the other subpopulations of V. cholerae studied.

The reference strain Aeromonas hydrophicla CIP 57.50 should be reclassified as Aeromonas salmonicida CIP 57.50.

The use of reference strains is a critical element for the quality control of different assays, from the development of molecular methods to the evaluation of antimicrobial activities. Most of the strains used in these assays are not type strains and some of them are cited erroneously because of subsequent reclassifications and descriptions of novel species. In this study, we propose that the reference strain Aeromonas hydrophila CIP 57.50 be reclassified as Aeromonas salmonicida CIP 57.50 based on phenotypic characterization and sequence analyses of the cpn60, dnaJ, gyrB and rpoD genes.

Buffering capacity and H+ membrane conductance of Gram-negative bacteria

Buffering capacity and membrane H+ conductance were examined in seven Gram-negative species: Aquaspirillum serpens, Pseudomonas aeruginosa, Alcaligenes faecalis, Escherichia coli, Salmonella typhimurium, Proteus mirabilis and Aeromonas hydrophila. All strains of Enterobacteriaceae studied here showed a decrease in both parameters as the external pH increased, over the pH range studied. The other four species presented an increase in buffering capacity and membrane conductance to protons as the external pH increased from 5.5 to 7.0.

Isolation from Urine of Two Serratia marcescens Strains Excreting a Diffusible Yellow Pigment

Two bacterial strains excreting a yellow pigment were isolated from human urine and identified as Serratia marcescens. The pigment was produced in the late exponential and early stationary phases of growth. Minimal media supplemented with tyrosine, phenylalanine, 3,4-dihydroxyphenylacetate or tryptophan, as well as complex media, induced pigment production. UV-visible spectra of the extracted pigment had peaks characteristic of 2-hydroxy-5-carboxymethylmuconate semialdehyde, produced from meta-cleavage of 3,4-dihydroxyphenylacetate by the enzyme 3,4-dihydroxyphenylacetate 2,3-dioxygenase (EC 1.13.11.15). This enzyme was active when the bacteria were grown under conditions promoting pigment production. The kinetics and factors affecting pigment production are also reported.

Acid-base response of bacterial suspensions

The response of suspensions of non-proliferating bacteria to external pH ranging from 3.6 to 9.9 was examined. The acid-base response is affected by the number of viable cells in the suspensions and culture conditions (aerobiosis or anaerobiosis) but not by culture time, composition of the culture medium or the age of suspensions. The results with carbonylcyanide m-chlorophenylhydrazone-and N, N′- dicyclohexylcarbodiimide-treated cells indicated that passive proton conductance and proton translocation by ATP could be involved in this response. pH profiles of suspensions of 18 chemoheterotrophic Gram-negative and Gram-positive bacteria support the hypothesis that the proton gradient generated by an alkali or an acid shift could be dissipated by bacteria with respiratory metabolism.