RESUMO
The definition of species boundaries constitutes an important challenge in biodiversity studies. In this work we applied the Generalized Mixed Yule Coalescent (GMYC) method, which determines a divergence threshold to delimit species in a phylogenetic tree. Based on the tree branching pattern, the analysis fixes the transition threshold between speciation and the coalescent process associated with the intra-species diversification. This approach has been widely used to delineate eukaryote species and establish their diversification process from sequence data. Nevertheless, there are few examples in which this analysis has been applied to a bacterial population. Although the GMYC method was originally designed to assume a constant (Yule) model of diversification at between-species level, it was later evaluated simulating other conditions. Our aim was therefore to determine the species delineation in Aeromonas using the GMYC method and asses which model best explains the speciation process in this bacterial genus. The application of the GMYC method allowed us to clearly delineate the Aeromonas species boundaries, even in the controversial groups, such as the A. veronii or A. media species complexes.
RESUMO
Despite the importance of diversification rates in the study of prokaryote evolution, they have not been quantitatively assessed for the majority of microorganism taxa. The investigation of evolutionary patterns in prokaryotes constitutes a challenge due to a very scarce fossil record, limited morphological differentiation and frequently complex taxonomic relationships, which make even species recognition difficult. Although the speciation models and speciation rates in eukaryotes have traditionally been established by analyzing the fossil record data, this is frequently incomplete, and not always available. More recently, several methods based on molecular sequence data have been developed to estimate speciation and extinction rates from phylogenies reconstructed from contemporary taxa. In this work, we determined the divergence time and temporal diversification of the genus Aeromonas by applying these methods widely used with eukaryotic taxa. Our analysis involved 150 Aeromonas strains using the concatenated sequences of two housekeeping genes (approximately 2,000 bp). Dating and diversification model analyses were performed using two different approaches: obtaining the consensus sequence from the concatenated sequences corresponding to all the strains belonging to the same species, or generating the species tree from multiple alignments of each gene. We used BEAST to perform a Bayesian analysis to estimate both the phylogeny and the divergence times. A global molecular clock cannot be assumed for any gene. From the chronograms obtained, we carried out a diversification analysis using several approaches. The results suggest that the genus Aeromonas began to diverge approximately 250 millions of years (Ma) ago. All methods used to determine Aeromonas diversification gave similar results, suggesting that the speciation process in this bacterial genus followed a rate-constant (Yule) diversification model, although there is a small probability that a slight deceleration occurred in recent times. We also determined the constant of diversification (λ) values, which in all cases were very similar, about 0.01 species/Ma, a value clearly lower than those described for different eukaryotes.
RESUMO
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.
Assuntos
Aeromonas/classificação , Aeromonas/metabolismo , Água Doce/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Moluscos/microbiologia , Aeromonas/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , Classificação/métodos , Cobaias , Humanos , Hibridização de Ácido Nucleico , Fenótipo , Reprodutibilidade dos Testes , EspanhaRESUMO
Comparative analysis of gene fragments of six housekeeping loci, distributed around the two chromosomes of Vibrio cholerae, has been carried out for a collection of 29 V. cholerae O139 Bengal strains isolated from India during the first epidemic period (1992 to 1993). A toxigenic O1 ElTor strain from the seventh pandemic and an environmental non-O1/non-O139 strain were also included in this study. All loci studied were polymorphic, with a small number of polymorphic sites in the sequenced fragments. The genetic diversity determined for our O139 population is concordant with a previous multilocus enzyme electrophoresis study in which we analyzed the same V. cholerae O139 strains. In both studies we have found a higher genetic diversity than reported previously in other molecular studies. The results of the present work showed that O139 strains clustered in several lineages of the dendrogram generated from the matrix of allelic mismatches between the different genotypes, a finding which does not support the hypothesis previously reported that the O139 serogroup is a unique clone. The statistical analysis performed in the V. cholerae O139 isolates suggested a clonal population structure. Moreover, the application of the Sawyer's test and split decomposition to detect intragenic recombination in the sequenced gene fragments did not indicate the existence of recombination in our O139 population.
Assuntos
Alelos , Cólera/epidemiologia , Surtos de Doenças , Variação Genética , Análise de Sequência de DNA , Vibrio cholerae/genética , Proteínas de Bactérias/genética , Sequência de Bases , Cólera/microbiologia , Humanos , Desequilíbrio de Ligação , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Recombinação Genética , Vibrio cholerae/classificaçãoRESUMO
Genetic diversity and genetic relationships among 42 Pseudomonas stutzeri strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this P. stutzeri sample exhibited the highest mean genetic diversity (H = 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (I(A)) for the P. stutzeri strains analyzed was 1.10. The I(A) values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of P. stutzeri is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium.
Assuntos
Variação Genética , Pseudomonas/genética , Alelos , Células Clonais , Eletroforese em Gel de Poliacrilamida/métodos , Enzimas/isolamento & purificação , Genótipo , Desequilíbrio de Ligação , Pseudomonas/classificação , Pseudomonas/enzimologiaRESUMO
The addition of glucose or other sugars to resting cells of Serratia maurcescens induced rapid acidification of the extracellular medium. This acidification was due to the catabolism of sugars. The rate of acidification depended on the carbon source and its concentration. HPLC analysis of the supernatants demonstrated that the progressive fall in pH resulted from the rapid production of lactic, acetic, pyruvic and citric acids. Other microorganisms were tested for their ability to produce this rapid acidification of the medium. This study may provide a rapid and simple method for metabolism studies.
Assuntos
Glucose/metabolismo , Concentração de Íons de Hidrogênio , Serratia marcescens/metabolismo , Ácido Acético/metabolismo , Metabolismo dos Carboidratos , Cromatografia Líquida de Alta Pressão , Ácido Cítrico/metabolismo , Meios de Cultivo Condicionados/química , Metabolismo Energético , Ácido Láctico/metabolismo , Prodigiosina/biossíntese , Ácido Pirúvico/metabolismo , Álcoois Açúcares/metabolismoRESUMO
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.
Assuntos
Cólera/microbiologia , Eletroforese em Gel de Poliacrilamida/métodos , Microbiologia Ambiental , Enzimas/análise , Vibrio cholerae/genética , Alelos , Variação Genética/genética , Genética Populacional , Humanos , Desequilíbrio de Ligação/genética , Vibrio cholerae/classificação , Vibrio cholerae/isolamento & purificaçãoRESUMO
Buffering capacity and membrane H+ conductance were examined in three gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis, and Bacillus alcalophilus. An acid pulse technique was used to measure both parameters. The buffering capacity and membrane H+ conductance of B. alcalophilus are influenced by the pH of the medium and the culture conditions. Suspensions of B. alcalophilus cells from both H. A. medium and L-malate medium cultures grown at pH 10.5 exhibited higher values for these parameters than cells grown at pH 8.5. B. alcalophilus grown aerobically had a lower buffering capacity and a lower membrane conductance for protons than the neutrophilic bacteria S. aureus and B. subtilis. Fermenting cells exhibited significantly higher values for both variables than respiring cells.
Assuntos
Bactérias Gram-Positivas/metabolismo , Bacillus/crescimento & desenvolvimento , Bacillus/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Soluções Tampão , Membrana Celular/metabolismo , Meios de Cultura , Condutividade Elétrica , Bactérias Gram-Positivas/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Transporte de Íons , Prótons , Especificidade da Espécie , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismoRESUMO
Strains of the facultative anaerobe Haemophilus influenzae, both type b and non typable strains, are frequently multiresistant. The measurement of the antibiotic permeability of Haemophilus influenzae outer membrane (OM) shows that antibiotics can cross through the OM easily. Thus, enzymatic activity or efflux pumps could be responsible for multiresistance. An efflux system closely related to AcrAB of Escherichia coli is present in Haemophilus influenzae. However, their role in multiresistance seems irrelevant. Classical mechanisms such as plasmid exchange seems to be playing a major role in the multidrug resistance in Haemophilus influenzae.
Assuntos
Proteínas de Transporte , Resistência Microbiana a Medicamentos/fisiologia , Resistência a Múltiplos Medicamentos/fisiologia , Proteínas de Escherichia coli , Haemophilus influenzae/efeitos dos fármacos , Antibacterianos/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Transporte Biológico , Transporte Biológico Ativo , Permeabilidade da Membrana Celular , Resistência Microbiana a Medicamentos/genética , Resistência a Múltiplos Medicamentos/genética , Haemophilus influenzae/genética , Haemophilus influenzae/fisiologia , Lipoproteínas/genética , Lipoproteínas/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Fatores RRESUMO
The genetic structure of a population of multidrug-resistant nontypeable (unencapsulated) Haemophilus influenzae strains isolated at a hospital in Barcelona, Spain, was investigated by using multilocus enzyme electrophoresis to determine the allelic variation in 15 structural loci. In our study we have also included some antimicrobial agent-susceptible strains isolated at the same hospital. All enzymes were polymorphic for two to eight electromorphs, and the analysis revealed 43 distinct electrophoretic types among the 44 isolates. The mean genetic diversity of the entire population was 0.55. Multilocus linkage disequilibrium analysis of the isolates revealed a strong association between alleles, suggesting little possibility of recombination. Furthermore, the dendrogram and the allele mismatch distribution are typical of a population with no extensive genetic mixing.
Assuntos
Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/genética , Alelos , Técnicas de Tipagem Bacteriana , Criança , Resistência a Múltiplos Medicamentos/genética , Enzimas/genética , Feminino , Variação Genética , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/classificação , Hospitais Pediátricos , Humanos , Desequilíbrio de Ligação , EspanhaRESUMO
Buffering capacity and membrane H+ conductance were measured in Halobacterium halobium suspensions in the light and in the dark over a wide range of external pH. The values of both variables for this archaeobacterium were significantly higher than those found for eubacteria in other reports. It appears from our results that the special chemical composition of the cell envelope and the movement of ions, mainly protons, may influence the magnitude of the buffering power and the H+ membrane conductance of these cells.
Assuntos
Halobacterium salinarum/química , Transporte Biológico , Soluções Tampão , Halobacterium salinarum/efeitos dos fármacos , Halobacterium salinarum/fisiologia , Halobacterium salinarum/efeitos da radiação , Concentração de Íons de Hidrogênio , Luz , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Prótons , Valinomicina/farmacologiaRESUMO
The permeability to cephaloridine was studied in five Haemophilus influenzae strains (four non-typable and one type b) using the Zimmermann and Rosselet method. The beta-lactamase activity was due to a plasmid-encoded TEM-1 enzyme. High permeability coefficients were measured in all strains examined. No great differences in permeability coefficients were found, even between strains with marked differences in OMP electrophoretic profiles.
Assuntos
Cefaloridina/farmacocinética , Haemophilus influenzae/metabolismo , Técnicas de Tipagem Bacteriana , Haemophilus influenzae/classificação , Haemophilus influenzae/ultraestrutura , Membranas/metabolismo , Testes de Sensibilidade Microbiana , Permeabilidade , Especificidade da Espécie , beta-Lactamases/metabolismoRESUMO
The influence of analgesics at low concentrations on permeability and beta-lactamase expression have been studied "in vitro". The effect of these drugs on major outer membrane proteins and the lipopolysaccharide was evaluated. Acetylsalicylate and paracetamol induced modifications in susceptibility to a large variety of antibiotics when tested at therapeutic concentrations. The results suggested that when analgesics and antibiotics are administered simultaneously, the interaction between both kinds of drugs can alter the response of microorganisms to antibiotics.
Assuntos
Analgésicos/administração & dosagem , Antibacterianos/administração & dosagem , Serratia marcescens/efeitos dos fármacos , Acetaminofen/administração & dosagem , Aspirina/administração & dosagem , Proteínas da Membrana Bacteriana Externa/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Interações Medicamentosas , Resistência Microbiana a Medicamentos , Humanos , Técnicas In Vitro , Salicilatos/administração & dosagem , Ácido Salicílico , Serratia marcescens/metabolismo , beta-Lactamases/metabolismoRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Bactérias Gram-Negativas/metabolismo , Hidrogênio/metabolismo , Soluções Tampão , Concentração de Íons de Hidrogênio , Transporte de ÍonsRESUMO
O-side chain-defective spontaneous mutants of Serratia marcescens, selected by phage resistance, showed lower MICs against various beta-lactams than did their parental strains. The recovery of their ability to produce O-antigen restored the original MIC values, as well as phage susceptibility. The permeability coefficients of wild-type, O- mutants, and revertants, demonstrated that O-antigen modifies the permeability of antibiotics in S. marcescens.
Assuntos
Antibacterianos/metabolismo , Polissacarídeos Bacterianos/fisiologia , Serratia marcescens/fisiologia , Antibacterianos/farmacologia , Permeabilidade da Membrana Celular/fisiologia , Testes de Sensibilidade Microbiana , Mutação , Antígenos O , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/metabolismo , beta-LactamasRESUMO
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.
Assuntos
Concentração de Íons de Hidrogênio , Prodigiosina/biossíntese , Serratia marcescens/metabolismo , Prolina Oxidase/metabolismo , Serratia marcescens/crescimento & desenvolvimentoRESUMO
The pigmented strain Serratia marcescens ATCC 274 had a higher buffering capacity and a higher membrane H conductance than S. marcescens GP, a spontaneous nonpigmented mutant of ATCC 274. The data suggest that mutations which apparently affect only the synthesis of a secondary metabolite can modify buffering capacity and passive H conductance.
RESUMO
The genetic relatedness of 42 penicillin-resistant Neisseria meningitidis isolates obtained during a 2-year period from a single hospital was studied by multilocus enzyme electrophoresis and by restriction fragment length polymorphism (RFLP) analysis of penicillin-binding protein (PBP) 2 genes. The PBP 2 genes of 7 susceptible strains gave identical RFLP profiles. Sixteen different PBP 2 RFLP profiles were found among the 42 resistant strains, but 4 were found in greater than 1 resistant isolate. Multilocus enzyme electrophoresis revealed a high level of genetic diversity. Four clusters of resistant strains could be distinguished at a genetic distance of 0.75. Resistant strains with the most common PBP 2 RFLP profile were restricted to one of these clusters and may be derived from a common ancestral strain. However, resistant strains with the 3 other common RFLP profiles were distributed in two or more of the clusters.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Variação Genética , Muramilpentapeptídeo Carboxipeptidase , Neisseria meningitidis/genética , Resistência às Penicilinas/genética , Adolescente , Resistência a Ampicilina/genética , Sequência de Bases , Criança , Pré-Escolar , Análise por Conglomerados , Sondas de DNA/química , DNA Bacteriano/química , Enzimas/análise , Hexosiltransferases/genética , Humanos , Lactente , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Neisseria meningitidis/efeitos dos fármacos , Neisseria meningitidis/enzimologia , Hibridização de Ácido Nucleico , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Polimorfismo de Fragmento de RestriçãoRESUMO
Kappa phage active on Serratia marcescens can form plaques on white and red strains with identical efficiencies. To identify the kappa phage receptor, the inactivation of the phage was studied after incubation with several bacterial subcellular fractions. The experiments demonstrated that kappa phage adsorbs to outer membrane fractions of susceptible cells. Proteinase K did not affect the rate of inactivation. Lipopolysaccharide proved to be the primary receptor for kappa phage. Prodigiosin content of the lipopolysaccharide fraction was low.
Assuntos
Bacteriófagos/metabolismo , Lipopolissacarídeos/metabolismo , Receptores Virais/metabolismo , Serratia marcescens/metabolismo , Adsorção , Membrana Celular/metabolismo , Eletroforese em Gel de PoliacrilamidaRESUMO
Serratia marcescens produced a brown pigment when grown in minimal medium in the presence of tyrosine and high concentrations of copper(II) ion. The pigment was not related to the melanin pigments, but was similar to the pigment produced by autooxidation and polymerization of 3,4-dihydroxyphenylacetate, which is synthesized in S. marcescens from tyrosine through the 3,4-dihydroxyphenylacetate catabolic pathway. The enzymes of this pathway were induced under pigment production conditions; however, 3,4-dihydroxyphenylacetate 2,3-dioxygenase remained at low activity levels, permitting the accumulation and excretion of the substrate. Mutants unable to use tyrosine as a sole carbon and energy source were able to produce brown pigments only if the step blocked by the mutation was after the synthesis of 3,4-dihydroxyphenylacetate. The ability to produce brown pigments was common to all the S. marcescens strains tested.