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Evolution of a vancomycin-intermediate Staphylococcus aureus strain in vivo: multiple changes in the antibiotic resistance phenotypes of a single lineage of methicillin-resistant S. aureus under the impact of antibiotics administered for chemotherapy.

A number of methicillin-resistant Staphylococcus aureus (MRSA) isolates were recovered over a period of several weeks from blood samples and from the heart valve of a patient who underwent extensive vancomycin chemotherapy for persistent S. aureus bacteremia. Consecutive isolates showed gradually decreasing growth rates during in vitro cultivation and increasing vancomycin MICs, from an MIC of 1 micro g/ml for the initial isolate to an MIC of 8 micro g/ml for the final MRSA isolates, which also became tolerant to vancomycin. Major changes were observed in the oxacillin resistance phenotype of several of the isolates-apparently related to in vivo exposure to imipenem, which was also used during a period of chemotherapy. Both the gradually increasing vancomycin MICs and the changes in oxacillin resistance could be reproduced by appropriate exposure of the initial MRSA isolate to antibiotics in vitro. All isolates had the same pulsed-field gel electrophoresis pattern, spaA type, and multilocus sequence type (MLST), which was identified as a single-locus variant of ST5, the MLST characteristic of previously characterized MRSA isolates with reduced susceptibility to vancomycin in the United States and Japan.

Anti-Bacterial Agents↗

Evolution of dnaQ, the gene encoding the editing 3' to 5' exonuclease subunit of DNA polymerase III holoenzyme in Gram-negative bacteria.

The nucleotide sequences of the dnaQ genes from Salmonella typhimurium and Buchnera aphidicola, encoding the epsilon-subunit of the DNA polymerase III holoenzyme, have been determined. The Salmonella typhimurium dnaQ protein consists of 243 amino acid residues with a calculated molecular weight of 27224. The Buchnera aphidicola dnaQ protein contains 233 amino acid residues with a calculated molecular weight of 27170. A multiple sequence alignment of the amino acid sequences of the dnaQ proteins and those of DNA polymerase IIIs from Gram-positive bacteria produced six homologous segments. These homologous segments contain highly conserved amino acid sequence motifs involved in catalytically important metal ion bindings (ligands 1, 2 and 3). However, metal ligand 4 is found to be altered in the 3'-5' exonuclease domain of the family C DNA polymerases and dnaQ proteins in Gram-negative bacteria. From these results, we propose that the last common ancestor of the dnaQ gene of Gram-negative bacteria and the DNA polymerase III gene (pol C gene) of Gram-positive bacteria was a single gene containing both 3'-5' exonuclease and DNA polymerase domains and then the dnaQ gene separated from the polymerase gene in Gram-negative bacteria.

Amino Acid Sequence↗

Change of pneumococcal resistance to antibiotics in adults between 1995 and 1997: a study in eight French counties.

The main object of this study was to describe the evolution of antibiotic resistance in pneumococci from adults, in eight French counties of France between 1995 and 1997. Despite the high and increasing prevalence (23-35%) of pneumococci with diminished susceptibility to penicillin G (PSDP), resistance to amoxycillin (0.8-0.5%) and to cefotaxime (0-0.3%) was rare in both 1995 and 1997 respectively. The percentage of pneumococci resistant to penicillin G (PRP, minimum inhibitory concentration >1 mg/l) remained stable between the two periods. PSDP showed increased resistance to macrolides (30-41%), to cotrimoxazole (28-34%) and to tetracycline (19-25%). These figures are lower than those obtained over the same periods and the same regions in children. The distribution of PSDP serotypes isolated in adults was the same as that seen in children: by descending order serotypes 23, 14, 9 and 6. This study by the Regional Pneumococcal Observatories confirms the high prevalence and the main characteristics of antibiotic resistance among pneumococci with variations in levels of resistance with the age of patients, with the site of sampling and from one Observatory to another.

Adolescent↗

Comparative molecular evolution of primary (Buchnera) and secondary symbionts of aphids based on two protein-coding genes.

A+T content, phylogenetic relationships, codon usage, evolutionary rates, and ratio of synonymous versus non-synonymous substitutions have been studied in partial sequences of the atpD and aroQ/pheA genes of primary ( Buchnera) and secondary symbionts of aphids and a set of selected non-symbiotic bacteria, belonging to the five subdivisions of the Proteobacteria. Compared to the homologous genes of the last group, both genes belonging to Buchnera behave in a similar way, showing a higher A+T content, forming a monophyletic group, a loss in codon bias, especially in third base position, an evolutionary acceleration and an increase in the number of non-synonymous substitutions, confirming previous results reported elsewhere for other genes. When available, these properties have been partly observed with the secondary symbionts, but with values that are intermediate between Buchnera and free living Proteobacteria. They show high A+T content, but not as high as Buchnera, a non-solved phylogenetic position between Buchnera, and the other gamma-Proteobacteria, a loss in codon bias, again not as high as in Buchnera and a significant evolutionary acceleration in the case of the three atpD genes, but not when considering aroQ/pheA genes. These results give support to the hypothesis that they are symbionts at different stages of the symbiotic accommodation to the host.

AT Rich Sequence↗

Ancestral population sizes and species divergence times in the primate lineage on the basis of intron and BAC end sequences.

The effective sizes of ancestral populations and species divergence times of six primate species (humans, chimpanzees, gorillas, orangutans, and representatives of Old World monkeys and New World monkeys) are estimated by applying the two-species maximum likelihood (ML) method to intron sequences of 20 different loci. Examination of rate heterogeneity of nucleotide substitutions and intragenic recombination identifies five outrageous loci (ODC1, GHR, HBE, INS, and HBG). The estimated ancestral polymorphism ranges from 0.21 to 0.96% at major divergences in primate evolution. One exceptionally low polymorphism occurs when African and Asian apes diverged. However, taking into consideration the possible short generation times in primate ancestors, it is concluded that the ancestral population size in the primate lineage was no smaller than that of extant humans. Furthermore, under the assumption of 6 million years (myr) divergence between humans and chimpanzees, the divergence time of humans from gorillas, orangutans. Old World monkeys, and New World monkeys is estimated as 7.2, 18, 34, and 65 myr ago, respectively, which are generally older than traditional estimates. Beside the intron sequences, three other data sets of orthologous sequences are used between the human and the chimpanzee comparison. The ML application to these data sets including 58,156 random BAC end sequences (BES) shows that the nucleotide substitution rate is as low as 0.6-0.8 x 10(-9) per site per year and the extent of ancestral polymorphism is 0.33-0.51%. With such a low substitution rate and short generation time, the relatively high extent of polymorphism suggests a fairly large effective population size in the ancestral lineage common to humans and chimpanzees.

Animals↗

Evolution of an autotransporter: domain shuffling and lateral transfer from pathogenic Haemophilus to Neisseria.

The genomes of pathogenic Haemophilus influenzae strains are larger than that of Rd KW20 (Rd), the nonpathogenic laboratory strain whose genome has been sequenced. To identify potential virulence genes, we examined genes possessed by Int1, an invasive nonencapsulated isolate from a meningitis patient, but absent from Rd. Int1 was found to have a novel gene termed lav, predicted to encode a member of the AIDA-I/VirG/PerT family of virulence-associated autotransporters (ATs). Associated with lav are multiple repeats of the tetranucleotide GCAA, implicated in translational phase variation of surface molecules. Laterally acquired by H. influenzae, lav is restricted in distribution to a few pathogenic strains, including H. influenzae biotype aegyptius and Brazilian purpuric fever isolates. The DNA sequence of lav is surprisingly similar to that of a gene previously described for Neisseria meningitidis. Sequence comparisons suggest that lav was transferred relatively recently from Haemophilus to Neisseria, shortly before the divergence of N. meningitidis and Neisseria gonorrhoeae. Segments of lav predicted to encode passenger and beta-domains differ sharply in G+C base content, supporting the idea that AT genes have evolved by fusing domains which originated in different genomes. Homology and base sequence comparisons suggest that a novel biotype aegyptius AT arose by swapping an unrelated sequence for the passenger domain of lav. The unusually mobile lav locus joins a growing list of genes transferred from H. influenzae to Neisseria. Frequent gene exchange suggests a common pool of hypervariable contingency genes and may help to explain the origin of invasiveness in certain respiratory pathogens.

Amino Acid Sequence↗

Molecular evolution perspectives on intraspecific lateral DNA transfer of topoisomerase and gyrase loci in Streptococcus pneumoniae, with implications for fluoroquinolone resistance development and spread.

Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.

Amino Acid Sequence↗

Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements.

BACKGROUND: Mobile elements are involved in genomic rearrangements and virulence acquisition, and hence, are important elements in bacterial genome evolution. The insertion of some specific Insertion Sequences had been associated with repetitive extragenic palindromic (REP) elements. Considering that there are a sufficient number of available genomes with described REPs, and exploiting the advantage of the traceability of transposition events in genomes, we decided to exhaustively analyze the relationship between REP sequences and mobile elements. RESULTS: This global multigenome study highlights the importance of repetitive extragenic palindromic elements as target sequences for transposases. The study is based on the analysis of the DNA regions surrounding the 981 instances of Insertion Sequence elements with respect to the positioning of REP sequences in the 19 available annotated microbial genomes corresponding to species of bacteria with reported REP sequences. This analysis has allowed the detection of the specific insertion into REP sequences for ISPsy8 in Pseudomonas syringae DC3000, ISPa11 in P. aeruginosa PA01, ISPpu9 and ISPpu10 in P. putida KT2440, and ISRm22 and ISRm19 in Sinorhizobium meliloti 1021 genome. Preference for insertion in extragenic spaces with REP sequences has also been detected for ISPsy7 in P. syringae DC3000, ISRm5 in S. meliloti and ISNm1106 in Neisseria meningitidis MC58 and Z2491 genomes. Probably, the association with REP elements that we have detected analyzing genomes is only the tip of the iceberg, and this association could be even more frequent in natural isolates. CONCLUSION: Our findings characterize REP elements as hot spots for transposition and reinforce the relationship between REP sequences and genomic plasticity mediated by mobile elements. In addition, this study defines a subset of REP-recognizer transposases with high target selectivity that can be useful in the development of new tools for genome manipulation.

Bacteria↗

Sequence diversity and molecular evolution of the heat-modifiable outer membrane protein gene (ompA) of Mannheimia(Pasteurella) haemolytica, Mannheimia glucosida, and Pasteurella trehalosi.

The OmpA (or heat-modifiable) protein is a major structural component of the outer membranes of gram-negative bacteria. The protein contains eight membrane-traversing beta-strands and four surface-exposed loops. The genetic diversity and molecular evolution of OmpA were investigated in 31 Mannheimia (Pasteurella) haemolytica, 6 Mannheimia glucosida, and 4 Pasteurella trehalosi strains by comparative nucleotide sequence analysis. The OmpA proteins of M. haemolytica and M. glucosida contain four hypervariable domains located at the distal ends of the surface-exposed loops. The hypervariable domains of OmpA proteins from bovine and ovine M. haemolytica isolates are very different but are highly conserved among strains from each of these two host species. Fourteen different alleles representing four distinct phylogenetic classes, classes I to IV, were identified in M. haemolytica and M. glucosida. Class I, II, and IV alleles were associated with bovine M. haemolytica, ovine M. haemolytica, and M. glucosida strains, respectively, whereas class III alleles were present in certain M. haemolytica and M. glucosida isolates. Class I and II alleles were associated with divergent lineages of bovine and ovine M. haemolytica strains, respectively, indicating a history of horizontal DNA transfer and assortative (entire gene) recombination. Class III alleles have mosaic structures and were derived by horizontal DNA transfer and intragenic recombination. Our findings suggest that OmpA is under strong selective pressure from the host species and that it plays an important role in host adaptation. It is proposed that the OmpA protein of M. haemolytica acts as a ligand and is involved in binding to specific host cell receptor molecules in cattle and sheep. P. trehalosi expresses two OmpA homologs that are encoded by different tandemly arranged ompA genes. The P. trehalosi ompA genes are highly diverged from those of M. haemolytica and M. glucosida, and evidence is presented to suggest that at least one of these genes was acquired by horizontal DNA transfer.

Adaptation, Biological↗

Molecular evolution of the pathogenicity island of enterotoxigenic Bacteroides fragilis strains.

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.

Amino Acid Sequence↗

Stimulation of DNA repair as an evolutionary drive for bacterial luminescence.

It was demonstrated recently that luminescence of a free-living marine bacterium, Vibrio harveyi, stimulates DNA repair, most probably by activation of the photoreactivation process. Here, we ask whether the stimulation of DNA repair could be an evolutionary drive that ensured maintenance and development of early bacterial luminescent systems. To test this hypothesis, we cultivated V. harveyi lux(+) bacteria and luxA mutants in mixed cultures. Initial cultures were mixed to obtain a culture consisting of roughly 50% lux(+) cells and 50% luxA mutants. Then bacteria were cultivated for several days and ratio of luminescent to dark bacteria was measured. Under these conditions, luxA mutants became highly predominant within a few days of cultivation. This indicates that, without a selective pressure, the luminescence is a disadvantage for bacteria, perhaps due to consumption of significant portion of cell energy. However, when the same experiments were repeated but cultures were irradiated with low UV doses, luminescent bacteria started to predominate shortly after the irradiation. Therefore, we conclude that stimulation of photoreactivation may be an evolutionary drive for bacterial bioluminescence.

DNA Repair↗

Parallel acceleration of evolutionary rates in symbiont genes underlying host nutrition.

The overproduction of essential amino acids by Buchnera aphidicola, the primary bacterial mutualist of aphids, is considered an adaptation for increased production of nutrients that are lacking in aphids' diet of plant sap. Given their shared role in host nutrition, amino acid biosynthetic genes of Buchnera are expected to experience parallel changes in selection that depend on host diet quality, growth rate, and population structure. This study evaluates the hypothesis of parallel selection across biosynthetic pathways by testing for correlated changes in evolutionary rates at biosynthetic genes of Buchnera. Previous studies show fast evolutionary rates at tryptophan biosynthetic genes among Buchnera associated with the aphid genus Uroleucon and suggest reduced purifying selection on symbiont nutritional functions in this aphid group. Here, we test for parallel rate acceleration at other amino acid biosynthetic genes of Buchnera-Uroleucon, including those for leucine (leuABC) and isoleucine/valine biosynthesis (ilvC). Ratios of nonsynonymous to synonymous substitutions (d(N)/d(S)) were estimated using codon-based maximum-likelihood methods that account for the extreme AT compositional bias of Buchnera sequences. A significant elevation in d(N)/d(S) at biosynthetic loci but not at two housekeeping genes sampled (dnaN and tuf) suggests reduced host-level selection on biosynthetic capabilities of Buchnera-Uroleucon. In addition, the discovery of trpEG pseudogenes in Buchnera-U. obscurum further supports reduced selection on amino acid biosynthesis.

Alcohol Oxidoreductases↗

Genome size in bacteria.

This manuscript examines genome size in bacteria. The opposing capability of bacteria to alter their genome sizes and order of genes within limits yet remain somewhat constant provides a mechanisms for diversity and evolution in bacterial populations. Bacteria may have evolved by increasing their genome size and changing gene orders with the assistance of restriction endonucleases cleaving foreign DNA and providing a diverse pool of DNA sequences for recombination. These aspects and selected information on physically mapping bacterial genomes will be discussed.

Chromosome Mapping↗

Phylogenetic analysis shows that the OXA beta-lactamase genes have been on plasmids for millions of years.

The OXA genes encode a class of beta-lactamases that confer resistance to a wide range of beta-lactam antibiotics. To determine whether the diversity of the OXA beta-lactamases is the result of recent or ancient events, and to determine whether mobilization of the OXA genes from chromosomes to plasmids occurred recently or long ago, we have constructed a Bayesian phylogeny of the OXA beta-lactamase genes. Analysis of that phylogeny shows that much of the diversity is the result of ancient events and that the OXA genes were mobilized from chromosomes to plasmids on at least two independent occasions that occurred millions of years ago. That observation contradicts the commonly held impression that mobilization of antibiotic resistance genes is strictly the result of modern use of antibiotics.

Bayes Theorem↗

Independent origins of subgroup Bl + B2 and subgroup B3 metallo-beta-lactamases.

The metallo-beta-lactamases constitute Class B in the Ambler classification of beta-lactamases and are divided into three subclasses: Bl, B2, and B3. Bayesian phylogenies of the Subclass B1 + B2 and Subclass B3 metallo-beta-lactamases and their homologs show that the beta-lactam-hydrolyzing function evolved independently within each group. In Subclass B1+B2 that function evolved about 1 billion years ago, and in Subclass B3 it evolved before the divergence of the Gram-positive and Gram-negative eubacteria, about 2 billion years ago. These results lend additional support to the proposal that the metallo-beta-lactamases should be divided into two distinct classes.

Archaea↗

Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori comprise two geographically widespread types, m1 and m2, and have evolved through limited recombination.

Vacuolating cytotoxin (vacA) alleles of Helicobacter pylori vary, particularly in their mid region (which may be type m1 or m2) and their signal peptide coding region (type s1 or s2). We investigated nucleotide diversity among vacA alleles in strains from several locales in Asia, South America, and the USA. Phylogenetic analysis of vacA mid region sequences from 18 strains validated the division into two main groups (m1 and m2) and showed further significant divisions within these groups. Informative site analysis demonstrated one example of recombination between m1 and m2 alleles, and several examples of recombination among alleles within these groups. Recombination was not sufficiently extensive to destroy phylogenetic structure entirely. Synonymous nucleotide substitution rates were markedly different between regions of vacA, suggesting different evolutionary divergence times and implying horizontal transfer of genetic elements within vacA. Non-synonymous/synonymous rate ratios were greater between m1 and m2 sequences than among m1 sequences, consistent with m1 and m2 alleles encoding functions fitting strains for slightly different ecological niches.

Alleles↗

Diversity of group II introns in the genome of Sinorhizobium meliloti strain 1021: splicing and mobility of RmInt1.

The number and diversity of known group II introns in eubacteria are continually increasing with the addition of new data from sequencing projects, but the significance of these introns in the evolution of bacterial genomes is unknown. We analyzed the main features of the group II introns present in the genome of the soil microorganism Sinorhizobium meliloti (strain 1021), the nitrogen-fixing symbiont of alfalfa, the DNA sequence of which was recently determined. Strain 1021 harbors three different classes of group II introns: RmInt1, of bacterial class D; SMb2147/SMb21167, which cluster within bacterial class C; and SMa1875, the phylogenetic class of which is uncertain. The group II introns SMb2147/SMb21167 and SMa1875 are widely distributed in S. meliloti, but are present in lower copy numbers than RmInt1. Strain 1021 harbors three copies of RmInt1, which is pSym-specific. Although RmInt1 is spliced in strain 1021, mobility assays suggested that, in contrast to other S. meliloti strains, the genetic background of strain 1021 does not support intron homing events.

Bacterial Proteins↗