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Is the nectar redox cycle a floral defense against microbial attack?

Many angiosperms use a remarkable reproductive strategy that relies on attracting animals (insect, avian or mammalian pollinators) to transfer pollen between plants. Relying on other organisms for sexual reproduction seems evolutionarily untenable, but the great diversity of angiosperms illustrates how highly successful this strategy is. To attract pollinators, plants offer a variety of rewards. Perhaps the primary floral reward is floral nectar. Plant nectar has long been considered a simple sugar solution but recent work has demonstrated that nectar is a complex biological fluid containing significant and important biochemistry with the potential function of inhibiting microbial growth. These results lead the way to novel insights into the mechanisms of floral defense and the co-evolution of angiosperms and their pollinators.

Bacteria↗

Evolution of susceptibility of non-typhi Salmonella in a Spanish hospital (1992-1994) and report of a Salmonella ser. Typhimurium isolate resistant to quinolones.

We report the evolution of the antimicrobial resistance of non-typhi Salmonella (1992-1994) in a Spanish hospital in relation with a case of infection due to a fluoroquinolone-resistant strain of Salmonella ser. Typhimurium. None of the isolates were resistant to ciprofloxacin. The resistance to ampicillin has increased from 20.6% (1992) to 29.5% (1994) whereas no significative differences in chloramphenicol and cotrimoxazole susceptibility were noted.

Anti-Infective Agents↗

Rapid evolution of a protein in vitro by DNA shuffling.

DNA shuffling is a method for in vitro homologous recombination of pools of selected mutant genes by random fragmentation and polymerase chain reaction (PCR) reassembly. Computer simulations called genetic algorithms have demonstrated the importance of iterative homologous recombination for sequence evolution. Oligonucleotide cassette mutagenesis and error-prone PCR are not combinatorial and thus are limited in searching sequence space. We have tested mutagenic DNA shuffling for molecular evolution in a beta-lactamase model system. Three cycles of shuffling and two cycles of backcrossing with wild-type DNA, to eliminate non-essential mutations, were each followed by selection on increasing concentrations of the antibiotic cefotaxime. We report here that selected mutants had a minimum inhibitory concentration of 640 micrograms ml-1, a 32,000-fold increase and 64-fold greater than any published TEM-1 derived enzyme. Cassette mutagenesis and error-prone PCR resulted in only a 16-fold increase.

Base Sequence↗

[Induction of direct antimicrobial activity through mammalian toll-like receptors].

Drosophila, the toll gene controls a powerful innate defense system against bacteria and fungi. Conserved through evolution, the mammalian innate immune system retains a family of homologous Toll-like receptors (TLRs) that are activated by microbial ligands to release cytokines that instruct the adaptive immune responses. Here we show that TLR2 activation leads to killing of intracellular Mycobacterium (M.) tuberculosis in both mouse and human macrophages. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli. In human monocytes and alveolar macrophages, bacterial lipoproteins similarly activated TLR2 to kill intracellular M. tuberculosis, however by an antimicrobial pathway that is nitric oxide independent. TLR2+CD14+CD68+ macrophages were detected in human lesions of tuberculous lymphadenitis within granulomas and surrounding foci of necrosis. These data provide evidence that mammalian TLRs have retained not only the structural features of Drosophila Toll that allow them to respond to microbial ligands, but also the ability directly to activate antimicrobial effector pathways at the site of infection.

Animals↗

Aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in Escherichia coli.

Bacillus circulans NRRL B-3312, a nonpathogenic bacterium that produces the aminoglycoside antibiotic butirosin, is known to contain an aminoglycoside phosphotransferase that is similar to the neomycin phosphotransferases of clinically isolated antibiotic-resistant bacteria. Purified DNAs from B. circulans and the plasmid ColE1-ApR were digested with EcoRI endonuclease and the resulting fragments covalently joined with polynucleotide ligase. The recombined DNA was used to transform E. coli and ampicillin-neomycin resistant colonies were selected. Analysis of several clones indicated that neomycin resistance in the E. coli transformants was due to the presence of the B. circulans phosphotransferase gene. This observation is consistent with the notion that anitbiotic-modifying enzymes from antibiotic-producing organisms may be the sources of antibiotic resistance in plasmid-containing bacteria.

Bacillus↗

Streptolydigin-resistant mutants in an evolutionarily conserved region of the beta' subunit of Escherichia coli RNA polymerase.

Mutations conferring streptolydigin resistance onto Escherichia coli RNA polymerase have been found exclusively in the beta subunit (Heisler, L. M., Suzuki, H., Landick, R., and Gross, C. A. (1993) J. Biol. Chem. 268, 25369-25375). We report here the isolation of a streptolydigin-resistant mutation in the E. coli rpoC gene, encoding the beta' subunit. The mutation is the Phe793-->Ser substitution, which occurred in an evolutionarily conserved segment of the beta' subunit. The homologous segment in the eukaryotic RNA polymerase II largest subunit harbors mutations conferring alpha-amanitin resistance. Both streptolydigin and alpha-amanitin are inhibitors of transcription elongation. Thus, the two antibiotics may inhibit transcription in their respective systems by a similar mechanism, despite their very different chemical nature.

Amino Acid Sequence↗

Evolution of erythromycin-resistant Streptococcus pneumoniae from Asian countries that contains erm(B) and mef(A) genes.

To investigate the genetic characteristics of erythromycin-resistant Streptococcus pneumoniae in Asian countries, 110 pneumococcal isolates were analyzed by multilocus sequence typing (MLST). MLST analysis showed that 2 clonal complexes--CC236 (Taiwan19F-14 clone) and CC81 (Spain23F-1 clone)--are the major lineages of erythromycin-resistant S. pneumoniae in the Asian region. Pneumococcal isolates containing both the erm(B) and the mef(A) genes are thought to have originated from the Taiwan19F-14 clone containing the mef(A) gene, after introduction of the erm(B) gene. Further evolution of this variant clone has generated resistant strains with different sequence types. Dissemination of these variant clones of the Taiwan19F-14 could be the main reason for the high frequency of pneumococcal isolates containing both erm(B) and mef(A) in some Asian countries. Data suggest that the high prevalence of erythromycin-resistant S. pneumoniae in the Asian region is partly due to the clonal spread of a few multidrug-resistant clones.

Asia↗

Evolutionary barriers to quinolone resistance in Streptococcus pneumoniae.

It is assumed that bacteria always pay a significant physiological price for the acquisition of resistance to antibiotics. To test whether this was the case for a strain of Streptococcus pneumoniae that develops resistance to fluoroquinolone antibiotics, we selected resistance to these agents in a wild-type strain and measured their fitness in comparative growth experiments. The relative growth rate of a mutant strain selected on ciprofloxacin (parC Serine 79 to Tyrosine) was compared with its susceptible isogenic parent and no significant deficit was found (relative fitness 1.15 95% C.I. +/- 0.2.). A double mutant, however, had a relative fitness of 0.81 (parC Serine 79 to Tyrosine gyrA Serine 81 to Tyrosine). Mutant strains selected on gemifloxacin had only a modest increase in minimum inhibitory concentration; thus, second-round mutants were competed with a first-round gyrA Serine 81 to Tyrosine or the susceptible isogenic parent. The growth rate of three double-mutant strains parC Serine 79 to Tyrosine gyrA Serine 81 to Phenylanine, parC Serine 79 to Tyrosine, and Asparagine 83 to Phenylalanine were similar to the isogenic susceptible parent 1.16 (95% C.I. +/- 0.17), 0.99 (95% C.I. +/- 0.05), and 0.95 (95% C.I. +/- 0.05), respectively. These data suggest that mutation in the parC and gyrA genes may, on some occasions, not be associated with a physiological deficit.

4-Quinolones↗

Multilocus sequence typing of hospital-associated Enterococcus faecium from Brazil reveals their unique evolutionary history.

We studied the genetic relationships between vancomycin-susceptible (n = 11) and -resistant Enterococcus faecium (VRE, n = 20) recovered from Brazil using a multilocus sequence typing (MLST) scheme. Grouping of allelic profiles revealed six clusters of related sequence types (STs) that differ in no more than two of the seven alleles. Of these, one cluster harbored 16 of the 20 isolates recovered during the first VRE outbreak in Brazil. The ampicillin and gentamicin resistance profiles were stable in the isolates that clustered within the groups I-III. Comparison with the allelic profiles of 139 E. faecium from different geographical regions and origins found in the international database http://www.mlst.net revealed that the Brazilian outbreak clone did not cluster in the previously named complex-17. This genetic complex contains hospital epidemic and clinical isolates recovered from different countries and continents. Twenty two of the 31 Brazilian isolates, including the VRE outbreak clone, clustered apart from the E. faecium isolates from the database, suggesting that these Brazilian isolates have a distinct evolutionary history.

Alleles↗

Genetic and biochemical basis of resistance of Enterobacteriaceae to beta-lactam antibiotics.

Resistance to beta-lactam drugs is usually determined by genes mediating the production of beta-lactamases. These genes can be located on resistance plasmids or on the chromosome. Resistance to drugs which have been available for many years is mostly transposable. Although the origin of these genes is not known, it is possible to draw a hypothetical flow diagram of the evolution of resistance genes in general. The mechanism of resistance although mediated in Gram-negative bacteria mostly by beta-lactamases cannot be simply described as the hydrolytic function of the enzyme. It is a complex interaction involving the affinity of the drug for the target and the lactamase, the amount of drug in the periplasmic space, the amount of enzyme and the number of lethal target sites. Usually one of these factors is predominant.

Anti-Bacterial Agents↗

Genetic basis for dissemination of armA.

OBJECTIVES AND METHODS: armA is a novel plasmid-borne 16S rRNA methyltransferase that confers high-level resistance to 4,6-disubstituted deoxystreptamines. Recently, we have isolated from a high-level broad-spectrum aminoglycoside-resistant Escherichia coli animal isolate a plasmid, pMUR050, that bore the armA gene. In order to elucidate the genetic basis for the spread of armA, we have determined the complete nucleotide sequence of pMUR050. RESULTS: armA was borne by a complex transposon composite flanked by two direct repeats of IS26. The transposon composite included a class one integron with sul1 for resistance to sulphonamides and ant3''9 conferring resistance to spectinomycin-streptomycin, and a macrolide efflux pump and mefE/mel conferring high-level resistance to erythromycin. We identified in GenBank that another plasmid, pCTX-M3, from a Polish Citrobacter freundii human isolate, bore the same genetic structure, including armA. CONCLUSIONS: armA is present in human and animal isolates within a novel transposon composite. Further spread of armA between bacteria of diverse origin is to be expected.

Aminoglycosides↗

Fusidic acid resistance, mediated by fusB, in bovine coagulase-negative staphylococci.

OBJECTIVES: The aim of this study was to determine the occurrence of fusidic acid resistance, mediated by the fusB gene, among coagulase-negative staphylococci (CoNS) isolated from bovine mastitis. METHODS: A total of 113 CoNS isolates were screened for susceptibility to fusidic acid by using a disc diffusion method. The fusB gene was detected by using PCR and subsequent DNA sequencing. The localization of fusB was determined by hybridization. RESULTS: The fusB gene was detected in 3 of 11 fusidic acid-resistant bovine CoNS isolates. The organization of the fusB downstream region on a 40 kb plasmid in a Staphylococcus haemolyticus isolate (288/96) was highly similar to the previously reported organization of fusB on plasmid pUB101 from a Staphylococcus aureus isolate of human origin. The fusB gene was chromosomally located in the remaining two isolates. CONCLUSIONS: Fusidic acid resistance mediated by fusB is not the dominant resistance mechanism in fusidic acid-resistant CoNS studied in this work. The similarity between the organization of the fusB downstream region in S. haemolyticus (isolate 288/96) and on plasmid pUB101 from an S. aureus isolate of human origin indicates a common ancestral origin of these genes.

Animals↗

Nucleotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase.

The nucleotide sequence of a transposon Tn7 DNA fragment encoding a 3"(9)-O-nucleotidyltransferase, an aminoglycoside-modifying enzyme, which mediates bacterial resistance to spectinomycin and streptomycin, was determined. The aadA structural gene was 786 bases long and predicted a polypeptide of 262 amino acids with a calculated molecular weight of 29,207. Comparison of the DNA sequences of Tn7 and plasmid R538-1 indicated that their aadA genes were nearly identical. Comparison of the polypeptides predicted by the aadA genes of Tn7 and Tn554 indicated that the genes were related.

Amino Acid Sequence↗

Molecular comparison of the IncX plasmids allows division into IncX1 and IncX2 subgroups.

We have investigated molecular relationships and evolution of plasmids classified genetically to incompatibility (Inc) group X, in particular by comparison of plasmids from the pre-antibiotic era (PAE) with contemporary R-plasmids. On the basis of restriction analysis, R6K, the best-described and 'prototype' plasmid of the IncX group, exhibited little similarity with the other plasmids in this Inc group. Other contemporary IncX R-plasmids exhibited a substantial degree of interrelationship, and were also related to PAE IncX plasmids. When the origin of plasmid replication of R6K was used as a replicon probe, R6K was the only plasmid tested which exhibited homology. Other contemporary and PAE IncX plasmids exhibited homology with the origin of plasmid R485. These data suggest that the IncX group should be subdivided. R485 may be regarded as representative of the major subgroup present before and after the advent of antibiotic selection pressure. Plasmids of this subgroup, IncX1, possess an internal region which yields five characteristic EcoRV fragments. R6K may be regarded as representative of subgroup IncX2, of which it is presently the sole well-described member. The antibiotic resistances encoded by contemporary IncX R-plasmids are due to insertion of identifiable transposons in progenitor plasmids identical to the R485 subgroup of PAE IncX plasmids.

Biological Evolution↗

Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants.

Gene transfer within bacterial communities has been recognized as a major contributor in the recent evolution of antibiotic resistance on a global scale. The linked strA-strB genes, which encode streptomycin-inactivating enzymes, are distributed worldwide and confer streptomycin resistance in at least 17 genera of gram-negative bacteria. Nucleotide sequence analyses suggest that strA-strB have been recently disseminated. In bacterial isolates from humans and animals, strA-strB are often linked with the suIII sulfonamide-resistance gene and are encoded on broad-host-range nonconjugative plasmids. In bacterial isolates from plants, strA-strB are encoded on the Tn3-type transposon Tn5393 which is generally borne on conjugative plasmids. The wide distribution of the strA-strB genes in the environment suggests that gene transfer events between human, animal, and plant-associated bacteria have occurred. Although the usage of streptomycin in clinical medicine and animal husbandry has diminished, the persistence of strA-strB in bacterial populations implies that factors other than direct antibiotic selection are involved in maintenance of these genes.

Animals↗

Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa.

A new integron, located on the R plasmid of Pseudomonas aeruginosa, was isolated in Japan. This integron was made up of two conserved segments (5'-and 3'-conserved segments) and a single streptomycin resistance gene as a gene cassette. The structure of this integron resembles that of integron InC, the existence of which was postulated by Bissonnette and Roy (J. Bacteriol. 174, 1248-1257, 1992).

Biological Evolution↗