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Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase.

In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.

Amino Acid Sequence↗

Co-composting of PAH-contaminated soil with poultry manure.

AIMS: To investigate the effects of co-composting poultry manure with soil contaminated with different concentrations of polycyclic aromatic hydrocarbons (PAHs), on the degradation of selected PAHs in a static-pile compost system. METHODS: Mispah form (Food & Agricultural Organisation, FAO : lithosol) soil contaminated with PAHs was co-composted with poultry manure for 19 months. The soil was mixed with wood chips in a ratio of 1:1 to improve aeration and then mixed with poultry manure in a ratio of 4:1. A data logger measured temperature monthly. Residual concentrations of selected PAHs in the compost were determined monthly by gas chromatography/flame ionisation detection (GC/FID). Moisture, pH, ash content and C:N ratios were also monitored monthly. Microbial activity was measured by measuring CO(2) evolution. CONCLUSIONS: The results obtained in this experiment have shown that co-composting poultry manure with PAH-contaminated soil is capable of removing large concentrations of high molecular weight PAH from contaminated soil to levels below 1 mg kg(-1) in 19 months. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper adds to the body of knowledge necessary for the development of a cost effective technology for the remediation of soil contaminated with high molecular weight PAHs by providing information on the behaviour of selected PAHs and factors such as nutrient ratio, temperature and pH during composting.

Animals↗

Weak mutators can drive the evolution of fluoroquinolone resistance in Escherichia coli.

Weak mutators are common among clinical isolates of Escherichia coli. We show that the relative mutation rate and the "evolvability of fluoroquinolone resistance" are related by a power law slope of 1.2 over 3 orders of magnitude. Thus, even weak mutators can drive the evolution of fluoroquinolone resistance under selection pressure.

Anti-Bacterial Agents↗

Biological cost of single and multiple norfloxacin resistance mutations in Escherichia coli implicated in urinary tract infections.

Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

Animals↗

Trophic relationships between Saccharomyces cerevisiae and Lactobacillus plantarum and their metabolism of glucose and citrate.

Glucose and citrate are two major carbon sources in fruits or fruit juices such as orange juice. Their metabolism and the microorganisms involved in their degradation were studied by inoculating with an aliquot of fermented orange juice a synthetic model medium containing glucose and citrate. At pH 3.6, their degradation led, first, to the formation of ethanol due to the activity of yeasts fermenting glucose and, eventually, to the formation of acetate resulting from the activity of lactobacilli. The yeast population always outcompeted the lactobacilli even when the fermented orange juice used as inoculum was mixed with fermented beet leaves containing a wider variety of lactic acid bacteria. The evolution of the medium remained similar between pH 3.3 and 5.0. At pH 3.0 or below, the fermentation of citrate was totally inhibited. Saccharomyces cerevisiae and Lactobacillus plantarum were identified as the only dominant microorganisms. The evolution of the model medium with the complex microbial community was successfully reconstituted with a defined coculture of S. cerevisiae and L. plantarum. The study of the fermentation of the defined model medium with a reconstituted microbial community allows us to better understand the behavior not only of fermented orange juice but also of many other fruit fermentations utilized for the production of alcoholic beverages.

Citrates↗

Natural horizontal transfer of a naphthalene dioxygenase gene between bacteria native to a coal tar-contaminated field site.

Horizontal transfer of genes responsible for pollutant biodegradation may play a key role in the evolution of bacterial populations and the adaptation of microbial communities to environmental contaminants. However, field evidence for horizontal gene transfer between microorganisms has traditionally been very difficult to obtain. In this study, the sequences of the 16S rRNA and naphthalene dioxygenase iron-sulfur protein (nahAc) genes of nine naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site, as well as a naphthalene-degrading bacterium from a contaminated site in Washington state and two archetypal naphthalene-degrading strains, were compared. Seven strains from the study site had a single nahAc allele, whereas the 16S rRNA gene sequences of the strains differed by as much as 7.9%. No nahAc alleles from the site were identical to those of the archetypal strains, although the predominant allele was closely related to that of Pseudomonas putida NCIB 9816-4, isolated in the British Isles. However, one site-derived nahAc allele was identical to that of the Washington state strain. Lack of phylogenetic congruence of the nahAc and 16S rRNA genes indicates that relatively recent in situ horizontal transfer of the nahAc gene has occurred, possibly as a direct or indirect consequence of pollutant contamination. Alkaline lysis plasmid preparations and pulsed-field gel electrophoresis have revealed the presence of plasmids ranging in size from 70 to 88 kb in all site isolates. Southern hybridizations with a 407-bp nahAc probe have suggested that the nahAc gene is plasmid borne in all the site isolates but one, a strain isolated from subsurface sediment 400 m upstream from the source of the other site isolates. In this strain and in the naphthalene-degrading strain from Washington state, nahAc appears to be chromosomally located. In addition, one site isolate may carry nahAc on both chromosome and plasmid. Within the group of bacteria with identical nahAc sequences the Southern hybridizations showed that the gene was distributed between plasmids of different sizes and a chromosome. This suggests that plasmid modification after transfer may have been effected by transposons. Horizontal transfer of catabolic genes may play a significant role in the acclimation of microbial communities to pollutants.

Alleles↗

Mutation patterns of the reverse transcriptase and protease genes in human immunodeficiency virus type 1-infected patients undergoing combination therapy: survey of 787 sequences.

The aim of the present study was to evaluate the resistance-associated mutations in 302 human immunodeficiency virus type 1 (HIV-1)-infected patients receiving combination therapy and monitored in Marseille, France, hospitals from January 1997 to June 1998. In the reverse transcriptase (RT) gene, the most frequent mutations were found at codons 215 (53%), 41 (34%), and 67, 70, 184, and 210 (>20%). One deletion and two insertions in the beta3-beta4 hairpin loop of the finger subdomain (codon 69) were detected. Interesting associations and/or exclusions of specific mutations were observed. In 96% of RT genes, a mutation at codon 70 (most frequently, K70R) was associated with a wild-type genotype at position 210 (P < 10(-5)). Similarly, a mutation at codon 210 (most frequently, L210W) was generally associated with mutations at codons 41 (92%) and 215 (96%) but not at codon 219 (16%) or codon 70 (4%) (P < 10(-5)). In the protease gene, the most prevalent mutations were at codons 63 (84%), followed by codons 10, 36, 71, 77, and 93 (ca. 20%). As for RT, pairwise associations of mutations were observed. Analysis of the mutation patterns for patients with undetectable HIV-1 loads revealed a high proportion (65%) of wild-type RT genotypes but only 18% wild-type protease genotypes. For patients with high viral loads (>100,000 copies/ml), more than 50% of the RT and protease genes displayed three or more mutations. The significant correlation between the level of viremia in plasma and the number of resistance mutations in the protease (P = 0.007) and RT (P = 0.00078) genes strengthens the importance of defining the genotype of the predominant HIV-1 quasispecies before initiating antiretroviral therapy.

Amino Acid Substitution↗

Drug resistance evolution of a Mycobacterium tuberculosis strain from a noncompliant patient.

The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.

Adult↗

Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.

A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.

Adult↗

Evolutionary explanations in medical and health profession courses: are you answering your students' "why" questions?

BACKGROUND: Medical and pre-professional health students ask questions about human health that can be answered in two ways, by giving proximate and evolutionary explanations. Proximate explanations, most common in textbooks and classes, describe the immediate scientifically known biological mechanisms of anatomical characteristics or physiological processes. These explanations are necessary but insufficient. They can be complemented with evolutionary explanations that describe the evolutionary processes and principles that have resulted in human biology we study today. The main goal of the science of Darwinian Medicine is to investigate human disease, disorders, and medical complications from an evolutionary perspective. DISCUSSION: This paper contrasts the differences between these two types of explanations by describing principles of natural selection that underlie medical questions. Thus, why is human birth complicated? Why does sickle cell anemia exist? Why do we show symptoms like fever, diarrhea, and coughing when we have infection? Why do we suffer from ubiquitous age-related diseases like arteriosclerosis, Alzheimer's and others? Why are chronic diseases like type II diabetes and obesity so prevalent in modern society? Why hasn't natural selection eliminated the genes that cause common genetic diseases like hemochromatosis, cystic fibrosis, Tay sachs, PKU and others? SUMMARY: In giving students evolutionary explanations professors should underscore principles of natural selection, since these can be generalized for the analysis of many medical questions. From a research perspective, natural selection seems central to leading hypotheses of obesity and type II diabetes and might very well explain the occurrence of certain common genetic diseases like cystic fibrosis, hemochromatosis, Tay sachs, Fragile X syndrome, G6PD and others because of their compensating advantages. Furthermore, armed with evolutionary explanations, health care professionals can bring practical benefits to patients by treating their symptoms of infection more specifically and judiciously. They might also help curtail the evolutionary arms race between pathogens and antibiotic defenses.

Adaptation, Biological↗

Immunotherapy and immunoprevention of cancer: where do we stand?

Although evolution has shaped the immune system to control microbial invasions, this does not necessarily mean that the immune system can not be triggered to eliminate tumour cells. The exploitation of the terrific potential of the immune system to recognise cell alterations and to selectively destroy large populations of neoplastic cells is a possibility made even more attractive by the advances in our understanding of the immune mechanisms and our ability to manipulate them. This review summarises the state of the different immunotherapy strategies available or in development today, and examines the future developments that hold out the promise of an effective control of cancer growth.

Animals↗

Plasmid-mediated antimicrobial resistance in Salmonella enterica.

The selective pressure imposed by the use of antimicrobials in both human and veterinary medicine promotes the spread of multiple antimicrobial resistance. The dissemination of antimicrobial resistance in Salmonella enterica strains, causing severe enteritis in human, has been reported worldwide and is largely attributed to conjugative DNA exchange. In the present review, the relevance of plasmids to the dissemination of antimicrobial resistance in S. enterica is discussed. Recent examples of plasmid-mediated resistance to expanded-spectrum cephalosporins are reported to illustrate the severity of current situation in enteric pathogens. The exchanges between plasmid(s) and the bacterial chromosome and the integration of resistance genes into specialised genetic elements, called integrons, play a major role in acquisition and dissemination of resistance genes. The evolution of a plasmid through the acquisition of integrons is reported, describing novel mechanisms for short-term accumulation of resistance determinants in plasmids circulating in Salmonella.

Animals↗

Communal use of integumental wounds in honey bee (Apis mellifera) pupae multiply infested by the ectoparasitic mite Varroa destructor.

The ectoparasitic bee mite, Varroa destructor, is highly adapted to its natural and adopted honey bee hosts, Apis cerana and Apis mellifera. Adult females perforate the integument of bee pupae in such a way that they and their progeny can feed. We examined the wounds that founder females made, and usually found one, and rarely up to three, integumental wounds on pupae of A. mellifera multiply infested by V. destructor. The punctures were mainly on the 2nd abdominal sternite of the host. These perforations are used repeatedly as feeding sites by these hemolymph-sucking mites and by their progeny. The diameter of the wounds increased during pupal development. In brood cells containing 4-5 invading female mites and their progeny, healing of the wound is delayed, normally occurring just before the imaginal moult of the bee pupa. These wounds are subject to microbial infections, and they are relevant to the evolution of behavioral traits in these parasitic mites and their relations to host bees.

Animals↗

[Control of the fermentation activity during ensilage of Lolium italicum A. Br. and Medicago sativa L].

Samples with different dry matter contents of Lolium italicum A. Br., Medicago sativa L., have been ensiled in hermetically sealed containers, in order to study the evolution of microflora and its activity. The principal microbial groups (coliforms, proteolytics , lactic acid bacteria, clostridia, yeasts) have been detected and enumerated, in anaerobic atmosphere, after ensiling periods of 3, 6, 10, 13, 20, 100 days. At the same time, the samples were chemically analyzed for the detection of: pH, moisture, ashes, total and ammoniacal nitrogen, total reducing sugars, lactic acid, volatile fatty acids, short chain alcohols. The relations found between chemical and microbiological data, show that a strong lactic fermentation is not enough for the inhibition of silage spoiling microorganisms, such as coliforms, yeasts, and (less) proteolytics . This phenomenon may be related to the "quality" of the lactic acid microflora, more than to the characteristics of the environment.

Animal Feed↗