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PubMed2005

Peracetic acid in the disinfection of a hospital water system contaminated with Legionella species.

OBJECTIVE: To assess the efficacy of an alternative disinfection method for hospital water distribution systems contaminated with Legionella. METHODS: Disinfection with peracetic acid was performed in a small hospital contaminated with L. pneumophila serotype 1. The disinfectant was used at concentrations of 50 ppm (first three surveillance phases) and 1,000 ppm (fourth surveillance phase) for 30 minutes. RESULTS: Environmental monitoring revealed that disinfection was maintained 1 week after treatment; however, levels of recontamination surpassing baseline values were detected after approximately 1 month. Comparison of water temperatures measured at the distal outlets showed a statistically significant association between temperature and bacterial load. The circulating water temperature was found to be lower in the two wards farthest away from the hot water production plant than in other wards. It was thought that the lower water temperature in the two wards promoted the bacterial growth even after disinfection. CONCLUSION: Peracetic acid may be useful in emergency situations, but does not provide definitive protection even if used monthly.

Colony Count, Microbial↗
PubMed2001

Can correlated mutations in protein domain families be used for protein design?

Evidence from diverse studies, such as protein design experiments and analysis of the emergence of drug resistance in human immunodeficiency virus 1 (HIV-1), indicates that protein function can be diminished or altered by mutations at positions distant from the classic 'functional' site. Furthermore, results from correlation analysis of the ligand-binding domain of nuclear receptors suggest that mutation events at positions distributed throughout a protein domain may be involved in functional diversification during the evolution of homologous domain families. This review explores potential applications for a protein design procedure based on correlated substitutions.

Computational Biology↗
PubMed1996

A library of organic landscapes on filamentous phage.

A billion-clone library of filamentous phage with different surface structures ("landscapes') was generated by fusing random octapeptides to the N-terminus of all 4000 copies of the major coat protein. Such a "landscape library' might include clones exhibiting emergent properties that inhere in the entire surface architecture, not in the peptides by themselves. Because the diverse surface landscapes are displayed on viable phage, they can be surveyed for exceedingly rare functions using microbiological selection methods. Clones with several emergent properties of the sort envisioned were successfully selected, suggesting that landscape libraries have promise as a novel source of nanomaterials with exploitable surface properties.

Amino Acid Sequence↗
PubMed2005

Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin.

An urgent need exists to prevent the sexual transmission of HIV-1. With prevalence rates exceeding 35% in parts of sub-Saharan Africa, increasing attention has been placed on developing and testing microbicidal agents capable of preventing virus transmission at mucosal sites. HIV-1 microbicides must meet several requirements before their widespread use. The drugs must be able to neutralize a diversity of HIV-1 strains, not induce mucosal inflammation, be associated with minimal side effects, and be effective for a prolonged period after a single application. Recent work has demonstrated the utility of recombinant lactic acid bacteria (LAB) as agents of mucosal drug delivery. Here, we describe the bioengineering of strains of LAB to secrete the prototypic virucidal compound cyanovirin (CV-N) and demonstrate the anti-HIV-1 activity of secreted CV-N. Our results suggest that recombinant LAB may serve as effective microbicidal compounds and deserve in vivo testing in simian immunodeficiency virus models of mucosal virus transmission.

Bacterial Proteins↗
PubMed1989

Turnover of extracellular DNA in eutrophic and oligotrophic freshwater environments of southwest Florida.

The turnover of extracellular DNA was investigated in oligotrophic springs of the Crystal River and the eutrophic Medard Reservoir of southwest Florida. The Medard Reservoir possessed large populations of bacterioplankton and phytoplankton (6.8 x 10 cells per liter and 28.6 mug of chlorophyll a per liter, respectively), while the Crystal River springs only contained a fraction of the microbial biomass found in the Medard Reservoir. Although dissolved DNA values were greater in the Medard Reservoir, higher rates of DNA removal resulted in similar extracellular DNA turnover times in both environments (9.62 +/- 3.6 h in the Crystal River and 10.5 +/- 2.1 h in the Medard Reservoir). These results indicate that regardless of trophic status or microbial standing stock, extracellular DNA turns over rapidly in subtropical planktonic freshwater environments. Therefore, recombinant DNA sequences from released genetically engineered microorganisms might not be expected to survive for long periods of time in freshwater planktonic environments.

Journal Article↗
PubMed2000

Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone.

Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.

Anaerobiosis↗
PubMed1984

Pseudomonas in the sinks in an intensive care unit: relation to patients.

Sink drains in a medical-surgical intensive care unit (ICU) were cultured during six consecutive weeks as part of a seven month prospective study of acquisition of Pseudomonas aeruginosa by ICU patients. Isolates were typed serologically and by aminoglycoside and chlorhexidine susceptibility patterns. All 11 sinks contained multiple strains of P aeruginosa; some strains persisted for weeks while others were isolated once. Of the sink isolates 56% had high level resistance to gentamicin and tobramycin whereas none of the strains found in patients. In sink isolates chlorhexidine resistance correlated with aminoglycoside resistance and with the presence of a chlorhexidine dispenser at a sink. The sequence of recovery of phenotypically similar isolates suggested that sinks were the source of at most two acquisitions of P aeruginosa by patients during the six weeks. Our study confirms that sinks may be reservoirs for large numbers of highly resistant P aeruginosa but are rarely the source of organisms colonising patients in our ICU.

Aminoglycosides↗
PubMed2000

Improvement of microbial strains and fermentation processes.

Improvement of microbial strains for the overproduction of industrial products has been the hallmark of all commercial fermentation processes. Conventionally, strain improvement has been achieved through mutation, selection, or genetic recombination. Overproduction of primary or secondary metabolites is a complex process, and successful development of improved strains requires a knowledge of physiology, pathway regulation and control, and the design of creative screening procedures. In addition, it requires mastery of the fermentation process for each new strain, as well as sound engineering know-how for mediaoptimization and the fine-tuning of process conditions. This review focuses on the various options that may be employed to improve microbial strains and addresses the complex problems of screening, the tools and technology behind the selection of targeted organisms, and the importance of process optimization. Furthermore, this review discusses new and emerging technologies and designing optimized media for tracking mutants with enhanced productivity or other desired attributes.

Bacteria↗
PubMed1991

Nuclear magnetic resonance spectroscopy of dense cell populations for metabolic studies and bioreactor engineering: a synergistic partnership.

Commercial exploitation of the fruits of recombinant DNA and cell fusion technologies is significantly limited by the lack of fundamental metabolic information on the cell lines of interest, whether these are plant, animal, insect, or microbial cells. NMR can help to provide this information and thereby improve bioreactor design and operation. However, in the case of on-line NMR of dense cell culture devices for metabolic studies, these devices are inherently heterogeneous bioreactors. To ensure that the metabolic information generated is reliable, a number of precautions should be taken. These are the same precautions that should be taken to ensure that commercial bioreactors operate in a reaction-controlled regime. Therefore, reactor engineering methodologies, particularly diffusion and reaction analyses and reaction monitoring by whole-cell NMR must go hand in hand, each extending, complementing, and validating the other.

Animals↗
PubMed2006

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

Animals↗
PubMed2004

Inverse metabolic engineering with phosphagen kinase systems improves the cellular energy state.

Inverse metabolic engineering attempts to identify or construct desired phenotypes of applied interest to endow them on appropriate host organisms. A particular desirable phenotype is the ATP homeostasis exhibited by animal cells with high and variable ATP turnover through temporal and spatial energy buffering. This buffering is achieved by phosphagen kinase systems that consist of a specific kinase and its cognate phosphagen, which functions as a large pool of 'high-energy phosphates' that are used to replenish ATP during periods of high energetic demand. This review discusses recent advances and potentials of inverse metabolic engineering of cell types that do not normally contain such systems--bacteria, yeast, plants, and liver--with creatine or arginine kinase systems. Examples are discussed that illustrate how microbial metabolism can be tailored for large-scale industrial processes with imperfect mixing and how the liver can be protected from metabolic insults or stimulated for better regeneration.

Adenosine Triphosphate↗
PubMed2002

Metabolic engineering towards biotechnological production of carotenoids in microorganisms.

Carotenoids are important natural pigments produced by many microorganisms and plants. Traditionally, carotenoids have been used in the feed, food and nutraceutical industries. The recent discoveries of health-related beneficial properties attributed to carotenoids have spurred great interest in the production of structurally diverse carotenoids for pharmaceutical applications. The availability of a considerable number of microbial and plant carotenoid genes that can be functionally expressed in heterologous hosts has opened ways for the production of diverse carotenoid compounds in heterologous systems. In this review, we will describe the recent progress made in metabolic engineering of non-carotenogenic microorganisms for improved carotenoid productivity. In addition, we will discuss the application of combinatorial and evolutionary strategies to carotenoid pathway engineering to broaden the diversity of carotenoid structures synthesized in recombinant hosts.

Bacteria↗
PubMed2002

[Microbial warfare and bioterrorism].

Infectious diseases have been used as warfares since ancient times. Since the 1920s military organizations have studied bacteria of anthrax, plague, tularemia, botulism, brucelloses, glander, Q-fever, and smallpox virus, Filo-, Arena-, Bunyaviruses causing hemorrhagic fever or Alphaviruses eliciting encephalitis. These can be dispersed by aerosol. Salmonellae, Shigellae, Vibrio cholerae, distinguished Escherichia coli strains are suitable to contaminate food, water, pharmaceutical products. Fanatical groups or terrorist individuals deploy microbe weapons. In the future, genetically engineered recombinant microbes could be used with genomes containing multiple resistance elements to antimicrobial compounds and additional virulence factors. These become resistant to all known treatment regimens, vaccination and the host immune response. Microbial terrorist attacks result in an outbrake on a restricted area with large number of casualties. The disease course is severe and unusual followed by high mortality. Identification of microbes is complicated and delayed. Most countries have neither laboratories at high biosafety level nor specially trained personnel. Physicians might misdiagnose these diseases. Health care systems with minimal elasticity face difficulties in maintaining mass quarantine. A considerable part of health care workers leave hospitals. No plan is available to stockpile medicines. Robust surveillance and laboratory systems coordinated at international level must be established. All health care personnel should be trained periodically to gain practical skills. Additional standards governing working conditions with selected microbes will be enforced by law. Related scientific data might be published with restricted access only.

Animals↗
PubMed2000

Increased sensitivity to quinolone antibacterials can be engineered in human topoisomerase IIalpha by selective mutagenesis.

A potential region of drug-DNA interaction in the A subunit of DNA gyrase has previously been identified from crystallographic studies. The local amino acid sequence has been compared with similar regions in yeast topoisomerase II and human topoisomerase IIalpha. Three non- conserved, potentially solvent-accessible residues at positions 762, 763 and 766 in human topoisomerase IIalpha lie between well-conserved regions. The corresponding residues in GyrA (83, 84 and 87) have a high frequency of mutation in quinolone-resistant bacteria. Mutations in human topoisomerase IIalpha have been generated in an attempt to engineer ciprofloxacin sensitivity into this enzyme: M762S, S763A and M766D (each mutated to the identical amino acid present in gyrase), along with an M762S/S763A double mutant and a triple mutant. These enzymes were introduced into a temperature-sensitive yeast strain, deficient in topoisomerase II, for in vivo studies, and were overproduced for in vitro studies. The M766D mutation renders the enzyme incapable of supporting the temperature-sensitive strain at a non-permissive temperature. However, both M766D and the triple mutant enzymes can be overproduced and are fully active in vitro. The double mutant was impaired in its ability to cleave DNA and had reduced catalytic activity. The triple mutation confers a three-fold increase in sensitivity to ciprofloxacin in vitro and similar sensitivities to a range of other quinolones. The activity of the quinolone CP-115,953, a bacterial and eukaryotic topoisomerase II poison, was unaffected by any of these mutations. Mutations in this region were found to increase the sensitivity of the enzyme to the DNA intercalating anti-tumour agents m-AMSA and ellipticine, but confer resistance to the non-intercalating agents etoposide, teniposide and merbarone, an effect that was maximal in the triple mutant. We have therefore shown the importance of this region in determining the sensitivity of topoisomerase II to drugs and have engineered increased sensitivity to quinolones.

Amino Acid Sequence↗
PubMed1989

Microbial metabolism of pesticides and structurally related compounds.

This chapter provides a review concerning the microbial metabolism of pesticides and substances that are either major metabolites from pesticides or have structural similarity to certain pesticides, and covers the period 1981 to 1987. While reference has only been made to work published during this period, it should be realized that in some instances the results cited may confirm or expand upon earlier findings rather than being entirely novel. Therefore, the reader is referred to earlier reviews. The metabolism of pesticides in natural environments, water and wastewater, mixed microbial cultures, and pure cultures has been discussed. Attention has been drawn to the meager amount of information concerning the biodegradation of pesticides in anaerobic and marine environments. Issues such as the importance of cometabolism of pesticides in natural environments and a clear understanding of enhanced degradation of pesticides in soil still remain unresolved. Separate sections have been devoted to methodology in biodegradation studies, bound residues and removal of pesticides from soil and water. While pure culture studies have an important place in investigations into microbial metabolism of pesticides, increasing emphasis has been placed on the use of microbial consortia, either natural or artificial and microcosms to provide an understanding of pesticide biodegradation in natural environments. Another dimension in bound residue formation, one of physical entrapment in humic materials has been described. Various questions regarding the bioavailability of bound residues and whether they pose an environmental problem have not been answered fully. The microbiological removal of pesticides from soil and water by selected or genetically-engineered strains is discussed. It has been emphasized that the future success of such methods for the decontamination of soil and water depends very heavily on an improved knowledge of microbial ecology.

Biodegradation, Environmental↗