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Chemical ecology of marine microbial defense.

Because marine animals and plants are continuously exposed to a large diversity of potentially harmful microorganisms, it seems reasonable to hypothesize that potential hosts might produce bioactive compounds to deter microbial attack. However, little is known about how host metabolites may defend against harmful microbes or facilitate the growth or colonization of helpful symbionts. While there is a large body of literature describing the antimicrobial activities of marine secondary metabolites, we are only now beginning to understand how these compounds function in an ecological context. For example, there is mounting evidence that nontoxic concentrations of secondary metabolites can have significant effects on microbial behavior, suggesting that certain host-microbe interactions are chemically mediated. Herein, we discuss the importance of employing ecologically relevant assays to elucidate microbiological effects and the need to develop a better understanding of host-microbe associations within an ecologically realistic context. Continued research in this field along with improved techniques will certainly provide further insight into how microbes have influenced the evolution of secondary metabolite production in marine organisms.

Animal Population Groups↗

Nutrition and oral infectious diseases: state of the science.

Immunological competence and nutritional status are two of the most important determinants of morbidity and mortality. Oral diseases, particularly inflammatory periodontal lesions, are caused by viruses and specific periodontopathic bacteria. Paradoxically, the production of cytokines and oxidant molecules, which are supposed to be part of a highly effective mechanism for combating the periodontal pathogens, may damage the host. Malnutrition consistently impairs innate and adaptive defenses of the host, including phagocytic function, cell-mediated immunity, complement system, secretory antibody, and cytokine production and function. In protein-energy malnutrition, there are marked changes in the oral microbial ecology resulting in a preponderance of pathogenic anaerobic organisms, increased propensity of bacteria to bind to oral mucosal cells, attenuation of acute phase protein response, and dysfunction of the cytokine system. Cellular depletion of antioxidant nutrients promotes immunosuppression, accelerated replication rate of ribonucleic acid viruses, and increased disease progression. Therefore, malnutrition can intensify the severity of oral infections and may lead to their evolution into life-threatening diseases.

Acute-Phase Proteins↗

Evolution of sucrose synthesis.

Cyanobacteria and proteobacteria (purple bacteria) are the only prokaryotes known to synthesize sucrose (Suc). Suc-P synthase, Suc-phosphatase (SPP), and Suc synthase activities have previously been detected in several cyanobacteria, and genes coding for Suc-P synthase (sps) and Suc synthase (sus) have been cloned from Synechocystis sp. PCC 6803 and Anabaena (Nostoc) spp., respectively. An open reading frame in the Synechocystis genome encodes a predicted 27-kD polypeptide that shows homology to the maize (Zea mays) SPP. Heterologous expression of this putative spp gene in Escherichia coli, reported here, confirmed that this open reading frame encodes a functional SPP enzyme. The Synechocystis SPP is highly specific for Suc-6(F)-P (K(m) = 7.5 microM) and is Mg(2+) dependent (K(a) = 70 microM), with a specific activity of 46 micromol min(-1) mg(-1) protein. Like the maize SPP, the Synechocystis SPP belongs to the haloacid dehalogenase superfamily of phosphatases/hydrolases. Searches of sequenced microbial genomes revealed homologs of the Synechocystis sps gene in several other cyanobacteria (Nostoc punctiforme, Prochlorococcus marinus strains MED4 and MIT9313, and Synechococcus sp. WH8012), and in three proteobacteria (Acidithiobacillus ferrooxidans, Magnetococcus sp. MC1, and Nitrosomonas europaea). Homologs of the Synechocystis spp gene were found in Magnetococcus sp. MC1 and N. punctiforme, and of the Anabaena sus gene in N. punctiforme and N. europaea. From analysis of these sequences, it is suggested that Suc synthesis originated in the proteobacteria or a common ancestor of the proteobacteria and cyanobacteria.

Archaea↗

Assay of cytomegalovirus susceptibility to ganciclovir in renal and heart transplant recipients.

Ganciclovir (GCV) prophylaxis or pre-emptive therapy significantly reduce the rate of cytomegalovirus (CMV) disease and viremia, but increase the potential for emergence of ganciclovir-resistant CMV strains. The inhibitor concentration at 50% (IC(50)) of GCV from 156 CMV isolates from 59 renal or heart transplant recipients was calculated by means of a rapid phenotypic susceptibility assay. Twenty-seven strains were from 14 patients undergoing GCV therapy. The IC(50) was higher in patients under the prophylaxis regimen. One CMV strain, from a heart transplant recipient, became GCV-resistant after 1 month of therapy (IC(50)=13.7 micromol/l). These data, together with clinical and virological markers, suggested that a switch to foscarnet was necessary, and good evolution was observed. Thus, assay of CMV susceptibility to GCV could be helpful in clinical management.

Antiviral Agents↗

Identification, structure and differential expression of novel pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum).

Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel pleurocidin-like antimicrobial peptides. Four genes and three pseudogenes (Psi) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune-related genes have been identified in upstream regions by comparative genomics. Using reverse transcription-PCR assays, we have shown for the first time that each gene is expressed in a tissue-specific and developmental stage-specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides.

Amino Acid Sequence↗

[Primary antibiotic treatment of corneal ulcers in contact lens patients].

Two patient histories show that the therapy of contact lens corneal ulcers is complicated by bacterial resistance and mixed infection. To start an effective therapy against pseudomonas, gram-negative as well as gram-positive germs, a primary combination of tobramycin with fusidic acid seems to us as reasonable. Because of possible resistance to these antibiotics a smear should be taken from the ulcer and the contact lens case before therapy is begun and the clinical evolution must be controlled. In the choice of the antibiotics, the actual state of resistance and commercial availability was taken into account.

Adult↗

Venus, Mars, and the ices on Mercury and the moon: astrobiological implications and proposed mission designs.

Venus and Mars likely had liquid water bodies on their surface early in the Solar System history. The surfaces of Venus and Mars are presently not a suitable habitat for life, but reservoirs of liquid water remain in the atmosphere of Venus and the subsurface of Mars, and with it also the possibility of microbial life. Microbial organisms may have adapted to live in these ecological niches by the evolutionary force of directional selection. Missions to our neighboring planets should therefore be planned to explore these potentially life-containing refuges and return samples for analysis. Sample return missions should also include ice samples from Mercury and the Moon, which may contain information about the biogenic material that catalyzed the early evolution of life on Earth (or elsewhere). To obtain such information, science-driven exploration is necessary through varying degrees of mission operation autonomy. A hierarchical mission design is envisioned that includes spaceborne (orbital), atmosphere (airborne), surface (mobile such as rover and stationary such as lander or sensor), and subsurface (e.g., ground-penetrating radar, drilling, etc.) agents working in concert to allow for sufficient mission safety and redundancy, to perform extensive and challenging reconnaissance, and to lead to a thorough search for evidence of life and habitability.

Atmosphere↗

Bacterial genomics as a potential tool for discovering new antimicrobial agents.

The past 30 years have witnessed the emergence of new infectious diseases as well as the re-emergence of those thought to be defeated or under control. It is likely that this threat will continue and that infectious micro-organisms will be found to be responsible for numerous diseases whose etiology had been previously unknown. Compounding this threat is the rapid evolution of drug resistance by micro-organisms that is rendering many existing antimicrobial agents obsolete. Thus, there is an urgent need for the development of new classes of antimicrobial agents and the identification of new drug targets. Over the past decade, advances in high-throughput automated DNA sequencing have delivered a wealth of genetic information in the form of whole genome sequences of microbial pathogens. Coupled with this advancement has been the development of new genetic tools and computational advances capable of selecting genes of particular interest as well as testing for the effects of candidate drugs. While no new drugs have yet been developed, further study into the application and limitations of these new approaches to the identification of novel targets will aid in overcoming the current problem of antimicrobial drug resistance.

Bacteria↗

[Spondylodiscitis. Analysis of 25 cases].

BACKGROUND: Spondylodiscitis is a rare but prolonged inflammation of two adjacent vertebral bodies and the disk in between. AIM: To report the clinical features of a series of patients with spondylodiscitis. MATERIAL AND METHODS: A retrospective analysis of medical records of patients with spondylitis, identified between 1989 and 2002. RESULTS: A total of 25 cases were identified, 15 female, aged 49.8 years as a mean. Their mean evolution before admission was 4.3 months. Main complaints were back or radicular pain. Mild anemia was present in most patients. Mean erythrocyte sedimentation rate and C reactive protein values were 66 mm/h and 60 mg/L, respectively. Forty four percent of patients had neurological complications. Vertebral computed tomography and scintigraphic studies were done in 72% of patients, but magnetic resonance imaging was done only in 4 (16%). In 18 patients, a tissue sample for pathological and microbiological analysis, was obtained by imaging guiding or surgically. Tuberculosis, diagnosed on pathology, was the leading cause of spondylitis in nine cases (36%), followed by Staphylococcus aureus infection in five (20%). Other agents found were E coli and group D Streptococcus (one each). Age, symptoms, evolution time and different laboratory parameters did not differ between patients with tuberculosis and patients with other causes. A microbiological cause was not established in 36% of cases. Most patients evolved satisfactorily and recovered from neurological complications (88%). One patient with tuberculosis did not improve after prolonged treatment and 2 patients infected with S aureus died (8%). CONCLUSIONS: Spondylodiscitis is associated to a diversity of microbial agents and in most cases has a favorable prognosis.

Adolescent↗

Evolutionary engineering of industrially important microbial phenotypes.

The tremendous complexity of dynamic interactions in cellular systems often impedes practical applications of metabolic engineering that are largely based on available molecular or functional knowledge. In contrast, evolutionary engineering follows nature's 'engineering' principle by variation and selection. Thus, it is a complementary strategy that offers compelling scientific and applied advantages for strain development and process optimization, provided a desired phenotype is amenable to direct or indirect selection. In addition to simple empirical strain development by random mutation and direct selection on plates, evolutionary engineering also encompasses recombination and continuous evolution of large populations over many generations. Two distinct evolutionary engineering applications are likely to gain more relevance in the future: first, as an integral component in metabolic engineering of strains with improved phenotypes, and second, to elucidate the molecular basis of desired phenotypes for subsequent transfer to other hosts. The latter will profit from the broader availability of recently developed methodologies for global response analysis at the genetic and metabolic level. These methodologies facilitate identification of the molecular basis of evolved phenotypes. It is anticipated that, together with novel analytical techniques, bioinformatics, and computer modeling of cellular functions and activities, evolutionary engineering is likely to find its place in the metabolic engineer's toolbox for research and strain development. This review presents evolutionary engineering of whole cells as an emerging methodology that draws on the latest advances from a wide range of scientific and technical disciplines.

Biological Evolution↗

Recovery of replication-competent HIV despite prolonged suppression of plasma viremia.

In evaluating current combination drug regimens for treatment of human immunodeficiency virus (HIV) disease, it is important to determine the existence of viral reservoirs. After depletion of CD8 cells from the peripheral blood mononuclear cells (PBMCs) of both patients and normal donors, activation of patient CD4 lymphocytes with immobilized antibodies to CD3 and CD28 enabled the isolation of virus from PBMCs of six patients despite the suppression of their plasma HIV RNA to fewer than 50 copies per milliliter for up to 2 years. Partial sequencing of HIV pol revealed no new drug resistance mutations or discernible evolution, providing evidence for viral latency rather than drug failure.

Anti-HIV Agents↗

Comparative analysis of Pseudomonas syringae pv. actinidiae and pv. phaseolicola based on phaseolotoxin-resistant ornithine carbamoyltransferase gene (argK) and 16S-23S rRNA intergenic spacer sequences.

Pseudomonas syringae pv. phaseolicola, which causes halo blight on various legumes, and pv. actinidiae, responsible for canker or leaf spot on actinidia plants, are known as phaseolotoxin producers, and the former possesses phaseolotoxin-resistant ornithine carbamoyltransferase (ROCT) which confers resistance to the toxin. We confirmed that the latter is also resistant to phaseolotoxin and possesses ROCT, and we compared the two pathovars by using sequence data of the ROCT gene and the intergenic spacer region located between the 16S and 23S rRNA genes (16S-23S spacer region) as an index. It was found that the identical ROCT gene (argK) is contained not only in bean isolates of P. syringae pv. phaseolicola in Mexico and the United States but also in bean isolates in Japan and Canada, and that it is also distributed in the kudzu (Pueraria lobata) isolates of P. syringae pv. phaseolicola. Moreover, the kiwifruit and tara vine isolates of P. syringae pv. actinidiae were also found to possess the identical argK. On the contrary, the 16S-23S spacer regions showed a significant level of sequence variation between P. syringae pv. actinidiae and pv. phaseolicola, suggesting that these two pathovars evolved differently from each other in the phylogenetic development. The fact that even synonymous substitution has not occurred in argK among these strains despite their extreme differences in phylogenetic evolution and geographical distribution suggests that it was only recently in evolutionary time that argK was transferred from its origin to P. syringae pv. actinidiae and/or pv. phaseolicola.

Base Sequence↗

Analysis of pol gene heterogeneity, viral quasispecies, and drug resistance in individuals infected with group O strains of human immunodeficiency virus type 1.

Nucleotide sequences of the reverse transcriptase (RT) coding region have been compared in four new human immunodeficiency virus type 1 (HIV-1) group O isolates. Phylogenetic analysis of this pol region highlights a cluster of these four HIV-1 group O sequences with seven other group O isolates (5% intracluster nucleotide sequence diversity) similar to clusters classified as subtypes in HIV-1 group M (an average of 4.9% intrasubtype sequence diversity). Based on these analyses, this group O cluster has been designated subtype A-O. A longitudinal study of a heterosexual couple infected with group O (ESP1 and ESP2) allowed a detailed analysis of RT sequences (amino acids 28 to 219). Directed evolution and a slightly higher mutation frequency was observed in the RT sequences of patient ESP2, treated with antiretroviral drugs, than that from the untreated patient ESP1. Antiretroviral treatment also selected for specific substitutions, M184V and T215Y in the RT coding region, conferring resistance to 3'-dideoxy-3'-thiacytidine and zidovudine, respectively. A Gly98 to Glu RT substitution identified in the treated patient suggests a possible reversion of a nonnucleoside RT inhibitor-resistant phenotype. Using RT clones from this longitudinal study, both heteroduplex tracking assay and cloning-sequencing techniques were employed for an extensive genetic analysis of pol gene quasispecies. Amino acid substitutions (i.e., Phe-77 to Leu, Lys-101 to Glu, and Val-106 to Iso) associated with antiretroviral resistance were identified in RT clones from HIV-1 group O-infected patients not subjected to drug therapy or treated with unrelated drugs. Finally, phylogenetic relationships between RT clones of the treated ESP2 patient and those of the untreated ESP1 patient show how drug pressure can direct evolution of viral pol gene quasispecies independently of direct drug-resistant substitutions.

Adolescent↗

Hospital-acquired pneumonia: risk factors, microbiology, and treatment.

Pneumonia complicates hospitalization in 0.5 to 2.0% of patients and is associated with considerable morbidity and mortality. Risk factors for hospital-acquired pneumonia (HAP) include mechanical ventilation for > 48 h, residence in an ICU, duration of ICU or hospital stay, severity of underlying illness, and presence of comorbidities. Pseudomonas aeruginosa, Staphylococcus aureus, and Enterobacter are the most common causes of HAP. Nearly half of HAP cases are polymicrobial. In patients receiving mechanical ventilation, P aeruginosa, Acinetobacter, methicillin-resistant S aureus, and other antibiotic-resistant bacteria assume increasing importance. Optimal therapy for HAP should take into account severity of illness, demographics, specific pathogens involved, and risk factors for antimicrobial resistance. When P aeruginosa is implicated, monotherapy, even with broad-spectrum antibiotics, is associated with rapid evolution of resistance and a high rate of clinical failures. For pseudomonal HAP, we advise combination therapy with an antipseudomonal beta-lactam plus an aminoglycoside or a fluoroquinolone (eg, ciprofloxacin).

Anti-Bacterial Agents↗

Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor.

AIMS: This paper attempts to provide visual evidence of how aerobic granulation evolves in sequential aerobic sludge blanket reactors. METHODS AND RESULTS: A series of experiments were conducted in two column-type sequential aerobic sludge reactors fed with glucose and acetate as sole carbon source, respectively. The evolution of aerobic granulation was monitored using image analysis and optical and scanning electron microscopy. The results indicated that the formation of aerobic granules was a gradual process from seed sludge to compact aggregates, further to granular sludge and finally to mature granules with the sequential operation proceeding. Glucose- and acetate-fed granules have comparable characteristics in terms of settling velocity, size, shape, biomass density and microbial activity. However, the microbial diversity of the granules was associated with the carbon source supplied. In this work, an important aerobic starvation phase was identified during sequential operation cycles. It was found that periodical aerobic starvation was an effective trigger for microbial aggregation in the reactor and further strengthened cell-cell interaction to form dense aggregates, which was an essential step of granulation. The periodical starvation-induced aggregates would finally be shaped to granules by hydrodynamic shear and flow. CONCLUSION: Aerobic granules can be formed within 3 weeks in the systems. The periodical starvation and hydrodynamic conditions would play a crucial role in the granulation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic granules have excellent physical characteristics as compared with conventional activated sludge flocs. This research could be helpful for the development of an aerobic granule-based novel type of reactor for handling high strength organic wastewater.

Aerobiosis↗

Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis.

Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.

Aminoglycosides↗

Differences in the evolution of imipenem susceptibility among Klebsiella pneumoniae and Escherichia coli isolates during a 6-year period in a tertiary care hospital.

The evolution of imipenem disk-diffusion susceptibility results of 2652 strains of Klebsiella pneumoniae and 7596 Escherichia coli isolated during the period 2000-2005 were analysed. Screening for production of metallo-beta-lactamases was performed using the EDTA-synergy method. The percentage rate of K. pneumoniae isolates having a zone diameter < or =25 mm increased from 20% in 2000 to 41% in 2005, whereas the respective rate of isolates having a zone diameter > or =30 mm decreased from 48 to 23%. These changes were more evident during 2000-2002, followed in 2003 by the isolation of the first imipenem-resistant strains. Regarding E. coli, a similar decrease was observed (the rates of isolates having a zone diameter < or =25 mm and > or =30 mm changed from 7% and 68% in 2000, to 32% and 36% in 2005, respectively) following the respective changes of K. pneumoniae. A total of 20 K. pneumoniae strains, but no E. coli, were confirmed as metallo-beta-lactamase producers. In conclusion, a decrease of the imipenem susceptibility prior to the isolation of the first resistant strains in a tertiary care hospital was detected, as well as differences in this decrease between the two species. These findings indicate that monitoring of the evolution of imipenem susceptibility in real-time may help in unveiling forthcoming resistance and in implementing the appropriate diagnostic techniques.

Anti-Bacterial Agents↗

How do we compare hundreds of bacterial genomes?

The genomic revolution is fully upon us in 2006 and the pace of discovery is set to accelerate with the emergence of ultra-high-throughput sequencing technologies. Our complete genome collection of bacteria and archaea continues to grow in number and diversity, as genome sequencing is applied to an array of new problems, from the characterization of the pan-genome to the detection of mutation after experimentation and the exploration of microbial communities in unprecedented detail. The benefits of large-scale comparative genomic analyses are driving the community to think about how to manage our public collections of genomes in novel ways.

Bacteria↗