Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase.

UNLABELLED: CcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group of Alphaproteobacteria. In Caulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, that C. crescentus can significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrM populations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulated ftsZ expression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrM mutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulates ftsZ and mipZ transcription, uncovering a previously unsuspected link between metabolic regulation and cell division in Alphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrM cells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM. IMPORTANCE: In bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness in Caulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of the ftsZ cell division gene. This finding suggests that the most critical function of CcrM in C. crescentus is to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to other Alphaproteobacteria.

Assessment of the levels of degradation in fat co- and by-products for feed uses and their relationships with some lipid composition parameters

This paper discusses the levels of degradation of some co- and byproducts of the food chain intended for feed uses. As the first part of a research project, 'Feeding Fats Safety', financed by the sixth Framework Programme-EC, a total of 123 samples were collected from 10 European countries, corresponding to fat co- and byproducts such as animal fats, fish oils, acid oils from refining, recycled cooking oils, and other. Several composition and degradation parameters (moisture, acid value, diacylglycerols and monoacylglycerols, peroxides, secondary oxidation products, polymers of triacylglycerols, fatty acid composition, tocopherols, and tocotrienols) were evaluated. These findings led to the conclusion that some fat by- and coproducts, such as fish oils, lecithins, and acid oils, show poor, nonstandardized quality and that production processes need to be greatly improved. Conclusions are also put forward about the applicability and utility of each analytical parameter for characterization and quality control.

Improvements in the Assessment of Bacterial Viability and Killing

It is axiomatic that our planet is extensively inhabited by diverse micro-organisms such as bacteria, yet the absolute diversity of different bacterial species is widely held to be unknown. Different bacteria can be found from the depths of the oceans to the top of the mountains; even the air is more or less colonized by bacteria. Most bacteria are either harmless or even advantageous to human beings but there are also bacteria, which can cause severe infectious diseases or spoil the supplies intended for human consumption. Therefore, it is vitally important not only to be able to detect and enumerate bacteria but also to assess their viability and possible harmfulness. Whilst the growth of bacteria is remarkably fast under optimum conditions and easy to detect by cultural methods, most bacteria are believed to lie in stationary phase of growth in which the actual growth is ceased and thus bacteria may simply be undetectable by cultural techniques. Additionally, several injurious factors such as low and high temperature or deficiency of nutrients can turn bacteria into a viable but non-culturable state (VBNC) that cannot be detected by cultural methods. Thereby, various noncultural techniques developed for the assessment of bacterial viability and killing have widely been exploited in modern microbiology. However, only a few methods are suitable for kinetic measurements, which enable the real-time detection of bacterial growth and viability. The present study describes alternative methods for measuring bacterial viability and killing as well as detecting the effects of various antimicrobial agents on bacteria on a real-time basis. The suitability of bacterial (lux) and beetle (luc) luciferases as well as green fluorescent protein (GFP) to act as a marker of bacterial viability and cell growth was tested. In particular, a multiparameter microplate assay based on GFP-luciferase combination as well as a flow cytometric measurement based on GFP-PI combination were developed to perform divergent viability analyses. The results obtained suggest that the antimicrobial activities of various drugs against bacteria could be successfully measured using both of these methods. Specifically, the data reliability of flow cytometric viability analysis was notably improved as GFP was utilized in the assay. A fluoro-luminometric microplate assay enabled kinetic measurements, which significantly improved and accelerated the assessment of bacterial viability compared to more conventional viability assays such as plate counting. Moreover, the multiparameter assay made simultaneous detection of GFP fluorescence and luciferase bioluminescence possible and provided extensive information about multiple cellular parameters in single assay, thereby increasing the accuracy of the assessment of the kinetics of antimicrobial activities on target bacteria.

Oxidative stress in gastric mucosa of asymptomatic humans infected with Helicobacter pylori: effect of bacterial eradication.

BACKGROUND: Inducible nitric oxide synthase (iNOS) and interleukin 8 (IL-8) are positive in approximately 50% of Helicobacter pylori-related diseases but it is not clear whether oxidative stress is also present in H. pylori asymptomatic humans. Our aim was to study the expression of iNOS, superoxide dismutase, catalase and IL-8 production in H. pylori-infected asymptomatic humans, and to investigate the effect of eradication of H. pylori. MATERIALS AND METHODS: Biopsies of corpus and antrum of asymptomatic H. pylori positive and negative humans served for determination of the gastritis score and H. pylori status; iNOS was measured by reverse transcriptase polymerase chain reaction and immunohistochemistry and superoxide dismutase and catalase by immunohistochemistry. IL-8 in biopsies was assessed by enzyme-linked immunosorbent assay. RESULTS: Immunostaining of iNOS, catalase and superoxide dismutase was significantly associated with H. pylori infection and was localized to inflammatory cells. IL-8 concentrations were greater in the H. pylori positive than H. pylori negative group and decreased after bacterial eradication. A decrease in staining for iNOS and catalase was observed after H. pylori eradication. CONCLUSIONS: INOS and antioxidant enzymes are present in gastric biopsies of asymptomatic H. pylori positive humans. Eradication caused a significant decrease in staining for iNOS and catalase. These results indicate that oxidative stress occurs in asymptomatic patients and can be modulated by H. pylori eradication.

CSF lactate for accurate diagnosis of community-acquired bacterial meningitis.

CSF lactate measurement is recommended when nosocomial meningitis is suspected, but its value in community-acquired bacterial meningitis is controversial. We evaluated the diagnostic performance of lactate and other CSF parameters in a prospective cohort of adult patients with acute meningitis. Diagnostic accuracy of lactate and other CSF parameters in patients with microbiologically documented episodes was assessed by receiver operating characteristic (ROC) curves. The cut-offs with the best diagnostic performance were determined. Forty-five of 61 patients (74%) had a documented bacterial (n = 18; S. pneumoniae, 11; N. meningitidis, 5; other, 2) or viral (n = 27 enterovirus, 21; VZV, 3; other, 3) etiology. CSF parameters were significantly different in bacterial vs. viral meningitis, respectively (p < 0.001 for all comparisons): white cell count (median 1333 vs. 143/mm(3)), proteins (median 4115 vs. 829 mg/l), CSF/blood glucose ratio (median 0.1 vs. 0.52), lactate (median 13 vs. 2.3 mmol/l). ROC curve analysis showed that CSF lactate had the highest accuracy for discriminating bacterial from viral meningitis, with a cutoff set at 3.5 mmol/l providing 100% sensitivity, specificity, PPV, NPV, and efficiency. CSF lactate had the best accuracy for discriminating bacterial from viral meningitis and should be included in the initial diagnostic workup of this condition.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Differential regulation of horizontally acquired and core genome genes by the bacterial modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization.

Comparison of differential gene expression to water stress among bacteria with relevant pollutant-degradation properties.

Resistance to semi-dry environments has been considered a crucial trait for superior growth and survival of strains used for bioaugmentation in contaminated soils. In order to compare water stress programmes, we analyse differential gene expression among three phylogenetically different strains capable of aromatic compound degradation: Arthrobacter chlorophenolicus A6, Sphingomonas wittichii RW1 and Pseudomonas veronii 1YdBTEX2. Standardized laboratory-induced water stress was imposed by shock exposure of liquid cultures to water potential decrease, induced either by addition of solutes (NaCl, solute stress) or by addition of polyethylene glycol (matric stress), both at absolute similar stress magnitudes and at those causing approximately similar decrease of growth rates. Genome-wide differential gene expression was recorded by micro-array hybridizations. Growth of P. veronii 1YdBTEX2 was the most sensitive to water potential decrease, followed by S. wittichii RW1 and A. chlorophenolicus A6. The number of genes differentially expressed under decreasing water potential was lowest for A. chlorophenolicus A6, increasing with increasing magnitude of the stress, followed by S. wittichii RW1 and P. veronii 1YdBTEX2. Gene inspection and gene ontology analysis under stress conditions causing similar growth rate reduction indicated that common reactions among the three strains included diminished expression of flagellar motility and increased expression of compatible solutes (which were strain-specific). Furthermore, a set of common genes with ill-defined function was found between all strains, including ABC transporters and aldehyde dehydrogenases, which may constitute a core conserved response to water stress. The data further suggest that stronger reduction of growth rate of P. veronii 1YdBTEX2 under water stress may be an indirect result of the response demanding heavy NADPH investment, rather than the presence or absence of a suitable stress defence mechanism per se.

Thermal Degradation of Small Molecules: A Global Metabolomic Investigation.

Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). In this study the effect of heating was examined on a set of 64 small molecule standards and, separately, on human plasma metabolite extracts. The samples, either derivatized or underivatized, were heated at three different temperatures (60, 100, and 250 °C) at different exposure times (30 s, 60 s, and 300 s). All the samples were analyzed by liquid chromatography coupled to electrospray ionization mass spectrometry (LC/MS) and the data processed by XCMS Online ( xcmsonline.scripps.edu ). The results showed that heating at an elevated temperature of 100 °C had an appreciable effect on both the underivatized and derivatized molecules, and heating at 250 °C created substantial changes in the profile. For example, over 40% of the molecular peaks were altered in the plasma metabolite analysis after heating (250 °C, 300s) with a significant formation of degradation and transformation products. The analysis of 64 small molecule standards validated the temperature-induced changes observed on the plasma metabolites, where most of the small molecules degraded at elevated temperatures even after minimal exposure times (30 s). For example, tri- and diorganophosphates (e.g., adenosine triphosphate and adenosine diphosphate) were readily degraded into a mono-organophosphate (e.g., adenosine monophosphate) during heating. Nucleosides and nucleotides (e.g., inosine and inosine monophosphate) were also found to be transformed into purine derivatives (e.g., hypoxanthine). A newly formed transformation product, oleoyl ethyl amide, was identified in both the underivatized and derivatized forms of the plasma extracts and small molecule standard mixture, and was likely generated from oleic acid. Overall these analyses show that small molecules and metabolites undergo significant time-sensitive alterations when exposed to elevated temperatures, especially those conditions that mimic sample preparation and analysis in GC/MS experiments.

Preparation of a blood culture pellet for rapid bacterial identification and antibiotic susceptibility testing.

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

Emerging bacterial pathogens: the past and beyond.

Since the 1950s, medical communities have been facing with emerging and reemerging infectious diseases, and emerging pathogens are now considered to be a major microbiologic public health threat. In this review, we focus on bacterial emerging diseases and explore factors involved in their emergence as well as future challenges. We identified 26 major emerging and reemerging infectious diseases of bacterial origin; most of them originated either from an animal and are considered to be zoonoses or from water sources. Major contributing factors in the emergence of these bacterial infections are: (1) development of new diagnostic tools, such as improvements in culture methods, development of molecular techniques and implementation of mass spectrometry in microbiology; (2) increase in human exposure to bacterial pathogens as a result of sociodemographic and environmental changes; and (3) emergence of more virulent bacterial strains and opportunistic infections, especially affecting immunocompromised populations. A precise definition of their implications in human disease is challenging and requires the comprehensive integration of microbiological, clinical and epidemiologic aspects as well as the use of experimental models. It is now urgent to allocate financial resources to gather international data to provide a better understanding of the clinical relevance of these waterborne and zoonotic emerging diseases.

Presence of Chlamydiales DNA in samples negative by broad-range bacterial 16S rRNA PCRs: new insights into chlamydial pathogenic role.

Since routine eubacterial 16S rRNA PCR does not amplify members of the Chlamydiales order, we tested all samples received in our laboratory during a 10 months period using a pan-Chlamydiales real-time PCR. 3 of 107 samples (2.8%) revealed to be positive, suggesting a role of some Chlamydiales in the pathogenesis of chronic bronchial stenosis or bronchial stenosis superinfection and as agents of orthopaedic prosthesis infections.