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Chokeberry (Aronia melanocarpa) fruit extracts (CE) are rich in polyphenols and usually exhibit immunomodulatory, anti-viral and anti-bacterial effects. We have previously shown that the CE used in this study activated macrophages and stimulated effector T cell differentiation in vitro. When applied orally to healthy mice, CE increased the proportion of CD11c+ dendritic cells in the gut-associated lymphoid tissue. CE-pretreated BALB/c mice readily eradicated orally ingested Listeria monocytogenes as evidenced by a slighter decrease in body weight and number of bacteria recovered from the spleen and reduced spleen size compared to the control infected mice. CE pretreatment in infected mice resulted in higher proportions of CD11b+ macrophages and CD8+ cytotoxic T cells both in the gut and the spleen. Phagocytosis, reactive oxygen species production and the proportions of activated CD86+ macrophages (CD11b+) and dendritic cells (CD11c+) was also enhanced in CE-pretreated infected mice. Further, the expression of inducible nitric oxide synthase and IL-6 was increased in CE-pretreated infected mice and the similar results were obtained in peritoneal macrophages in vitro. This effect of CE was associated with increased phosphorylation of IκB and Notch1 production. Finally, CE pretreatment elevated the proportion of perforin-producing cells in the spleen compared to control infected mice. This study demonstrates that prophylactic treatment with CE leads to more rapid eradication of bacterial infection with Listeria monocytogenes predominantly through increased activity of myeloid cells in the gut and in the spleen. This is the peer reviewed version of the following article: Gajić D, Saksida T, Koprivica I, Šenerović L, Morić I, Šavikin K, Menković N, Pejnović N, Stojanović I. Immunomodulatory activity and protective effects of chokeberry fruit extract on Listeria monocytogenes infection in mice. Food Funct. 2020;[DOI:10.1039/d0fo00946f].

The rapid emergence of antibiotic-resistant pathogens presents a global healthcare challenge. Bacteria control their virulence, motility, and biofilm formation, all of them being required for establishing pathogenicity, through a cell density-dependent communication system known as quorum sensing (QS). QS comprises production of extracellular signaling molecules, their detection, and population-wide response involving regulation of the virulence genes expression. Inhibition of QS affects virulence and reduces harmful effects to the host and as such presents a promising strategy to fight antibiotic-resistant infections. Multiresistant Pseudomonas aeruginosa belogns to the group of most critical pathogens for which the introduction of new therapeutics is imperative. In the search for novel therapeutics nature continues to be inexhaustible source of bioactive scaffolds, which provide the bases for structure-based rational drug design enabling further exploitation of diverse natural structures. This review describes bacterial QS systems, highlights strategies for their manipulation, overviews nature-inspired antivirulence molecules primarily against P. aeruginosa, and discusses their perspectives.

This study discusses utilization of supercritical carbon dioxide for impregnation of cellulose acetate films with thymol in order to produce material with anti-biofilm activity against Pseudomonas aeruginosa and Staphylococcus aureus. Analysis of anti-biofilm activity of cellulose acetate beads impregnated with thymol suggested that optimal thymol loading was in the range from 26% to 30% for efficient reduction of biofilm formation and eradication of pre-formed biofilms. Polymer films were fabricated by the solvent casting method from polymer solutions of different contents, and loaded with thymol using supercritical carbon dioxide at 15.5 MPa and 35 °C. The film containing 30% of thymol (F1 30%) exhibited substantial anti-adhesion properties inhibiting biofilm formation on its surface and considerably reduced formation of biofilms on the surrounding surfaces (up to 80%) by all tested strains including antibiotic resistant P. aeruginosa DM50 and methicillin-resistant S. aureus. This is the peer-reviewed version of the article: Zizovic, I.; Senerovic, L.; Moric, I.; Adamovic, T.; Jovanovic, M.; Krusic, M. K.; Misic, D.; Stojanovic, D.; Milovanovic, S. Utilization of Supercritical Carbon Dioxide in Fabrication of Cellulose Acetate Films with Anti-Biofilm Effects against Pseudomonas Aeruginosa and Staphylococcus Aureus. The Journal of Supercritical Fluids 2018, 140, 11–20. [https://doi.org/10.1016/j.supflu.2018.05.025]

Selected plants of white fir and lime, damaged during severe weather episode on the mountain Avala (Serbia) in summer 2014, were analyzed and characterized (including their spatial soil samples) by inductively coupled plasma optical emission spectroscopy (ICP-OES), infrared (IR) thermography, and microbiological method such as enumeration of cultivable microorganisms. The results obtained from chemical and microbiological analyses provided valuable information on possible biotic and abiotic stressors such as soil fungi and heavy metals, which could affect the health status of trees, while IR thermography visualized this status in a very specific and effective way. The results of ICP-OES analysis clearly showed that the investigated heavy metals (Cu, Zn, Pb, As, Cd, and Ni) were less likely crucial factors responsible for ruined health status of damaged trees. The role of soil fungi was not clear, since the results of microbiological analysis only provided evidence that their amounts in all investigated soil samples were within normal ranges as well as that their amounts in the corresponding samples of the uprooted trees were much greater than in the case of snapped trees. Therefore, further molecular characterization of microorganisms should be performed to identify if pathogenic species are present and clarify their role. Nevertheless, all used methods, especially IR thermal imaging as a totally non-invasive, fast and very comfortable technique, can be recommended as very useful in preventive screening of the trees’ health status and for early detection of tissue decay that usually hamper trees survival or resistance to extreme weather events.

The study reports the application of supercritical solvent impregnation to deliver antibacterial substance thymol to the defined polymeric structure of commercial polyamide microfiltration membranes. The process performed at a temperature of 40 °C and pressures of 10, 15, and 20 MPa was characterized by the fast impregnation in the first 30 min with thymol loadings up to 29 wt%. The maximal achieved thymol loading in the process was around 42 wt%, regardless the pressure. The scanning electron and ion microscopy analyses revealed that the membranes’ microstructure was preserved with a thymol content of 20 wt%, while a more prominent swelling effect was observed in samples with 35 wt% of thymol. The cross-flow filtration tests showed that the impregnation process did not harm the membranes’ functionality in samples with 20 wt% of thymol. These membranes exhibited strong anti-biofilm activity, and were less prone to the blockage with bacteria in comparison to neat membranes.

Antibacterial resistance is a growing public health threat of major concern around the world so development of new therapeutic approaches to prevent bacterial multidrug resistance has become a primary consideration for medicinal chemistry research. QSAR models for the dihydrofolate reductase inhibition with 2,4-diamino-5-(substituted-benzyle)-pyramidine derivatives were developed with further computer-aided design of new derivatives with desired activity. The Monte Carlo method has been used as a computational tool for QSAR modeling. For the representation of molecular structure and optimal descriptor calculation, the simplified molecular input line entry system (SMILES) together with the molecular graph (hydrogen-suppressed graph—HSG, hydrogen-filled graph—HFG, and the graph of atomic orbitals-GAO) was used. One-variable models have been calculated for one data split into training, test, and validation set. The impact of Morgan’s extended connectivity index on built QSAR models and outliers was determined. Statistical parameters for the best QSAR model are satisfying. Structural indicators (molecular fragments) responsible for the increase and the decrease of the stated activity are defined, and with the application of defined structural alerts, the computer-aided design of new derivatives with desired activity is presented. Computational experiments presented and applied in this research can satisfactorily predict desired endpoint and can be used further for computer-aided antibacterial drug design.

Antibiotic resistance has become a serious global threat to public health; therefore, improved strategies and structurally novel antimicrobials are urgently needed to combat infectious diseases. Here we report a new type of highly potent 4-aminoquinoline derivatives as quorum sensing inhibitors in Serratia marcescens and Pseudomonas aeruginosa, exhibiting weak bactericidal activities (minimum inhibitory concentration (MIC) gt 400 mu M). Through detailed structure-activity study, we have identified 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines (5 and 10) as biofilm formation inhibitors with 50% biofilm inhibition at 69 mu M and 63 mu M in S. marcescens and P. aeruginosa, respectively. These two compounds, 5 and 10, are the first quinoline derivatives with anti-biofilm formation activity reported in S. marcescens. Quantitative structure-activity relationship (QSAR) analysis identified structural descriptors such as Wiener indices, hyper-distance-path index (HDPI), mean topological charge (MTC), topological charge index (TCI), and log D(o/w)exp as the most influential in biofilm inhibition in this bacterial species. Derivative 10 is one of the most potent quinoline type inhibitors of pyocyanin production described so far (IC50 = 2.5 mu M). While we have demonstrated that 5 and 10 act as Pseudomonas quinolone system (PQS) antagonists, the mechanism of inhibition of S. marcescens biofilm formation with these compounds remains open since signaling similar to P. aeruginosa PQS system has not yet been described in Serratia and activity of these compounds on acylhomoserine lactone (AHL) signaling has not been detected. Our data show that 7-Cl and 7-CF3 substituted N-dodecylamino-4-aminoquinolines present the promising scaffolds for developing antivirulence and anti-biofilm formation agents against multidrug-resistant bacterial species. This is the peer-reviewed version of the following article: Aleksić, I.; Šegan, S.; Andrić, F.; Zlatović, M.; Moric, I.; Opsenica, D. M.; Senerovic, L. Long-Chain 4-Aminoquinolines as Quorum Sensing Inhibitors in Serratia Marcescens and Pseudomonas Aeruginosa. ACS Chemical Biology 2017, 12 (5), 1425–1434. [https://doi.org/10.1021/acschembio.6b01149] Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3090]

Infective diseases have become health threat of a global proportion due to appearance and spread of microorganisms resistant to majority of therapeutics currently used for their treatment. Therefore, there is a constant need for development of new antimicrobial agents, as well as novel therapeutic strategies. Quinolines and quinolones, isolated from plants, animals, and microorganisms, have demonstrated numerous biological activities such as antimicrobial, insecticidal, anti-inflammatory, antiplatelet, and antitumor. For more than two centuries quinoline/quinolone moiety has been used as a scaffold for drug development and even today it represents an inexhaustible inspiration for design and development of novel semi-synthetic or synthetic agents exhibiting broad spectrum of bioactivities. The structural diversity of synthetized compounds provides high and selective activity attained through different mechanisms of action, as well as low toxicity on human cells. This review describes quinoline and quinolone derivatives with antibacterial, antifungal, anti-virulent, antiviral, and anti-parasitic activities with the focus on the last 10 years literature.

Infective diseases have become health threat of a global proportion due to appearance and spread of microorganisms resistant to majority of therapeutics currently used for their treatment. Therefore, there is a constant need for development of new antimicrobial agents, as well as novel therapeutic strategies. Quinolines and quinolones, isolated from plants, animals, and microorganisms, have demonstrated numerous biological activities such as antimicrobial, insecticidal, anti-inflammatory, antiplatelet, and antitumor. For more than two centuries quinoline/quinolone moiety has been used as a scaffold for drug development and even today it represents an inexhaustible inspiration for design and development of novel semi-synthetic or synthetic agents exhibiting broad spectrum of bioactivities. The structural diversity of synthetized compounds provides high and selective activity attained through different mechanisms of action, as well as low toxicity on human cells. This review describes quinoline and quinolone derivatives with antibacterial, antifungal, anti-virulent, antiviral, and anti-parasitic activities with the focus on the last 10 years literature.

Many traditional remedies represent potential candidates for integration with modern medical practice, but credible data on their activities are often scarce. For the first time, the anti-virulence potential and the safety for human use of the ethanol extracts of two medicinal plants, Persicaria maculosa (PEM) and Bistorta officinalis (BIO), have been addressed. Ethanol extracts of both plants exhibited anti-virulence activity against the medically important opportunistic pathogen Pseudomonas aeruginosa. At the subinhibitory concentration of 50 &micro, g/mL, the extracts demonstrated a maximal inhibitory effect (approx. 50%) against biofilm formation, the highest reduction of pyocyanin production (47% for PEM and 59% for BIO) and completely halted the swarming motility of P. aeruginosa. Both extracts demonstrated better anti-quorum sensing and antibiofilm activities, and a better ability to interfere with LasR receptor, than the tested dominant extracts&rsquo, constituents. The bioactive concentrations of the extracts were not toxic in the zebrafish model system. This study represents an initial step towards the integration of P. maculosa and B. officinalis for use in the treatment of Pseudomonas infections.

Bioleaching of samples taken from depths of 10, 15, and 20 meters from old flotation tailings of the Copper Mine Bor was conducted in shaken flasks using extremely acidic water of Lake Robuleas lixiviant. Yield of copper after five weeks of the bioleaching experiment was 68.34±1.21% for 15 m sample, 72.57±0.57% for 20 m sample and 97.78±5.50% for 10 m sample. The obtained results were compared to the results of acid leaching of the same samples and it was concluded that bioleaching was generally more efficient for the treatment of samples taken from depths of 10 m and 20 m. The content of pyrite in the 20 m sample, which contained the highest amount of this mineral, was reduced after bioleaching. Benefits of this approach are: recovery of substantial amounts of copper, reducing the environmental impact of flotation tailings and the application of abundant and free water from the Robule acidic lake as lixiviant. Results of the experiment showed that bioleaching can be more efficient than acid leaching for copper extraction from flotation tailings with higher sulfide contents. [Projekat Ministarstva nauke Republike Srbije, br. 176016 i br. 173048]

The aim of the study was to evaluate and adapt the PCR-based protocol that utilizes the developed serotype-specific primers to identify Salmonella enterica species and its serotypes that are most frequently isolated from poultry samples in Vojvodina. Using the slide agglutination test, 64 and 33 out of 107 Salmonella isolates were identified as S. Infantis and S. Enteritidis, respectively, while ten isolates were identified as eight different Salmonella serovars. Using the same isolates, presence of 993-bp (bcfC gene), 636-bp (steB gene) and 293-bp (sdf locus) amplicons in multiplex PCR unambiguously identified 31 isolates as S. Enteritidis. Two isolates identified as Enteritidis in slide agglutination test were not identified as such in PCR-based approach since they both were missing 293-bp long PCR product. Thirty-nine isolates produced a 727-bp amplicon in the specific simplex PCR, and thus were identified as S. Infantis. The greatest discrepancy in comparison to the results of conventional serotyping has been observed in the case of S. Infantis, since 25 more isolates were noted as S. Infantis by conventional serotyping. Seven isolates, with unexpected PCR profiles stayed unidentified by molecular typing, although they were serotyped as S. Typhimurium (1) and S. Infantis (6). S. Gallinarum serovar has to be additionally confirmed, since it shares the same PCR profile with S. Livingstone. Clearly, PCR-based identification has to be thoroughly checked, verified and adapted if it is to be applied as the routine identification protocol.

rRNA Methyltransferases and their Role in Resistance to AntibioticsMethyltransferases (MTases), a large protein superfamily, commonly use S-adenosyl-L-methionine (SAM) as the methyl group donor. SAM-dependant MTases methylate both nucleic acids (DNA, RNA) and proteins, and thus modulate their activity, function and folding. Methylation of G1405 or A1408 nucleotides of 16S rRNA in aminoglycoside-producing microorganisms confers the resistance to their own toxic product(s). This mechanism of resistance has been considered as unique to antibiotics producers until recently. Since 2003, methylation of 16S rRNA as a mechanism of resistance is increasingly emerging in pathogenic bacteria. This represents a major threat towards the usefulness of aminoglycosides in the clinical practice. A potential solution to the problem involves the design of novel compounds that would act against new ribosomal targets. The second approach to the issue includes the development of resistance MTases' inhibitors, with the idea to prevent them from modifying the bacterial rRNA, and thus reinstate the therapeutic power of existing aminoglycosides. As the latter approach has considerable potential, it is obvious that fundamental research related to protein expression, in-depth understanding of the mechanism of action and resolving a tertiary structure of 16S rRNAs MTases are prerequisites for application in medicine.

Sgm methyltransferase from Micromonospora zionensis and KgmB methyltransferase from Streptoalloteichus tenebrarius are resistant to aminoglycoside antibiotics as a result of their ability to specifically methylate G1405 within the bacterial 16S rRNA A-site. The (C)CGCCC motif, assumed to be a regulatory sequence responsible for the autoregulation of the sgm gene, could most likely also be responsible for the autoregulation of the kgmB gene. This sequence, found within the 5' untranslated region of both sgm and kgmB mRNAs, as indicated by in silico prediction, may be involved in the formation of a specific stem-loop structure. Sgm and KgmB are mutually down-regulated and it is likely that they share the same cis-acting elements. Structure probing experiments confirmed the existence of a stable secondary structure within the 5' UTR of the sgm mRNA, while the analysis of kgmB mRNA failed to confirm the predicted structure. .

The aminoglycoside resistance genes sgm from Micromonospora zionensis and kgmB from Streptomyces tenebrarius were cloned into a yeast expression vector to test whether the encoded prokaryotic methylases can modify the 18S rRNA A-site and thus confer resistance to G-418. Despite the detectable presence of mRNAs in yeast cells, neither G-418-resistant yeast transformants nor positive western blot signals were obtained. Neither methylase was capable of methylating 40S subunits despite very high conservation of the antibiotic rRNA binding sites. However, the results provide novel insight into the action of Sgm by showing that it methylates the same site as KgmB, i.e. G1405 in 16S rRNA.

Pathogenic mycobacteria survive within phagosomes which are thought to represent a nutrient-restricted environment. Divalent cation transporters of the Nramp family in phagosomes and mycobacteria (Mramp) may compete for metals that are crucial for bacterial survival. The elemental concentrations in phagosomes of macrophages infected with wild-typeMycobacterium tuberculosis(M. tuberculosisstrain H37Rv) and aM. tuberculosis Mrampknockout mutant (Mramp-KO), derived from a clinical isolate isogenic to the strain MT103, were compared. Time points of 1 and 24 h after infection of mouse peritoneal macrophages (bcgS) were compared in both cases. Increased concentrations of P, Ni and Zn and reduced Cl concentration in Mramp-KO after 1 h of infection were observed, compared toM. tuberculosisvacuoles. After 24 h of infection, significant differences in the P, Cl and Zn concentrations were still present. The Mramp-KO phagosome showed a significant increase of P, Ca, Mn, Fe and Zn concentrations between 1 and 24 h after infection, while the concentrations of K and Ni decreased. In theM. tuberculosisvacuole, the Fe concentration showed a similar increase, while the Cl concentration decreased. The fact that the concentration of several divalent cations increased in the Mramp-KO strain suggests that Mramp may have no impact on the import of these divalent cations into the mycobacterium, but may function as a cation efflux pump. The concordant increase of Fe concentrations withinM. tuberculosis, as well as within the Mramp-KO vacuoles, implies that Mramp, in contrast to siderophores, might not be important for the attraction of Fe and its retention in phagosomes of unstimulated macrophages.

Bacterial pathogens survive in different environments in the human host by responding with expression of virulence factors that enable them to adapt to changing conditions. Trace elements regulate the expression of many virulence genes in bacteria and are thus important for their survival in the host. Mycobacteria are intracellular pathogens that can cause diseases such as tuberculosis or secondary infections in immunocompromised patients We have used a hard x-ray microprobe to study the trace element distribution in the mycobacterial phagosome after infection of macrophages. We have studied phagosomes with virulent (M. avium) and nonvirulent (M. smegmatis) mycobacteria. In this article, we will show that the iron concentration in phagosomes with macrophages infected with nonvirulent M. smegmatis is reduced 24 hours after infection but increased in phagosomes in cells infected with virulent M. avium. In addition, we will show the effect activation of macrophages with tumor necrosis factor (TNF-α) or interferon (IFN-γ) has on the iron concentration in M. avium.

Background: Influenza (Flu) and respiratory syncytial virus (RSV) are important viral pathogens that cause lower respiratory tract infections and severe exacerbations of asthma. Molecular biological techniques are permitting a rapid and accurate diagnosis of infections caused by respiratory pathogens, and have typically been applied to upper respiratory samples. Sputum induction provides an opportunity to directly sample secretions from the lower respiratory tract. Objectives/study design: To determine the role of induced sputum reverse-transcription polymerase chain reaction (RT-PCR) in the detection of respiratory pathogens and compare this with detection using serology and immunofluorescent antigen (IFA) testing, we recruited 49 adults from emergency room with exacerbations of asthma. After a medical assessment and spirometry, sputum was induced using ultrasonically nebulised normal saline. Sputum was assayed using IFA and RT-PCR for flu and RSV. Flu serology was performed acutely and at convalescence, 4 � /5 weeks later. Results: Influenza A or B was detected in 24% of the samples by PCR, significantly more than the nine cases detected using serology and the one case using IFA (P B/0.05). RSV was detected in 37% of samples using PCR and 20% by IFA (P B/0.05). Conclusion: The combination of induced sputum and RT-PCR provides a useful means of detecting respiratory infection. The technique is safe in both adults and children, and RT-PCR is more sensitive than conventional serology and IFA. The improved sensitivity of induced sputum RT-PCR also permits a more rapid diagnosis and the opportunity of early administration of effective treatments. # 2002 Elsevier Science B.V. All rights reserved.

An investigation of the microbial diversity of the extremely acidic, metal-rich Lake Robule was carried out using culture-dependant and culture-independent (T-RFLP) methods, and the ability of indigenous bacteria from the lake water to leach copper from a mineral concentrate was tested. T-RFLP analysis revealed that the dominant bacteria in lake water samples were the obligate heterotroph Acidiphilium cryptum (~50% of total bacteria) and the iron-oxidizing autotroph Leptospirillum ferrooxidans (~40%) The iron/sulfur-oxidizing autotroph Acidithiobacillus ferrooxidans had been reported to be the most abundant bacteria in the lake in an earlier study by other authors, but it was not detected in the present study using T-RFLP. Although it was isolated on solid media and detected in enrichment (bioleaching) cultures. The presence of the two bacterial species detected by T-RFLP (L. ferrooxidans and A. cryptum) was also confirmed by cultivation on solid media. The presence and relative abundance of bacteria inhabiting Lake Robule was explained by the physiological characteristics of the bacteria and the physico-chemical characteristics of the lake water. [Projekat Ministarstva nauke Republike Srbije, br. 176016 i br.173048]

Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.

BackgroundMycobacterium aviumsubsphominissuis(previouslyMycobacterium aviumsubspavium) is an environmental organism associated with opportunistic infections in humans.Mycobacterium hominissuisinfects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.ResultsMAV_2928 gene is primarily upregulated upon phagocytosis. The transcriptional profile of macrophages infected with the wild-type bacterium and the mutant were examined using DNA microarray, which showed that the two bacteria interact uniquely with mononuclear phagocytes. Based on the results, it was hypothesized that the phagosome environment and vacuole membrane of the wild-type bacterium might differ from the mutant. Wild-type bacterium phagosomes expressed a number of proteins different from those infected with the mutant. Proteins on the phagosomes were confirmed by fluorescence microscopy and Western blot. The environment in the phagosome of macrophages infected with the mutant differed from the environment of vacuoles withM. hominissuiswild-type in the concentration of zinc, manganese, calcium and potassium.ConclusionThe results suggest that the MAV_2928 gene/operon might participate in the establishment of bacterial intracellular environment in macrophages.

The mechanism of resistance to aminoglycosides based on methylation of their target, 16S rRNA, was until recently described only in antibiotic producing microorganisms. However, equivalent methyltransferases have now also been identified among numerous clinical Gram-negative pathogenic isolates. We have cloned, expressed, and purified GrmA, the aminoglycoside-resistance methyltransferase from Micromonospora purpurea, producer of gentamicin complex. Two vectors were created that express protein with an N-terminal 6x histidine tag with and without an enterokinase recognition producing proteins His(6)-EK-GrmA and His(6)-GrmA, respectively. The activity of both recombinant proteins was demonstrated in vivo. After optimized expression and native purification both protein variants proved to be active in in vitro methylation assays. This work lays a foundation for future detailed biochemical, structural and pharmacological studies with this member of an important group of aminoglycoside-resistance enzymes.

Acute exacerbations of asthma are frequently caused by viral infections, but the inflammatory mechanisms in virus-induced asthma are poorly understood. The aim of the present study was to determine whether viral infection in acute asthma was associated with increased sputum neutrophil degranulation and increased cellular lysis and whether these changes are related to clinical severity. Adults (n=49) presenting to the emergency department with acute asthma were examined for infection by means of sputum direct-fluorescence antigen detection, sputum culture, and sputum polymerase chain reaction for Mycoplasma, Chlamydia and Legionella pneumophila, and all common respiratory viruses. Subjects infected with one of these agents were classed as having an infective exacerbation. Spirometry and sputum induction were performed on presentation and 4-5 weeks later. Thirty-seven subjects (76%) had virus infection and acute asthma. Those with virus infection had increased sputum neutrophils (p

The effects of respiratory viral infection on the time course of chronic obstructive pulmonary disease (COPD) exacerbation were examined by monitoring changes in systemic inflammatory markers in stable COPD and at exacerbation. Eighty-three patients with COPD (mean [SD] age, 66.6 [7.1] yr, FEV(1), 1.06 [0.61] L) recorded daily peak expiratory flow rate and any increases in respiratory symptoms. Nasal samples and blood were taken for respiratory virus detection by culture, polymerase chain reaction, and serology, and plasma fibrinogen and serum interleukin-6 (IL-6) were determined at stable baseline and exacerbation. Sixty-four percent of exacerbations were associated with a cold occurring up to 18 d before exacerbation. Seventy-seven viruses (39 [58.2%] rhinoviruses) were detected in 66 (39.2%) of 168 COPD exacerbations in 53 (64%) patients. Viral exacerbations were associated with frequent exacerbators, colds with increased dyspnea, a higher total symptom count at presentation, a longer median symptom recovery period of 13 d, and a tendency toward higher plasma fibrinogen and serum IL-6 levels. Non-respiratory syncytial virus (RSV) respiratory viruses were detected in 11 (16%), and RSV in 16 (23.5%), of 68 stable COPD patients, with RSV detection associated with higher inflammatory marker levels. Respiratory virus infections are associated with more severe and frequent exacerbations, and may cause chronic infection in COPD. Prevention and early treatment of viral infections may lead to a decreased exacerbation frequency and morbidity associated with COPD.

The transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase (pac) gene was studied by modifying DNA sequences responsible for promoter activation by cyclic AMP receptor protein (CRP). The nucleotide sequence of the 5′-flanking region of the pac gene contains putative tandem CRP binding sites positioned at −69/−70 and at −111/−112 with respect to the transcriptional start site. Our results obtained with either point mutations or insertion or deletion mutants (each of which rotated the helix structure at the CRP binding site one-half turn) showed significant decrease of penicillin amidase (PA) activity, suggesting the CRP as a major activator. In this study, the evidence for the importance of spacing between tandem binding sites for CRP as well as for their location related to the promoter core sequence has been provided. Involvement of integration host factor (IHF) as an additional regulatory protein in the pac gene transcription regulation was also analyzed. It is shown that activation of the pac gene transcription is elevated by IHF.