Large Number Of Bacterial Strains Research Articles

Features of the Skin Microbiota in Common Inflammatory Skin Diseases.

Many relatively common chronic inflammatory skin diseases manifest on the face (seborrheic dermatitis, rosacea, acne, perioral/periorificial dermatitis, periocular dermatitis, etc.), thereby significantly impairing patient appearance and quality of life. Given the yet unexplained pathogenesis and numerous factors involved, these diseases often present therapeutic challenges. The term “microbiome” comprises the totality of microorganisms (microbiota), their genomes, and environmental factors in a particular environment. Changes in human skin microbiota composition and/or functionality are believed to trigger immune dysregulation, and consequently an inflammatory response, thereby playing a potentially significant role in the clinical manifestations and treatment of these diseases. Although cultivation methods have traditionally been used in studies of bacterial microbiome species, a large number of bacterial strains cannot be grown in the laboratory. Since standard culture-dependent methods detect fewer than 1% of all bacterial species, a metagenomic approach could be used to detect bacteria that cannot be cultivated. The skin microbiome exhibits spatial distribution associated with the microenvironment (sebaceous, moist, and dry areas). However, although disturbance of the skin microbiome can lead to a number of pathological conditions and diseases, it is still not clear whether skin diseases result from change in the microbiome or cause such a change. Thus far, the skin microbiome has been studied in atopic dermatitis, seborrheic dermatitis, psoriasis, acne, and rosacea. Studies on the possible association between changes in the microbiome and their association with skin diseases have improved the understanding of disease development, diagnostics, and therapeutics. The identification of the bacterial markers associated with particular inflammatory skin diseases would significantly accelerate the diagnostics and reduce treatment costs. Microbiota research and determination could facilitate the identification of potential causes of skin diseases that cannot be detected by simpler methods, thereby contributing to the design and development of more effective therapies.

Antibacterial activity of human defensins against Staphylococcus aureus and Escherichia coli.

BackgroundThe global problem of antibiotic resistance requires the search for and development of new methods of treatment. One of the promising strategies is the use of low doses of antimicrobial peptides, in particular, human defensins HNP-1, hBD-1, and hBD-3, in combination with antibacterial drugs already used in clinical practice. This approach may be used to increase the effectiveness of conventional antibiotics. However, this requires thorough study of the effectiveness of defensins in combination with antibiotics against a large number of bacterial strains with known phenotypes of antibiotic resistance. The aim of this work was to study the antibacterial effect of HNP-1, hBD-1 and hBD-3 in combination with rifampicin or amikacin against clinical isolates of Staphylococcus aureus (n = 27) and Escherichia coli (n = 24) collected from hospitalized patients.MethodsThe standard checkerboard assay was used to determine minimum inhibitory concentrations (MICs) of antimicrobials. The combined microbicidal effects of two substances (defensin + conventional antibiotic) were assessed by the fractional inhibitory concentration index (FICI).ResultsThe highest anti-staphylococcal activity (including methicillin-resistant strains) among defensins was demonstrated by hBD-3 that had MIC of 1 (0.5–4) mg/L (hereinafter, MIC values are presented as median and interquartile range). The MIC of HNP-1 against S. aureus was 4 (2–8) mg/L; the MIC of hBD-1 was 8 (4–8) mg/L. Against E. coli, the most effective was also found to be hBD-3 that had MIC of 4 (4–8) mg/L; the MIC of HNP-1 was 12 (4–32) mg/L. The combinations of HNP-1 + rifampicin and hBD-3 + rifampicin demonstrated synergistic effects against S. aureus. Against E. coli, combinations of HNP-1 + amikacin and hBD-3 + amikacin also showed synergy of action.

Evaluation of assays for screening polycyclic aromatic hydrocarbon-degrading potential of bacteria

Within a 30-day incubation laboratory study, the polycyclic aromatic hydrocarbon (PAH) degradation profile of two bacteria, Planomicrobium sp. RNP01 and Rhodococcus sp. RNP05 were studied by three microtiter plate assays to reveal the combination of certain biological and biochemical characteristics which are reliable indicators in evaluation of bacterial biodegradation abilities. The three assays, which are hydrocarbon growth assay, 2,6-DCPIP assay and dehydrogenase activity assay revealed that Rhodococcus sp. RNP05 exhibited better potential for PAH degradation than Planomicrobium sp. RNP01. Differences between initial and final optical density and specific growth rate constants were significantly higher (r = 0.995, P < 0.05) in case of Rhodococcus sp. RNP05 on all tested substrates, as compared to Planomicrobium sp. RNP01. This was confirmed by GC-FID analyses. Dehydrogenase activity of Rhodococcus sp. RNP05 was higher (r = 0.9995, P < 0.05) than Planomicrobium sp. RNP01 and correlated positively with the hydrocarbon growth assay (r = 0.999, P < 0.05, for Rhodococcus sp. RNP05, r = 0.986, P < 0.05 for Planomicrobium sp. RNP01). This study has shown that the combination of these assays could be used for determining the bioremediation potential of PAHs in petroleum contaminated soil with the ability of screening a large number of bacterial strains.

Multi-Approach Analysis for the Identification of Proteases within Birch Pollen.

Birch pollen allergy is highly prevalent, with up to 100 million reported cases worldwide. Proteases in such allergen sources have been suggested to contribute to primary sensitisation and exacerbation of allergic disorders. Until now the protease content of Betula verrucosa, a birch species endemic to the northern hemisphere has not been studied in detail. Hence, we aim to identify and characterise pollen and bacteria-derived proteases found within birch pollen. The pollen transcriptome was constructed via de novo transcriptome sequencing and analysis of the proteome was achieved via mass spectrometry; a cross-comparison of the two databases was then performed. A total of 42 individual proteases were identified at the proteomic level. Further clustering of proteases into their distinct catalytic classes revealed serine, cysteine, aspartic, threonine, and metallo-proteases. Further to this, protease activity of the pollen was quantified using a fluorescently-labelled casein substrate protease assay, as 0.61 ng/mg of pollen. A large number of bacterial strains were isolated from freshly collected birch pollen and zymographic gels with gelatinase and casein, enabled visualisation of proteolytic activity of the pollen and the collected bacterial strains. We report the successful discovery of pollen and bacteria-derived proteases of Betula verrucosa.

Sequence analysis of pooled bacterial samples enables identification of strain variation in group A streptococcus

Knowledge of the genomic variation among different strains of a pathogenic microbial species can help in selecting optimal candidates for diagnostic assays and vaccine development. Pooled sequencing (Pool-seq) is a cost effective approach for population level genetic studies that require large numbers of samples such as various strains of a microbe. To test the use of Pool-seq in identifying variation, we pooled DNA of 100 Streptococcus pyogenes strains of different emm types in two pools, each containing 50 strains. We used four variant calling tools (Freebayes, UnifiedGenotyper, SNVer, and SAMtools) and one emm1 strain, SF370, as a reference genome. In total 63719 SNPs and 164 INDELs were identified in the two pools concordantly by at least two of the tools. Majority of the variants (93.4%) from six individually sequenced strains used in the pools could be identified from the two pools and 72.3% and 97.4% of the variants in the pools could be mined from the analysis of the 44 complete Str. pyogenes genomes and 3407 sequence runs deposited in the European Nucleotide Archive respectively. We conclude that DNA sequencing of pooled samples of large numbers of bacterial strains is a robust, rapid and cost-efficient way to discover sequence variation.

Prediction of antimicrobial peptides based on sequence alignment and support vector machine-pairwise algorithm utilizing LZ-complexity.

This study concerns an attempt to establish a new method for predicting antimicrobial peptides (AMPs) which are important to the immune system. Recently, researchers are interested in designing alternative drugs based on AMPs because they have found that a large number of bacterial strains have become resistant to available antibiotics. However, researchers have encountered obstacles in the AMPs designing process as experiments to extract AMPs from protein sequences are costly and require a long set-up time. Therefore, a computational tool for AMPs prediction is needed to resolve this problem. In this study, an integrated algorithm is newly introduced to predict AMPs by integrating sequence alignment and support vector machine- (SVM-) LZ complexity pairwise algorithm. It was observed that, when all sequences in the training set are used, the sensitivity of the proposed algorithm is 95.28% in jackknife test and 87.59% in independent test, while the sensitivity obtained for jackknife test and independent test is 88.74% and 78.70%, respectively, when only the sequences that has less than 70% similarity are used. Applying the proposed algorithm may allow researchers to effectively predict AMPs from unknown protein peptide sequences with higher sensitivity.

Batch production and media optimization of anti-leukemic L-asparaginase from Pseudomonas fluorescens by Taguchi DOE methodology

L-Asparaginase isolated from bacterial sources has been shown to possess antileukemic characteristic, mainly against the acute lymphoblastic leukemia. Large number of bacterial strains have been reported which can produce it. The production level of the enzyme by the bacterial sources is very low and hence the factors have to be distinguished which effect the growth of cells and production of enzyme directly or indirectly, in order to decrease the cost of treatment. The production of L-Asparaginase was optimized in classical fermentation process by the use of Taguchi DOE methodology. L-18 array was selected for the purpose of media optimization. The five factors at three levels were considered for the optimization. L-Asparaginase production was significantly affected by the interaction of two factors present in the culture broth. The factors interaction viz. energy source-nitrogen source, energy source-phosphate source etc. having pronounced effect on the production while individually they have the minimum impact on L-Asparaginase production. So the interactions of different parameters were studied and they were used to formulate the optimized condition for the production of L-Asparaginase. After the validation of result by performing the experiment under optimized condition there was about 28.48% increase in the production of the enzyme.

Experimental approach for bacteriophage susceptibility testing of planktonic and sessile bacterial populations - Study protocol.

Antimicrobial resistance is a growing threat for all clinical branches. This phenomenon poses important challenges in controlling infectious diseases. However, multidrug resistance is not the only issue, as bacteria that are otherwise susceptible to common antibiotics express other patterns for evading antibiotherapy, for example they can aggregate within a self-produced matrix to form biofilm. We intend to perform a prospective laboratory study of the germs isolated from different samples collected from patients admitted with infectious pathology in reference hospitals in Romania. We will perform antibiotic resistance testing as well as phage testing, both on solid and liquid growth medium, for Staphylococcus spp., Enterococcus spp., and Pseudomonas spp. We intend to collect data for 150 patients with different infections with these identified pathogens. Phage susceptibility testing will be performed using 5 types of strain-specific bacteriophage mixtures: PYO, INTESTI, STAPHYLOCOCCAL (Eliava BioPreparations, Tbilisi, Georgia), PHAGYO, PHAGESTI (JSC "Biochimpharm", Tbilisi, Georgia). For phage-susceptible strains, we will evaluate biofilm formation in the presence of phages, as well as phage effect on already formed biofilm. Through this study, we intend to provide the first set of results on bacteriophage-susceptibility of bacteria isolated from patients with hard to treat infections, from reference hospitals in Romania. By evaluating a large number of bacterial strains we aim to predict and project biofilm kinetics, while adding binary phage dilutions at key timepoints during biofilm formation. POSDRU/159/1.5/S/141531; Carol Davila University of Medicine and Pharmacy, Young Researchers Grant no. 28341/2013.

Modelling the emergent dynamics and major metabolites of the human colonic microbiota.

We present here a first attempt at modelling microbial dynamics in the human colon incorporating both uncertainty and adaptation. This is based on the development of a Monod-equation based, differential equation model, which produces computer simulations of the population dynamics and major metabolites of microbial communities from the human colon. To reduce the complexity of the system, we divide the bacterial community into 10 bacterial functional groups (BFGs) each distinguished by its substrate preferences, metabolic pathways and its preferred pH range. The model simulates the growth of a large number of bacterial strains and incorporates variation in microbiota composition between people, while also allowing succession and enabling adaptation to environmental changes. The model is shown to reproduce many of the observed changes in major phylogenetic groups and key metabolites such as butyrate, acetate and propionate in response to a one unit pH shift in experimental continuous flow fermentors inoculated with human faecal microbiota. Nevertheless, it should be regarded as a learning tool to be updated as our knowledge of bacterial groups and their interactions expands. Given the difficulty of accessing the colon, modelling can play an extremely important role in interpreting experimental data and predicting the consequences of dietary modulation.

Cyanide measurements in bacterial culture and sputum.

Cyanide is produced by a few bacterial species, including Pseudomonas aeruginosa, and it has a role in the opportunistic infections of this bacterium including in cystic fibrosis lung infections. We describe two methods for determining cyanide in culture and patient sputum samples. One uses an ion-selective electrode to provide a convenient, rapid method of cyanide quantitation in culture or sputum, and the second is a semiquantitative method using Feigl-Anger paper that is useful for screening large numbers of bacterial strains for cyanide production.

Evaluation of Antibacterial Activities of Compounds Isolated From Sida rhombifolia Linn. (Malvaceae)

The main objective of this study was to isolate compounds from roots of Sida rhombifolia and subsequently evaluate their antibacterial activities. Crude gradient extracts were obtained from three solvents (petroleum ether, chloroform and methanol) with increasing solvent polarity using cold maceration technique. The in vitro antibacterial activity evaluation of gradient extracts and isolated compounds was done on four different pathogenic bacterial strains (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella typhimurium) using agar disc diffusion technique. The results showed that antibacterial activities were comparable to each other. But their activities were relatively weaker as compared to that of the reference compound (ciprofloxacin). Among the three crude extracts, the chloroform extract was subjected to column chromatographic separation that led to isolation of SRL-1, SRL-2 and SRL-3. The chemical structures of the compounds were found to be n-hexacos-11-enoic acid, stigmasterol and β-sitosterol, respectively, based on physical properties and spectroscopic (IR and NMR) data as well as literature reports. The observed antibacterial activities of the crude extracts and the isolated compounds could justify the traditional use of the plant for the treatment of different bacterial infections. Thus, further test is recommended on large number of bacterial strains to decide the potentials of the compounds as candidates in development of antibacterial drugs.

Resazurin-based assay for screening bacteria for radiation sensitivity

We report a simple and efficient colorimetric method to screen large numbers of bacterial strains for UV- and X-radiation sensitivity. We used reference radiation-sensitive and control strains of Escherichia coli K-12 to compare our colorimetric method to a standard clonogenic plating method. Our colorimetric method was as accurate as the standard method and was superior in terms of savings in supplies and man-hours.

Real-time PCR optimization to identify environmental Vibrio spp. strains

To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V.vulnificus, V.cholerae and V.alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V.alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V.alginolyticus. Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.

Chemoorganotrophic bacteria isolated from biodeteriorated surfaces in cave and catacombs

The main objective of this work was the comparative analysis of a large number of bacterial strains isolated from biodeteriorated surfaces in three different sites, namely the catacombs of St. Callistus in Rome, Italy, the catacombs dedicated to St. Agatha in Rabat, Malta and the Cave of Bats in Zuheros, Spain. Our results showed that even considering only culturable chemoorganotrophic bacteria the variability is very high, reflecting the great variety of microhabitats present. Hence any strategies to prevent, control or eliminate the biofilm-embedded microbiota from an archeological surface should take into account a number of considerations as stipulated in our study.

Atomic resolution structure of EhpR: phenazine resistance in Enterobacter agglomerans Eh1087 follows principles of bleomycin/mitomycin C resistance in other bacteria

BackgroundThe phenazines are redox-active secondary metabolites that a large number of bacterial strains produce and excrete into the environment. They possess antibiotic activity owing to the fact that they can reduce molecular oxygen to toxic reactive oxygen species. In order to take advantage of this activity, phenazine producers need to protect themselves against phenazine toxicity. Whereas it is believed that phenazine-producing pseudomonads possess highly active superoxide dismutases and catalases, it has recently been found that the plant-colonizing bacterium Enterobacter agglomerans expresses a small gene ehpR to render itself resistant towards D-alanyl-griseoluteic acid, the phenazine antibiotic produced by this strain.ResultsTo understand the resistance mechanism installed by EhpR we have determined its crystal structure in the apo form at 2.15 Å resolution and in complex with griseoluteic acid at 1.01 Å, respectively. While EhpR shares a common fold with glyoxalase-I/bleomycin resistance proteins, the ligand binding site does not contain residues that some related proteins employ to chemically alter their substrates. Binding of the antibiotic is mediated by π-stacking interactions of the aromatic moiety with the side chains of aromatic amino acids and by a few polar interactions. The dissociation constant KD between EhpR and griseoluteic acid was quantified as 244 ± 45 μM by microscale thermophoresis measurements.ConclusionsThe data accumulated here suggest that EhpR confers resistance by binding D-alanyl-griseoluteic acid and acting as a chaperone involved in exporting the antibiotic rather than by altering it chemically. It is tempting to speculate that EhpR acts in concert with EhpJ, a transport protein of the major facilitator superfamily that is also encoded in the phenazine biosynthesis operon of E. agglomerans. The low affinity of EhpR for griseoluteic acid may be required for its physiological function.

Antibacterial properties of compounds isolated from Carpobrotus edulis

Several compounds isolated from the plant Carpobrotus edulis were evaluated for their activity against multidrug-resistant (MDR) bacteria and their efflux pump systems. Amongst the compounds isolated, six compounds were tested, namely uvaol, β-amyrin, oleanolic acid, catechin, epicatechin and monogalactosyldiacylglycerol. Oleanolic acid presented high antibacterial activity against a large number of bacterial strains. The triterpene uvaol was the most active compound for modulation of efflux activity by MDR Gram-positive strains.

Repetitive Extragenic Palindromic PCR for Study of Streptococcus mutans Diversity and Transmission in Human Populations

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular epidemiological study. Repetitive extragenic palindromic PCR (rep-PCR) is less time-consuming and more suitable for analyzing large numbers of bacterial strains in human populations. PFGE and rep-PCR provide comparable genotyping results for investigating Streptococcus mutans diversity and transmission.

Modeling Pseudomonas aeruginosa pathogenesis in plant hosts.

A pathogenic model in which both the pathogen and its host are amenable to genetic manipulation can greatly facilitate the understanding of bacterial pathogenesis. Plants are genetically tractable and can be used as experimental models for human microbial pathogenesis. We present protocols for both lettuce and Arabidopsis leaf infection models using the opportunistic human bacterial pathogen, Pseudomonas aeruginosa. The lettuce model allows for high-throughput qualitative analysis of virulence and is suitable for screening large numbers of bacterial strains, whereas the Arabidopsis model provides a quantitative approach and permits the tracking of bacterial cell proliferation in planta. The lettuce model takes approximately 24 h including bacterial growth using store-bought lettuce, and the Arabidopsis model takes 4-6 weeks to grow the plants and a similar time as with lettuce to infect the plants. Both models are monitored for up to 5 d post-infection. These methodologies can and have been used to identify novel and critical P. aeruginosa pathogenicity agents, as virulence factors are often conserved across phylogeny.

Activities of synthetic hybrid peptides against anaerobic bacteria: aspects of methodology and stability.

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. One line of investigation is the synthesis of antimicrobial hybrid peptides. The aim of the present investigation was to determine the in vitro activities of 16 cecropin-melittin hybrid peptides (CAMEL analogues) against 60 anaerobic bacterial strains, to compare their activities with those of seven clinically used antimicrobial agents, and to compare different methods for anaerobic susceptibility testing of these peptides. The stability of one of the peptides, temporin B, with different stereoisomeric configurations was investigated in a fecal milieu. The CAMEL analogues showed antimicrobial activity against the anaerobic bacteria, with MICs ranging from 0.125 to 32 microg/ml. The overall activities (the MICs at which 90% of isolates are inhibited) of the CAMEL analogues against anaerobic bacteria were mainly inferior to those of imipenem, clindamycin, and piperacillin but were equal to or superior to those of metronidazole, cefoxitin, ciprofloxacin, and chloramphenicol. The agarose dilution method was found to be an accurate method for the testing of large numbers of bacterial strains. The D isomer of temporin B was inactivated more slowly in feces than the L isomer. This study shows that the CAMEL analogues are potential agents for the treatment of anaerobic infections.

Detection of two isotypically different antibodies produced by a murine hybridoma.

A hybridoma, F31P46B, secreting monoclonal antibodies (mAbs) comprised of mu and gamma heavy chains in association with a single kappa light chain, has been characterized. This hybridoma was prepared by fusing splenocytes, derived from a BALB/c mouse immunized with Vibrio vulnificus and SP2/O-Ag-14 mouse myeloma cells. The specificity of this hybridoma was determined by ELISA screening on a large number of bacterial strains. Hybridoma cells of F31P46B were cloned by limiting dilution to an average cell density of 0.1 cells/well and repeated 3 times to ensure monoclonality. Isotyping of 7 subclones was then performed by Ouchterlony gel double diffusion, as well as a Bio-Rad isotyping kit, and both methods showed that both IgM and IgG2b were secreted. PAGE and immunoblotting showed the presence of mu, gamma, and kappa chains with respective molecular weights of 80, 50, and 25kDa. A series of fractions, collected from F31P46B ascites during Superose 12 gel chromatography, were tested by the two isotyping methods and each confirmed the presence of two immunoglobulin products. These data indicated that the hybridoma secreted two separate immunoglobulins, IgM/kappa and IgG2b/kappa.