Adaptive remodeling of the bacterial proteome by specific ribosomal modification regulates Pseudomonas infection and niche colonisation

Post-transcriptional control of protein abundance is a highly important, underexplored regulatory process by which organisms respond to their environments. Here we describe an important and previously unidentified regulatory pathway involving the ribosomal modification protein RimK, its regulator proteins RimA and RimB, and the widespread bacterial second messenger cyclic-di-GMP (cdG). Disruption of rimK affects motility and surface attachment in pathogenic and commensal Pseudomonas species, with rimK deletion significantly compromising rhizosphere colonisation by the commensal soil bacterium P. fluorescens, and plant infection by the pathogens P. syringae and P. aeruginosa. RimK functions as an ATP-dependent glutamyl ligase, adding glutamate residues to the C-terminus of ribosomal protein RpsF and inducing specific effects on both ribosome protein complement and function. Deletion of rimK in P. fluorescens leads to markedly reduced levels of multiple ribosomal proteins, and also of the key translational regulator Hfq. In turn, reduced Hfq levels induce specific downstream proteomic changes, with significant increases in multiple ABC transporters, stress response proteins and non-ribosomal peptide synthetases seen for both ΔrimK and Δhfq mutants. The activity of RimK is itself controlled by interactions with RimA, RimB and cdG. We propose that control of RimK activity represents a novel regulatory mechanism that dynamically influences interactions between bacteria and their hosts; translating environmental pressures into dynamic ribosomal changes, and consequently to an adaptive remodeling of the bacterial proteome

Studying formation of <I>Pseudomonas aeruginosa</I> biofilms grown under different cultivation conditions

The purpose of the present study is to assess how cultivation conditions influence growth and formation of Pseudomonas aeruginosa biofilms. The topic is of great importance due to high incidence of P. aeruginosa-caused infections and P. aeruginosa resistance associated with its ability to form biofilms. The paper analyzes factors that influence biofilm formation, i.e.: growth phase used for inoculation (log, stationary), volume of the growth medium (0.2 and 1.0 ml) and concentration of nutrients (liquid nutrient media diluted to concentrations of 50; 25; 12.5 and 6%) in the cultivation volume. As the research demonstrates, all these factors influence biofilm formation; and a P. aeruginosa growth phase before inoculation is a determining factor in the biofilm formation. When P. aeruginosa is inoculated at a stationary phase, biofilm formation shows non-linear dependence on concentration of nutrients and on their total amount in the cultivation volume. The linear dependence of biofilm formation on concentration of nutrients in the culture medium is more pronounced, when P. aeruginosa is inoculated at a log phase. The study shows that lower concentrations of nutrient media components lead to more noticeable differences in biofilm formation, and such differences are statistically significant. Two-fold dilution of the liquid nutrient medium does not affect the intensity of biofilm formation; however, a 4 to 8-folddecrease in concentration of nutrients in 0.2 ml of cultivation volume in habited the biofilms formation. In 1.0 ml of the culture medium, the biofilm forms evenly, and in 0.2 ml of 4–8-fold dilution of nutrient medium it grows slower. The slow growth rate is statistically significant. The cultivation volume is also of great importance. For example, cultures grown in 0.2 ml of nutrient medium at different concentrations of nutrients formed fewer biofilms than microorganisms cultivated in 1.0 ml. At the same time, when inoculating P. aeruginosa both at log and stationary growth phases, biofilm formation is more pronounced in wells containing more cultivation volume

Comparative Expression Profiling of the Chlamydia trachomatis pmp Gene Family for Clinical and Reference Strains

Chlamydia trachomatis, an obligate intracellular pathogen, is a leading worldwide cause of ocular and urogenital diseases. Advances have been made in our understanding of the nine-member polymorphic membrane protein (Pmp) gene (pmp) family of C. trachomatis. However, there is only limited information on their biologic role, especially for biological variants (biovar) and clinical strains.We evaluated expression for pmps throughout development for reference strains E/Bour and L2/434, representing different biovars, and for clinical E and L2 strains. Immunoreactivity of patient sera to recombinant (r)Pmps was also determined. All pmps were expressed at two hours. pmpA had the lowest expression but was up-regulated at 12 h for all strains, indicating involvement in reticulate body development. For pmpD, expression peaked at 36 h. Additionally, 57.7% of sera from infected and 0% from uninfected adolescents were reactive to rPmpD (p = 0.001), suggesting a role in immunogenicity. pmpF had the highest expression levels for all clinical strains and L2/434 with differential expression of the pmpFE operon for the same strains. Sera were nonreactive to rPmpF despite immunoreactivity to rMOMP and rPmpD, suggesting that PmpF is not associated with humoral immune responses. pmpFE sequences for clinical strains were identical to those of the respective reference strains. We identified the putative pmpFE promoter, which was, surprisingly, 100% conserved for all strains. Analyses of ribosomal binding sites, RNase E, and hairpin structures suggested complex regulatory mechanism(s) for this >6 Kb operon.The dissimilar expression of the same pmp for different C. trachomatis strains may explain different strain-specific needs and phenotypic distinctions. This is further supported by the differential immunoreactivity to rPmpD and rPmpF of sera from patients infected with different strains. Furthermore, clinical E strains did not correlate with the E reference strain at the gene expression level, reinforcing the need for expansive studies of clinical strains

ЗАВИСИМОСТЬ УРОВНЯ ИНФИЦИРОВАННОСТИ САЛЬМОНЕЛЛАМИ В ПОПУЛЯЦИЯХ КУР ОТ АНТАГОНИСТИЧЕСКОЙ АКТИВНОСТИ LACTOBACILLACEAE И ENTEROCOCCACEAE В ОТНОШЕНИИ SALMONELLA ENTERICA

The antagonistic activity of lactobacilli in the intestine in relation to various enteropathogenic microorganisms can vary within wide limits, including depending on the species composition of the lactobiota of the intestine. The purpose of this work was to determine the antagonistic activity of representatives of the order Lactobacillales isolated from chickens in poultry farms with different levels of Salmonella infection. The test object was the chickens of the parent herd and broiler chickens of crosses Ross 308 and Hubbard F - 15 from five poultry farms. Three poultry farms were characterized by a low level of salmonella infection in birds (less than 5% for cloacal swabs in PCR and the absence of salmonella isolation from food products). Two poultry farms were characterized by a high level of Salmonella infection (poultry infection by cloacal swabs of more than 10% and official salmonellosis disadvantage due to isolation of Salmonella cultures in food products). The level of infection was evaluated by real-time PCR after preliminary subculture of cloacal swabs on Shadler’s broth. The antagonistic activity of lactobacilli and related bacterial species isolated from the same chickens was carried out in co-cultivation tests on the Shadler broth with subsequent identification of salmonella on the RVS broth. Poultry farms with low Salmonella infection were characterized by the presence of L. reuteri as a major component of intestinal lactobiota and had a higher antagonistic activity against more Salmonella cultures (odds ratio (OR) 17.33 (CI 95 = 5.99-50.07776))Антагонистическая активность лактобактерий в кишечнике в отношении различных энтеропатогенных микроорганизмов может варьировать в широких пределах, в т. ч. в зависимости от видового состава лактобиоты кишечника. Цель настоящей работы заключалась в определении антагонистической активности представителей отряда Lactobacillales, выделенных от кур на птицефабриках с разными уровнями инфицированности сальмонеллами. Тест-объектом являлись куры родительского стада и цыплята-бройлеры кроссов Ross 308 и Hubbard F-15 с пяти птицефабрик. Три птицефабрики характеризовались низким уровнем инфицированности птицы сальмонеллами (менее 5 % по клоакальным смывам в ПЦР и отсутствие выделения сальмонелл из пищевой продукции). Две птицефабрики отличались высоким уровнем инфицированности сальмонеллами (инфицированность птицы по клоакальным смывам более 10 % и официальное неблагополучие по сальмонеллезу ввиду выделения культур сальмонелл в пищевой продукции). Уровень инфицированности оценивали методом ПЦР в режиме реального времени после предварительного субкультивирования клоакальных смывов на бульоне Шэдлера. Антагонистическую активность лактобактерий и родственных видов бактерий, выделенных от этих же кур, проводили в тестах по сокультивировнию на бульоне Шэдлера с последующим выявлением сальмонелл на RVS-бульоне. Птицефабрики с низким уровнем инфицированности сальмонеллами характеризовались наличием L. reuteri в качестве мажорного компонента лактобиоты кишечника и обладали более высокой антагонистической активностью в отношении большего числа культур сальмонелл (отношение шансов (OR) 17,33 (СI 95= 5,990–50,077))

Experimental study of the biocompatibility of the implant based on bacterial cellulose

This work is devoted to an experimental study of the reaction of body tissues to an implant based on bacterial cellulose synthesized by the bacterial strain Gluconacetobacter xylinus. The influence of the internal environment of the body on the characteristics of the implanted material was also studied to determine the prospects for its use in veterinary medicine.Currently, the issue of creating new implantable materials for a long time, or improving existing ones, does not lose its relevance. Based on this, it can be concluded that there are shortcomings in the previously developed materials. Certain requirements are imposed on implantable materials, such as elasticity, strength, porosity, non-toxicity, and others. The most important property of such materials can be called biocompatibility, as well as resistance to the effects of the biological environment.This study is aimed at establishing the possibility of using bacterial cellulose (BC) as an implantable material, which combines all the necessary properties for implants. The study of the reaction of body tissues to the implant was carried out on laboratory rats of the Wistar line. The implanted material was placed on the outer layer of the muscles of the abdominal wall of laboratory animals, after which, on the 14th, 30th and 90th days, a visual examination of the state of cellulose and nearby tissues was carried out, and tissues were selected for histological examination.The experimental results indicate that the implant based on bacterial cellulose does not cause negative reactions from nearby tissues, does not collapse during the observation period, and is reliably fixed on the muscle layer by a capsule of collagen fibers.</jats:p

Study of gentamicin deposition in cellulose with albumin

Methods of binding antibacterial drugs to the surface of cellulose without the use of oxidizing agents to prevent the occurrence of wound infections have been studied. The immobilization of gentamicin in the complex of partially denatured albumin in the composition with bacterial cellulose has been analyzed. The study was carried out on samples of cellulose synthesized byGluconacetobacter hansenii. Albumin served as a binding agent, which was used to impregnate cellulose samples, which were then denatured. Using PCR amplifi cation CFX (BioRad), the optimal denaturation temperature was selected. The effectiveness of the immobilization of albumin in the thickness of the cellulose was assessed by staining it with the luminescent dye SYPRO® Ruby Protein Gel Stain, followed by transilluminator detection. Bacterial cellulose impregnated with undenatured albumin was used as a control. Albumin immobilization in bacterial cellulose was observed at temperatures of 65– 95 °C. The antibacterial activity of the complex “cellulose + albumin + gentamicin” was evaluated using a test strain of bacteriaStaphylococcus aureusATCC 25923. The growth inhibition of the test strain of bacteria was observed in all tests with bacterial cellulose in combination with partially denatured albumin and gentamicin. In control samples, in which gentamicin was not immobilized as part of partially denatured albumin, growth inhibition zones ofStaphylococcus aureusATCC 25923 were not noted. It was concluded that by partial denaturation of albumin it is possible to delay antibacterial drugs in the thickness of bacterial cellulose for their further release. A new version of the material suitable for the production of implants and bandages based on bacterial cellulose gel with antibacterial properties is proposed. Dressings based on a composite of bacterial cellulose, albumin and gentamicin are most relevant for the treatment of burns. The presence of gentamicin in their composition is also relevant for the prevention of bacterial infections.</jats:p

Using &lt;i&gt;Bacillus subtilis&lt;/i&gt; as an oral vaccine carrier against &lt;i&gt;Streptococcus suis&lt;/i&gt;

Due to the progressive growth of the bacteria caused by the widespread use of antibiotics, treatment of streptococcosis is becoming increasingly difficult. Reliable vaccination against Streptococcus suis is necessary. Modern molecular diagnostic and genetic engineering capabilities create prospects for direct cloning of the protective epitopes of the Lmb gene of the local S. suis strain into the proposed delivery system of the pig immune system antigen. Among oral vaccine carriers, Bacillus subtilis is recognized as a relatively environmentally friendly carrier with an efficient protein secretion system and adaptive metabolism capable of spore production under relatively harsh conditions. This spore property can be used to increase the stability and reusability of vaccines. The possibility of using the protective Lmb epitopes of S. suis in B. subtilis as a carrier of an oral recombinant vaccine against Streptococcus suis was studied. The nucleotide sequences of S. suis were obtained from the GenBank database after a preliminary analysis of literature data on the known protective antigens of S. suis of various serotypes. Nucleotide sequence analysis was performed using Unipro UGENE v. 43.0. The Immune Epitope Database (IEDB) was used to search for T (CTL and Th) and B dependent epitopes of the Lmb gene. A computer-designed vaccine in which localization of CTL, B, and Th epitopes is predicted is described. The results of cloning the sequence of the antigenically active epitope of the S. suis Lmb protein in B. subtilis for subsequent oral administration and study of changes in immunological reactions and adverse reactions in animals are described. The possibility to clone the epitopes of recombinant S. suis Lmb protein into the pBE-S polylinker vector was revealed. In the long term, it seems possible to create a new inexpensive and easy-to-use vaccine against S. suis that does not require injection.</jats:p