Bacterial Growth In Liquid Medium Research Articles

Multilayer dextran derivative based capsules fighting bacteria resistant to Antibiotic: Case of Kanamycin-Resistant Escherichia coli

Bacteria resistance to antibiotics has emerged as a major health problem. Developing new antibacterial systems is then of major interest. In this sense, we present biocapsules presenting inherent antibacterial capacity. The self-assembly of charged biopolymer, namely diethylaminoethyl-dextran hydrochloride (dex+) and dextran sulfate (dex-), were done on calcium carbonate microparticles, used as a template. Zeta potential measurements have shown the successful alternate adsorption of these biopolymers and related charge reversal upon the multilayer film construction onto the particles surface. The shape of the capsules was characterized by scanning electron microscopy (SEM). These particles were tested against bacteria resistant to antibiotics, namely kanamycin-resistant Escherichia coli. An inhibitory effect of the particles was observed during bacterial growth in liquid medium, i.e. in the range of 10 % for (dex+/dex-)n coated CaCO3 materials and of 50% for (dex+/dex-)n capsules. These findings evidence the high potential of capsules to act as antimicrobial agents in future and in treatments against infections.

The Cysteine Protease MaOC1, a Prokaryotic Caspase Homolog, Cleaves the Antitoxin of a Type II Toxin-Antitoxin System.

The bloom-forming cyanobacterium Microcystis aeruginosa is known for its global distribution and for the production of toxic compounds. In the genome of M. aeruginosa PCC 7806, we discovered that the gene coding for MaOC1, a caspase homolog protease, is followed by a toxin-antitoxin module, flanked on each side by a direct repeat. We therefore investigated their possible interaction at the protein level. Our results suggest that this module belongs to the ParE/ParD-like superfamily of type II toxin-antitoxin systems. In solution, the antitoxin is predominantly alpha-helical and dimeric. When coexpressed with its cognate toxin and isolated from Escherichia coli, it forms a complex, as revealed by light scattering and affinity purification. The active site of the toxin is restricted to the C-terminus of the molecule. Its truncation led to normal cell growth, while the wild-type form prevented bacterial growth in liquid medium. The orthocaspase MaOC1 was able to cleave the antitoxin so that it could no longer block the toxin activity. The most likely target of the protease was the C-terminus of the antitoxin with two sections of basic amino acid residues. E. coli cells in which MaOC1 was expressed simultaneously with the toxin-antitoxin pair were unable to grow. In contrast, no effect on cell growth was found when using a proteolytically inactive MaOC1 mutant. We thus present the first case of a cysteine protease that regulates the activity of a toxin-antitoxin module, since all currently known activating proteases are of the serine type.

A Label-Free, Non-Intrusive, and Rapid Monitoring of Bacterial Growth on Solid Medium Using Microwave Biosensor.

Microwave resonator sensors are attractive for their contactless and label-free capability of monitoring bacterial growth in liquid media. This paper outlines a new label-free microwave biosensor based on a pair of planar split ring resonators for non-invasive monitoring of bacterial growth on a solid agar media. The sensor is comprised of two split ring resonators with slightly different resonant frequencies for differential operation. The transmission coefficient (S21) of the sensor is considered as the sensor's response with a designed and measured quality factor above 200 to ensure a high-resolution operation of the biosensor. Two resonant frequencies of 1.95 and 2.11 GHz represent the sensing signal and the reference signal, respectively. The developed sensor demonstrates high performance in monitoring the growth dynamics of Escherichia coli (E. coli) on Luria-Bertani (LB) agar with 4 mm thickness. The sensor's resonant amplitude response demonstrated 0.5 dB variation corresponding to the bacterial growth over 48 hours when bacteria were spread on LB agar starting with initial OD600 = 1.5. Moreover, 0.6 dB change in the sensor's response was observed over 96 hours of bacterial growth starting with an initial OD600 = 1.17 spotted on LB agar. The measured results fit well to the curves created using Richards' bacterial growth model, showing the strength of the sensor as a potential candidate for use in predictive food microbiology systems.

Antioxidant and antibacterial evaluation of polysaccharides sequentially extracted from onion (Allium cepa L.)

We investigated the antioxidant and antimicrobial potential of sequentially extracted onion polysaccharide fractions namely HBSS, CHSS, DASS and CASS. The different antioxidant assays indicated that ACLPs exhibited potentially appreciable antioxidant activity in a dose-dependent manner. Among all the fractions, CHSS rendered the highest antioxidant action towards ABTS radical cations (97.52%), Fe2+ chelating (98.94%) and superoxide anion radical scavenging (76.27%). Whereas, HBSS possessed the highest potential for DPPH radicals (93.68%), hydroxyl radicals (65.12%) as well as for reducing power (0.559). CASS exhibited the highest lipid peroxidation inhibition (86.43%), while, DASS showed the best β-carotene bleaching inhibition (92.26%). Furthermore, regardless of the bacterial strain, DASS represented the strongest antibacterial activity on the basis of largest inhibition zone, the lowest minimal inhibitory concentration and maximum inhibition of bacterial growth in liquid medium. Overall results indicated that ACLPs hold a promise as potential natural antioxidant additives and antimicrobial agents for formulating the functional foods with potential applications in the medical and food industries.

Vision Marker-Based In Situ Examination of Bacterial Growth in Liquid Culture Media.

The detection of bacterial growth in liquid media is an essential process in determining antibiotic susceptibility or the level of bacterial presence for clinical or research purposes. We have developed a system, which enables simplified and automated detection using a camera and a striped pattern marker. The quantification of bacterial growth is possible as the bacterial growth in the culturing vessel blurs the marker image, which is placed on the back of the vessel, and the blurring results in a decrease in the high-frequency spectrum region of the marker image. The experiment results show that the FFT (fast Fourier transform)-based growth detection method is robust to the variations in the type of bacterial carrier and vessels ranging from the culture tubes to the microfluidic devices. Moreover, the automated incubator and image acquisition system are developed to be used as a comprehensive in situ detection system. We expect that this result can be applied in the automation of biological experiments, such as the Antibiotics Susceptibility Test or toxicity measurement. Furthermore, the simple framework of the proposed growth measurement method may be further utilized as an effective and convenient method for building point-of-care devices for developing countries.

THE ROLE OF mutR GENE IN METABOLISM AND VIRULENCE OF emm12 GENOTYPE STREPTOCOCCUS PYOGENES STRAINS

In the present study the functional role of the mutR regulatory protein gene of Streptococcus pyogenes (emm12) was studied. The mutR gene was inactivated in the strains no. 97 and no. 152 by insertional mutagenesis. Inactivation of the mutR gene was found to affect the dynamic and characteristics of bacterial growth in liquid medium. Expression of secreted nucleases was significantly lower in the mutant strains compared to the wild-type strains. Two-dimensional electrophoresis and mass-spectrometry revealed differences in expression of number of the proteins in mutant strainscompared to the wild-type strains. Inactivation of the mutR gene negatively affected capacity of S. pyogenes to adhere to human epithelial cells. Finally, the virulence properties of the no. 152[mutR] mutant strains were found to be 4,7-fold less compared to the strain no. 152, while the no. 97[mutR] mutant strain became avirulent compared to the strain no. 97 due to insertional inactivation of the mutR gene.

A small-molecule inhibitor of the bacterial type III secretion system protects against in vivo infection with Citrobacter rodentium

The type III secretion system (T3SS) is highly conserved in many Gram-negative pathogenic bacteria and functions as an injector of bacterial proteins (effectors) into host cells. T3SSs are involved in establishing disease processes, but this machinery is not essential for bacterial growth or homeostasis. Thus, T3SS is expected to be a candidate therapeutic target, and inhibitors of T3SSs could potentially reduce virulence without causing bacterial death, thereby avoiding any subsequent development of resistance. We identified a linear polyketide compound, aurodox, as a specific T3SS inhibitor from the culture broth of Streptomyces sp. using a screening system for the T3SS-mediated hemolysis of enteropathogenic Escherichia coli (EPEC) established by our group. Aurodox strongly inhibited T3SS-mediated hemolysis with an IC(50) value of 1.5 μg ml(-1) without affecting bacterial growth in liquid media. We also demonstrated that aurodox specifically inhibits the secretion of type III-secreted proteins such as EspB, EspF and Map, without affecting the expression of the housekeeping protein GroEL. Furthermore, an in vivo infection study using mice clearly indicated that the administration of aurodox allowed the mice to survive a lethal dose of Citrobactor rodentium, a model bacterium for human pathogens such as EPEC. Thus, our in vivo study directly demonstrated for the first time that this putative T3SS inhibitor can be applied as a novel class of anti-infective agents.

Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm

The 16S rRNA methyltransferase Sgm from "Micromonospora zionensis" confers resistance to aminoglycoside antibiotics by specific modification of the 30S ribosomal A site. Sgm is a member of the FmrO family, distant relatives of the S-adenosyl-L-methionine (SAM)-dependent RNA subfamily of methyltransferase enzymes. Using amino acid conservation across the FmrO family, seven putative key amino acids were selected for mutation to assess their role in forming the SAM cofactor binding pocket or in methyl group transfer. Each mutated residue was found to be essential for Sgm function, as no modified protein could effectively support bacterial growth in liquid media containing gentamicin or methylate 30S subunits in vitro. Using isothermal titration calorimetry, Sgm was found to bind SAM with a K(D) (binding constant) of 17.6 microM, and comparable values were obtained for one functional mutant (N179A) and four proteins modified at amino acids predicted to be involved in catalysis in methyl group transfer. In contrast, none of the G135, D156, or D182 Sgm mutants bound the cofactor, confirming their role in creating the SAM binding pocket. These results represent the first functional characterization of any FmrO methyltransferase and may provide a basis for a further structure-function analysis of these aminoglycoside resistance determinants.

Tracking bacterial growth in liquid media and a new bacterial life model

By increasing viscosity of liquid media above 8.4 centipoise (cp) i.e. 0.084 g * cm(-1) * s(-1), individual growth and family formation of Escherichia coli was continuously observed in real-time for up to 6 h. The observations showed primarily unidirectional growth and reproduction of E. coli and suggested more than one reproduction in the observed portion of E. coli life span. A new bacterial life model is proposed: each bacterium has a stable cell polarity that ultimately transforms into two bacteria of different generations; the life cycle of a bacterium can contain more than one reproduction cycle; and the age of a bacterium should be defined by its experienced chronological time. This new bacterial life model differs from the dominant concepts of bacterial life but complies with all basic life principles based on direct observation of macroorganisms.