Nanobody-Dependent Detection of Microcystis aeruginosa by ELISA and Thermal Lens Spectrometry.

Nanobodies against cell surface antigens of toxic cyanobacteria Microcystis aeruginosa were recovered by whole-cell biopanning of a naïve phage display library of nanobodies. Six unique sequences were identified and three sub-cloned and purified as fusion immunoreagents together with either green fluorescent protein or AviTag to be used for diagnostics. The yields of nanobody constructs were in the range of 5-10 mg/l and their specificity and sensitivity was initially evaluated by immunofluorescence and by fluorescent enzyme-linked immunosorbent assay (ELISA) using fluorescent nanobodies. The ELISA data confirmed the nanobody specificity but showed that the saturation of the fluorescence signal already in the presence of few hundreds of cells limited the dynamic range of the method. As an alternative, Avi-tagged nanobodies were used in combination with streptavidin-linked horseradish peroxidase for developing a diagnostic colorimetric cell ELISA, the limit-of-detection of which was 3.2 and 4.5 cells/ml for the two tested cyanobacteria strains, whereas the linear range of the assay was expanded from 10 to 10,000 cells. The fluorescent nanobodies were finally exploited for quantifying cyanobacteria by thermal lens spectrometry (TLS) that enabled to reach a limit-of-detection of 1.2 cells/ml and provided a linear range of measurement between 0 and 10,000 cells. No cross-reactivity with unrelated microalgae was detected and both colorimetric ELISA and TLS provided a linear range of detection of few logs. The data indicate that nanobodies are suitable capture reagents and that both TLS and colorimetric ELISA are reliable to monitor variations of cyanobacteria populations.

Molecules involved in motility regulation in Escherichia coli cells: a review.

The initial colonization of the host organism by commensal, probiotic, and pathogenic Escherichia coli strains is an important step in the development of infections and biofilms. Sensing and colonization of host cell surfaces are governed by flagellar and fimbriae/pili appendages, respectively. Biofilm formation confers great advantages on pathogenic E. coli cells such as protection against the host immune system, antimicrobial agents, and several environmental stress factors. The transition from planktonic to sessile physiological states involves several signaling cascades and factors responsible for the regulation of flagellar motility in E. coli cells. These regulatory factors have thus become important targets to control pathogenicity. Hence, attenuation of flagellar motility is considered a potential therapy against pathogenic E. coli. The present review describes signaling pathways and proteins involved in direct or indirect regulation of flagellar motility. Furthermore, application strategies for antimotility natural or synthetic compounds are discussed also.

Treatment strategies targeting persister cell formation in bacterial pathogens.

Persister cells are transiently antibiotic-tolerant and dormant subpopulations that are produced to escape the effects of antibiotics within biofilms or planktonic cell populations. Persister cells are of high clinical importance due to their tolerance to antimicrobial agents and subsequent failure in antibiotic treatments. Understanding persister cell formation mechanisms is therefore highly important for developing effective therapeutic strategies against pathogenic bacterial persisters. Several anti-persister compounds have been previously identified via isolation from natural resources or chemical synthesis. Furthermore, a combination of these compounds with antibiotics or non-antibiotic drugs also allows action on multiple targets while reducing the administration frequency. Here, we present a comprehensive overview of the clinical importance and formation mechanisms of persister cells as well as the current treatment strategies against persister cell formations in chronic infections.

Regulation and controlling the motility properties of Pseudomonas aeruginosa.

Chronic infections caused by Pseudomonas aeruginosa have been a major concern as their spread and mortality continue to be on the rise. These infections are majorly attributed to biofilm formation via sequential steps where motility plays an essential role in initial attachment of bacterial cells onto biotic and abiotic surfaces, thereby contributing to multi-drug resistance among pathogens. Therefore, attenuating motility properties can be considered as highly potential for controlling P. aeruginosa biofilm formation. This strategy has employed the use of various natural and chemically synthesized compounds. The present review article explained the importance and regulation of different types of motilities properties. Furthermore, it also covered several important alternative approaches using anti-motility agents which could be helpful for controlling P. aeruginosa biofilm-associated infections. Further studies are required for in-depth understandings about the mechanisms of motilities controlling of these molecules at molecular levels.

Chitosan and their derivatives: Antibiofilm drugs against pathogenic bacteria.

Biofilm formed by several pathogenic bacteria results in the development of resistance against antimicrobial compounds. The polymeric materials present in the biofilm architecture hinder the entry of antimicrobial compounds through the surface of bacterial cells which are embedded as well as enclosed beneath the biofilm matrix. Recent and past studies explored the alternative approaches to inhibit the formation of biofilm by different agents isolated from plants, animals, and microbes. Among these agents, chitosan and its derivatives have got more attention due to their properties such as biodegradability, biocompatibility, non-allergenic and non-toxicity. Recent researches have focused on employing chitosan and its derivatives as effective agents to inhibit biofilm formation and attenuate virulence properties by various pathogenic bacteria. Such antibiofilm activity of chitosan and its derivatives can be further enhanced by conjugation with a wide range of bioactive compounds. The present review describes the antibiofilm properties of chitosan and its derivatives against the pathogenic bacteria. This review also summarizes the mechanisms of biofilm inhibition exhibited by these molecules. The knowledge of the antibiofilm activities of chitosan and its derivatives as well as their underlying mechanisms provides essential insights for widening their applications in the future.

Diversity of Bacteria and Bacterial Products as Antibiofilm and Antiquorum Sensing Drugs Against Pathogenic Bacteria.

The increase in antibiotic resistance of pathogenic bacteria has led to the development of new therapeutic approaches to inhibit biofilm formation as well as interfere quorum sensing (QS) signaling systems. The QS system is a phenomenon in which pathogenic bacteria produce signaling molecules that are involved in cell to cell communication, production of virulence factors, biofilm maturation, and several other functions. In the natural environment, several non-pathogenic bacteria are present as mixed population along with pathogenic bacteria and they control the behavior of microbial community by producing secondary metabolites. Similarly, non-pathogenic bacteria also take advantages of the QS signaling molecule as a sole carbon source for their growth through catabolism with enzymes. Several enzymes are produced by bacteria which disrupt the biofilm architecture by degrading the composition of extracellular polymeric substances (EPS) such as exopolysaccharide, extracellular- DNA and protein. Thus, the interference of QS system by bacterial metabolic products and enzymatic catalysis, modification of the QS signaling molecules as well as enzymatic disruption of biofilm architecture have been considered as the alternative therapeutic approaches. This review article elaborates on the diversity of different bacterial species with respect to their metabolic products as well as enzymes and their molecular modes of action. The bacterial enzymes and metabolic products will open new and promising perspectives for the development of strategies against the pathogenic bacterial infections.

Bacteria and bacterial products: Foe and friends to Caenorhabditis elegans.

Caenorhabditis elegans is a model organism for the study of different molecular, biochemical, microbial and immunity-related mechanisms. In its natural habitat, C. elegans survives by feeding microorganisms (mainly bacteria), though majorly on Escherichia coli OP50 when grown in the laboratory. Numerous bacteria are shown to influence the lifespan, behavioural responses and innate immunity of C. elegans. The secondary metabolites produced by bacteria have shown to play key role in C. elegans longevity. This behaviour provides insights for potential development of new strategies for the treatment of diseases in other species, including humans. This review explains the concept of C. elegans microbiome, different mechanisms employed in its longevity and resistance against bacterial pathogens and the effects of various bacteria (both beneficial and harmful) as well as their products on the life cycle of C. elegans.

Strategies for Biofilm Inhibition and Virulence Attenuation of Foodborne Pathogen-Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (E. coli) O157:H7, a gram-negative bacteria identified as a foodborne pathogen causing severe disease is of great concern worldwide. The pathogenicity of E. coli O157:H7 is due to the presence of some virulence factors and its ability to form biofilm which resist antimicrobial compounds, withstand harsh environmental condition and protects from the host immune responses. Formation of biofilm is a multistep process such as adhesion, cellular aggregation and productions of extracellular matrix in which colonies are embedded. There are high numbers of research in the discovery of natural and synthetic compounds which can attenuate the E. coli O157:H7 biofilm formation as well as suppress virulence-related genes. The present review article focuses on the steps involved in E. coli O157:H7 biofilm formation, factors associated with virulence and attenuation.