A comparison between bright field and phase-contrast image analysis techniques in activated sludge morphological characterization

Different approaches using microscopy image analysis procedures were employed for characterization of activated sludge systems. The approaches varied mainly on the type of visualization and acquisition method used for collection of data. In this context, this study focused on the comparison of the two most common acquisition methods: bright field and phase-contrast microscopy. Images were acquired from seven different wastewater treatment plants for a combined period of two years. Advantages and disadvantages of each acquisition technique and the results are discussed. Bright field microscopy proved to be more simple and inexpensive and provided the best overall results.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/32329/2006, POCI/AMB/57069/200

Monitoring of activated sludge settling ability through image analysis : validation on full-scale wastewater treatment plants

In recent years, a great deal of attention has been focused on the research of activated sludge processes, where the solid–liquid separation phase is frequently considered of critical importance, due to the different problems that severely affect the compaction and the settling of the sludge. Bearing that in mind, in this work, image analysis routines were developed in Matlab environment, allowing the identification and characterization of microbial aggregates and protruding filaments in eight different wastewater treatment plants, for a combined period of 2 years. The monitoring of the activated sludge contents allowed for the detection of bulking events proving that the developed image analysis methodology is adequate for a continuous examination of the morphological changes in microbial aggregates and subsequent estimation of the sludge volume index. In fact, the obtained results proved that the developed image analysis methodology is a feasible method for the continuous monitoring of activated sludge systems and identification of disturbances.Empresa de Águas, Efluentes e Resíduos de Braga, Portugal - EM (AGERE)Fundação para a Ciência e a Tecnologia (FCT

Art-175 is a highly efficient antibacterial against multidrug-resistant strains and persisters of Pseudomonas aeruginosa

Artilysins constitute a novel class of efficient enzyme-based antibacterials. Specifically, they covalently combine a bacteriophage-encoded endolysin, which degrades the peptidoglycan, with a targeting peptide that transports the endolysin through the outer membrane of Gram-negative bacteria. Art-085, as well as Art-175, its optimized homolog with increased thermostability, are each composed of the sheep myeloid 29-amino acid (SMAP-29) peptide fused to the KZ144 endolysin. In contrast to KZ144, Art-085 and Art-175 pass the outer membrane and kill Pseudomonas aeruginosa, including multidrug-resistant strains, in a rapid and efficient (similar to 5 log units) manner. Time-lapse microscopy confirms that Art-175 punctures the peptidoglycan layer within 1 min, inducing a bulging membrane and complete lysis. Art-175 is highly refractory to resistance development by naturally occurring mutations. In addition, the resistance mechanisms against 21 therapeutically used antibiotics do not show cross-resistance to Art-175. Since Art-175 does not require an active metabolism for its activity, it has a superior bactericidal effect against P. aeruginosa persisters (up to > 4 log units compared to that of the untreated controls). In summary, Art-175 is a novel antibacterial that is well suited for a broad range of applications in hygiene and veterinary and human medicine, with a unique potential to target persister-driven chronic infections

Engineered endolysin-based "artilysins" to combat multidrug-resistant gram-negative pathogens

The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans).M.W. held a predoctoral fellowship of the "Instituut voor aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen" (IWT Flanders). Y.B. and M. W. were supported by IWT Flanders and Y.B. by a postdoctoral fellowship of the "Bijzonder Onderzoeksfonds-KU Leuven." S. M. is an employee of Lisando GmbH. R. L. acts as scientific adviser to Lisando GmbH

The bacteriophage carrier state of Campylobacter jejuni features changes in host non-coding RNAs and the acquisition of new host-derived CRISPR spacer sequences

Incorporation of self-derived CRISPR DNA protospacers in Campylobacter jejuni PT14 occurs in the presence of bacteriophages encoding a CRISPR-like Cas4 protein. This phenomenon was evident in carrier state infections where both bacteriophages and host are maintained for seemingly indefinite periods as stable populations following serial passage. Carrier state cultures of C. jejuni PT14 have greater aerotolerance in nutrient limited conditions, and may have arisen as an evolutionary response to selective pressures imposed during periods in the extra-intestinal environment. A consequence of this is that bacteriophage and host remain associated and able to survive transition periods where the chances of replicative success are greatly diminished. The majority of the bacteriophage population do not commit to lytic infection, and conversely the bacterial population tolerates low-level bacteriophage replication. We recently examined the effects of Campylobacter bacteriophage/C. jejuni PT14 CRISPR spacer acquisition using deep sequencing strategies of DNA and RNA-Seq to analyze carrier state cultures. This approach identified de novo spacer acquisition in C. jejuni PT14 associated with Class III Campylobacter phages CP8/CP30A but spacer acquisition was oriented toward the capture of host DNA. In the absence of bacteriophage predation the CRISPR spacers in uninfected C. jejuni PT14 cultures remain unchanged. A distinct preference was observed for incorporation of self-derived protospacers into the third spacer position of the C. jejuni PT14 CRISPR array, with the first and second spacers remaining fixed. RNA-Seq also revealed the variation in the synthesis of non-coding RNAs with the potential to bind bacteriophage genes and/or transcript sequences

Bacteria-Killing Type IV Secretion Systems

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I–IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitor

Bacteria-Killing Type IV Secretion Systems

Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I–IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors

Quantitative image analysis for the characterization of microbial aggregates in biological wastewater treatment : a review

Quantitative image analysis techniques have gained an undeniable role in several fields of research during the last decade. In the field of biological wastewater treatment (WWT) processes, several computer applications have been developed for monitoring microbial entities, either as individual cells or in different types of aggregates. New descriptors have been defined that are more reliable, objective, and useful than the subjective and time-consuming parameters classically used to monitor biological WWT processes. Examples of this application include the objective prediction of filamentous bulking, known to be one of the most problematic phenomena occurring in activated sludge technology. It also demonstrated its usefulness in classifying protozoa and metazoa populations. In high-rate anaerobic processes, based on granular sludge, aggregation times and fragmentation phenomena could be detected during critical events, e.g., toxic and organic overloads. Currently, the major efforts and needs are in the development of quantitative image analysis techniques focusing on its application coupled with stained samples, either by classical or fluorescent-based techniques. The use of quantitative morphological parameters in process control and online applications is also being investigated. This work reviews the major advances of quantitative image analysis applied to biological WWT processes.The authors acknowledge the financial support to the project PTDC/EBB-EBI/103147/2008 and the grant SFRH/BPD/48962/2008 provided by Fundacao para a Ciencia e Tecnologia (Portugal)