Resistome Diversity in Escherichia coli Isolates of Global Wastewaters.

Antimicrobial resistance (AMR) is a global health threat requiring urgent attention and effective strategies for containment. AMR is fueled by wastewater mismanagement and global mobility, disseminating multidrug-resistant (MDR) strains worldwide. While global estimates of AMR burden have been informative, community-level understanding has received little attention despite reports of high AMR prevalence in healthy communities. We assessed the "invasion" of antibiotic resistance genes (ARGs) into the normal human flora by characterizing AMR Escherichia coli in local wastewaters contributed by a healthy youth population. This study estimated 26% (out of 300 isolates) resistant and 59% plasmid-bearing E. coli in local wastewater. Of the 78 AMR isolates, the frequency of mono-resistance was higher against tetracycline (32%), followed by kanamycin (17%) and chloramphenicol (9%). Five isolates were potentially MDR. We further sequenced four MDRs and four sensitive strains to comprehend the genome and resistome diversity in comparison to the global wastewater E. coli (genomes from the PATRIC database). The whole-genome analysis revealed extensive genome similarity among global isolates, suggesting global dissemination and colonization of E. coli. Global wastewater resistome majorly comprised ARGs against aminoglycosides (26%), beta-lactam (17%), sulfonamide (11%), and trimethoprim (8%). Resistance to colistin, a last-resort antibiotic, was prevalent in MDRs of European and South Asian isolates. A systems approach is required to address the AMR crisis on a global scale, reduce antibiotic usage, and increase the efficiency of wastewater management and disinfection.

An Eco-evolutionary Model on Surviving Lysogeny Through Grounding and Accumulation of Prophages.

Temperate phages integrate into the bacterial genomes propagating along with the bacterial genomes. Multiple phage elements, representing diverse prophages, are present in most bacterial genomes. The evolutionary events and the ecological dynamics underlying the accumulation of prophage elements in bacterial genomes have yet to be understood. Here, we show that the local wastewater had 7% of lysogens (hosting mitomycin C-inducible prophages), and they showed resistance to superinfection by their corresponding lysates. Genomic analysis of four lysogens and four non-lysogens revealed the presence of multiple prophages (belonging to Myoviridae and Siphoviridae) in both lysogens and non-lysogens. For large-scale comparison, 2180 Escherichia coli genomes isolated from various sources across the globe and 523 genomes specifically isolated from diverse wastewaters were analyzed. A total of 15,279 prophages were predicted among 2180 E. coli genomes and 2802 prophages among 523 global wastewater isolates, with a mean of ~ 5 prophages per genome. These observations indicate that most putative prophages are relics of past bacteria-phage conflicts; they are "grounded" prophages that cannot excise from the bacterial genome. Prophage distribution analysis based on the sequence homology suggested the random distribution of E. coli prophages within and between E. coli clades. The independent occurrence pattern of these prophages indicates extensive horizontal transfers across the genomes. We modeled the eco-evolutionary dynamics to reconstruct the events that could have resulted in the prophage accumulation accounting for infection, superinfection immunity, and grounding. In bacteria-phage conflicts, the bacteria win by grounding the prophage, which could confer superinfection immunity.

Modeling endonuclease colicin-like bacteriocin operons as 'genetic arms' in plasmid-genome conflicts.

Plasmids are acellular propagating entities that depend on bacteria, as molecular parasites, for propagation. A 'tussle' between bacteria and plasmid ensues; bacteria for riddance of the plasmid and plasmid for persistence within a live host. Plasmid-maintenance systems such as endonuclease Colicin-Like Bacteriocins (CLBs) ensure plasmid propagation within the population; (i) the plasmid-cured cells are killed by the CLBs; (ii) damaged cells lyse and release the CLBs that eliminate the competitors, and (iii) the released plasmids invade new bacteria. Surprisingly, endonuclease CLB operons occur on bacterial genomes whose significance is unknown. Here, we study genetics, eco-evolutionary drive, and physiological relevance of genomic endonuclease CLB operons. We investigated plasmidic and genomic endonuclease CLB operons using sequence analyses from an eco-evolutionary perspective. We found 1266 genomic and plasmidic endonuclease CLB operons across 30 bacterial genera. Although 51% of the genomes harbor endonuclease CLB operons, the majority of the genomic endonuclease CLB operons lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the endonuclease CLB operon protects the plasmid-cured host, eliminating the metabolic burden. We show mutual exclusivity of endonuclease CLB operons on genomes and plasmids. We propose an anti-addiction hypothesis for genomic endonuclease CLB operons. Using a stochastic hybrid agent-based model, we show that the endonuclease CLB operons on genomes confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. The conflict between bacterial genome and plasmids allows the emergence of 'genetic arms' such as CLB operons that regulate the ecological interplay of bacterial genomes and plasmids.

Engineered Whole-Cell-Based Biosensors: Sensing Environmental Heavy Metal Pollutants in Water-a Review.

The frequent exposure and accumulation of heavy metals in organisms cause serious health issues affecting a range of organs such as the brain, liver, and reproductive organs in adults, infants, and children. Several parts of the world have high levels of heavy metals affecting millions of people, costing millions of dollars for improving the potability of water and medical treatment of the affected. Hence, water quality assessment is required to monitor the degree of heavy metal contamination in potable water. In nature, organisms respond to various environmental pollutants such as heavy metals, allowing their survival in a diverse environmental niche. With the advent of recombinant DNA technology, it is now possible to manipulate these natural bioreporters into controlled systems which either turn on or off gene expression or activity of enzymes in the presence of specific heavy metals (compound-specific biosensors) otherwise termed as whole-cell biosensors (WCBs). WCBs provide an upper hand compared to other immunosensors, enzyme-based sensors, and DNA-based sensors since microbes can be relatively easily manipulated, scaled up with relative ease, and can detect only the bioavailable heavy metals. In this review, we summarize the current knowledge of the various mechanisms of toxicity elicited by various heavy metals, thence emphasizing the need to develop heavy metal sensing platforms. Following this, the biosensor-based platforms including WCBs for detecting heavy metals developed thus far have been briefly elaborated upon, emphasizing the challenges and solutions associated with WCBs.

'Bacterial Programmed Cell Death': cellular altruism or genetic selfism?

Cell-dependent propagation of the 'self' is the driver of all species, organisms and even genes. Conceivably, elimination of these entities is caused by cellular death. Then, how can genes that cause the death of the same cell evolve? Programmed cell death (PCD) is the gene-dependent self-inflicted death. In multicellular organisms, PCD of a cell confers fitness to the surviving rest of the organism, which thereby allows the selection of genes responsible for PCD. However, PCD in free-living bacteria is intriguing; the death of the cell is the death of the organism. How can such PCD genes be selected in unicellular organisms? The bacterial PCD in a population is proposed to confer fitness to the surviving kin in the form of sporulation, nutrition, infection-containment and matrix materials. While the cell-centred view leading to propositions of 'altruism' is enticing, the gene-centred view of 'selfism' is neglected. In this opinion piece, we reconceptualize the PCD propositions as genetic selfism (death due to loss/mutation of selfish genes) rather than cellular altruism (death for the conferment of fitness to kin). Within the scope and the available evidence, we opine that some of the PCD-like observations in bacteria seem to be the manifestation of genetic selfism by Restriction-Modification systems and Toxin-Antitoxin systems.

Bacterial 'Grounded' Prophages: Hotspots for Genetic Renovation and Innovation.

Bacterial genomes are highly plastic allowing the generation of variants through mutations and acquisition of genetic information. The fittest variants are then selected by the econiche thereby allowing the bacterial adaptation and colonization of the habitat. Larger genomes, however, may impose metabolic burden and hence bacterial genomes are optimized by the loss of frivolous genetic information. The activity of temperate bacteriophages has acute consequences on the bacterial population as well as the bacterial genome through lytic and lysogenic cycles. Lysogeny is a selective advantage as the prophage provides immunity to the lysogen against secondary phage attack. Since the non-lysogens are eliminated by the lytic phages, lysogens multiply and colonize the habitat. Nevertheless, all lysogens have an imminent risk of lytic cycle activation and cell lysis. However, a mutation in the attachment sites or in the genes that encode the specific recombinase responsible for prophage excision could result in 'grounding' of the prophage. Since the lysogens with grounded prophage are immune to respective phage infection as well as dodge the induction of lytic cycle, we hypothesize that the selection of these mutant lysogens is favored relative to their normal lysogenic counterparts. These grounded prophages offer several advantages to the bacterial genome evolution through propensity for genetic variations including inversions, deletions, and insertions via horizontal gene transfer. We propose that the grounded prophages expedite bacterial genome evolution by acting as 'genetic buffer zones' thereby increasing the frequency as well as the diversity of variations on which natural selection favors the beneficial variants. The grounded prophages are also hotspots for horizontal gene transfer wherein several ecologically significant genes such as those involved in stress tolerance, antimicrobial resistance, and novel metabolic pathways, are integrated. Moreover, the high frequency of genetic changes within prophages also allows proportionate probability for the de novo genesis of genetic information. Through sequence analyses of well-characterized E. coli prophages we exemplify various roles of grounded prophages in E. coli ecology and evolution. Therefore, the temperate prophages are one of the most significant drivers of bacterial genome evolution and sites of biogenesis of genetic information.

Endoribonuclease type II toxin-antitoxin systems: functional or selfish?

Most bacterial genomes have multiple type II toxin-antitoxin systems (TAs) that encode two proteins which are referred to as a toxin and an antitoxin. Toxins inhibit a cellular process, while the interaction of the antitoxin with the toxin attenuates the toxin's activity. Endoribonuclease-encoding TAs cleave RNA in a sequence-dependent fashion, resulting in translational inhibition. To account for their prevalence and retention by bacterial genomes, TAs are credited with clinically significant phenomena, such as bacterial programmed cell death, persistence, biofilms and anti-addiction to plasmids. However, the programmed cell death and persistence hypotheses have been challenged because of conceptual, methodological and/or strain issues. In an alternative view, chromosomal TAs seem to be retained by virtue of addiction at two levels: via a poison-antidote combination (TA proteins) and via transcriptional reprogramming of the downstream core gene (due to integration). Any perturbation in the chromosomal TA operons could cause fitness loss due to polar effects on the downstream genes and hence be detrimental under natural conditions. The endoribonucleases encoding chromosomal TAs are most likely selfish DNA as they are retained by bacterial genomes, even though TAs do not confer a direct advantage via the TA proteins. TAs are likely used by various replicons as 'genetic arms' that allow the maintenance of themselves and associated genetic elements. TAs seem to be the 'selfish arms' that make the best use of the 'arms race' between bacterial genomes and plasmids.

Escherichia coli MazEF toxin-antitoxin system does not mediate programmed cell death.

Toxin-antitoxins systems (TAS) are prokaryotic operons containing two small overlapping genes which encode two components referred to as toxin and antitoxin. Involvement of TAS in bacterial programmed cell death (PCD) is highly controversial. MazEF, a typical type II TAS, is particularly implicated in mediating PCD in Escherichia coli. Hence, we compared the metabolic fitness and stress tolerance of E. coli strains (MC4100 and its mazEF-derivative) which were extensively used by proponents of mazEF-mediated PCD. We found that both the strains are deficient in relA gene and that the ΔmazEF strain has lower fitness and stress tolerance compared to wild type MC4100. We could not reproduce mazEF mediated PCD which emphasizes the need for skeptic approach to the PCD hypothesis.

Horizontal gene transfer of chromosomal Type II toxin-antitoxin systems of Escherichia coli.

Type II toxin-antitoxin systems (TAs) are small autoregulated bicistronic operons that encode a toxin protein with the potential to inhibit metabolic processes and an antitoxin protein to neutralize the toxin. Most of the bacterial genomes encode multiple TAs. However, the diversity and accumulation of TAs on bacterial genomes and its physiological implications are highly debated. Here we provide evidence that Escherichia coli chromosomal TAs (encoding RNase toxins) are 'acquired' DNA likely originated from heterologous DNA and are the smallest known autoregulated operons with the potential for horizontal propagation. Sequence analyses revealed that integration of TAs into the bacterial genome is unique and contributes to variations in the coding and/or regulatory regions of flanking host genome sequences. Plasmids and genomes encoding identical TAs of natural isolates are mutually exclusive. Chromosomal TAs might play significant roles in the evolution and ecology of bacteria by contributing to host genome variation and by moderation of plasmid maintenance.

mazEF-mediated programmed cell death in bacteria: "what is this?".

Toxin-antitoxin (TA) systems consist of a bicistronic operon, encoding a toxin and an antitoxin. They are widely distributed in the prokaryotic kingdom, often in multiple numbers. TAs are implicated in contradicting phenomena of persistence and programmed cell death (PCD) in bacteria. mazEF TA system, one of the widely distributed type II toxin-antitoxin systems, is particularly implicated in PCD of Escherichia coli. Nutrient starvation, antibiotic stress, heat shock, DNA damage and other kinds of stresses are shown to elicit mazEF-mediated-PCD. ppGpp and extracellular death factor play a central role in regulating mazEF-mediated PCD. The activation of mazEF system is achieved through inhibition of transcription or translation of mazEF loci. Upon activation, MazF cleaves RNA in a ribosome-independent fashion and subsequent processes result in cell death. It is hypothesized that PCD aids in perseverance of the population during stress; the surviving minority of the cells can scavenge the nutrients released by the dead cells, a kind of "nutritional-altruism." Issues regarding the strains, reproducibility of experimental results and ecological plausibility necessitate speculation. We review the molecular mechanisms of the activation of mazEF TA system, the consequences leading to cell death and the pros and cons of the altruism hypothesis from an ecological perspective.