Novel Acinetobacter baumannii Myovirus TaPaz Encoding Two Tailspike Depolymerases: Characterization and Host-Recognition Strategy

Acinetobacter baumannii, one of the most significant nosocomial pathogens, is capable of producing structurally diverse capsular polysaccharides (CPSs) which are the primary receptors for A. baumannii bacteriophages encoding polysaccharide-degrading enzymes. To date, bacterial viruses specifically infecting A. baumannii strains belonging to more than ten various capsular types (K types) were isolated and characterized. In the present study, we investigate the biological properties, genomic organization, and virus–bacterial host interaction strategy of novel myovirus TaPaz isolated on the bacterial lawn of A. baumannii strain with a K47 capsular polysaccharide structure. The phage linear double-stranded DNA genome of 93,703 bp contains 178 open reading frames. Genes encoding two different tailspike depolymerases (TSDs) were identified in the phage genome. Recombinant TSDs were purified and tested against the collection of A. baumannii strains belonging to 56 different K types. One of the TSDs was demonstrated to be a specific glycosidase that cleaves the K47 CPS by the hydrolytic mechanism.

Phage φAB6-Borne Depolymerase Combats Acinetobacter baumannii Biofilm Formation and Infection

Biofilm formation is one of the main causes of increased antibiotic resistance in Acinetobacter baumannii infections. Bacteriophages and their derivatives, such as tail proteins with depolymerase activity, have shown considerable potential as antibacterial or antivirulence agents against bacterial infections. Here, we gained insights into the activity of a capsular polysaccharide (CPS) depolymerase, derived from the tailspike protein (TSP) of φAB6 phage, to degrade A. baumannii biofilm in vitro. Recombinant TSP showed enzymatic activity and was able to significantly inhibit biofilm formation and degrade formed biofilms; as low as 0.78 ng, the inhibition zone can still be formed on the bacterial lawn. Additionally, TSP inhibited the colonization of A. baumannii on the surface of Foley catheter sections, indicating that it can be used to prevent the adhesion of A. baumannii to medical device surfaces. Transmission and scanning electron microscopy demonstrated membrane leakage of bacterial cells treated with TSP, resulting in cell death. The therapeutic effect of TSP in zebrafish was also evaluated and the results showed that the survival rate was significantly improved (80%) compared with that of the untreated control group (10%). Altogether, we show that TSP derived from φAB6 is expected to become a new antibiotic against multi-drug resistant A. baumannii and a biocontrol agent that prevents the formation of biofilms on medical devices.

Bacteria elicit a phage tolerance response subsequent to infection of their neighbors

Plaque occurrence on a bacterial lawn manifests successive rounds of bacteriophage infection. Yet, mechanisms evolved by bacteria to limit plaque spread have been hardly explored. Here we investigated the dynamics of plaque development by lytic phages infecting the bacterium Bacillus subtilis. We report that plaque expansion is followed by a constriction phase owing to bacterial growth into the plaque zone. This phenomenon is caused by an adaptive process, herein termed “phage tolerance response”, elicited by non-infected bacteria located at the plaque rim upon sensing infection of their neighbors. The temporary phage-tolerance is executed by the stress response RNA polymerase sigma factor σX, primarily through activation of the dlt operon, encoding enzymes that catalyze D-alanylation of cell wall teichoic acid polymers, the major attachment sites for phages infecting Gram-positive bacteria. D-alanylation impedes phage binding and hence infection, thus enabling the uninfected bacteria to form a protective shield opposing plaque spread.

Surface culture of Steinernema sp. on two solid media and their pathogenicity against Galleria mellonella

The use of native entomopathogenic nematodes as biocontrol agents is a strategy to decrease the environmental impact of insecticides and achieve sustainable agriculture crops. In this study, the effect of the surface culture of Steinernema sp. JAP1 over two solid media at 23–27°C on infective juvenile (IJ) production and pathogenicity against Galleria mellonella larvae were investigated. First, the bacterial lawn on the surface of the media with egg yolk (P2) or chicken liver (Cl) were incubated in darkness at 30°C for 48 and 72 h, and 100 surface-sterilized IJs were added. Four harvests were conducted within the next 35 days and the mean accumulated production was superior on Cl (210 × 103 IJs) than on P2 (135 × 103 IJs), but the productivity decreased up to 10% when the incubation time of the bacterial lawn was of 72 h. The mean pathogenicity of in vitro- and in vivo-produced IJs were of 47–64% and 31%, respectively. It is worth noting that none of the two solid media had a statistically significant difference in IJ pathogenicity. Considering that the maximum multiplication factor of IJs on solid media was 2108 and that the pathogenicity against G. mellonella was outstanding, Steinernema sp. has a good potential for in vitro mass production.

Multimaterial bioprinting—minus the printer: Synthetic bacterial patterning with UV-responsive genetic circuits

In this paper, we argue that synthetic biology can help us employ living systems’ unique capacity for self-construction and biomaterial production toward developing novel architectural fabrication paradigms, in which both the raw material production and its refinement into a target structure can be merged into a single computational process embedded in the living structure itself. To demonstrate, here we introduce bioPheme, a novel biofabrication method for engineering bacteria to build biomaterial(s) of designer’s choice into arbitrary 2D geometries specified via transient UV tracing. To this end, we present the design, construction, and testing of the enabling synthetic DNA circuit, which, once inserted into a bacterial colony, allows the bacteria to execute spatial computation by interacting with one another based on the if-then rules encoded in this circuit. At the heart of this genetic circuit is a pair of UV sensor – actuator, and a pair of cell-to-cell signal transmitter – receptor modules, created with genes extracted from the virus λ Phage and marine bacterium Vibrio fischeri, respectively. These modules are wired together to help designers engineer bacteria to build macro-scale structures with seamlessly integrated biomaterials, thereby bridge the molecular and architectural scales. In this way, a bacterial lawn can be programmed to produce different objects with complementary biomaterial compositions, such as a biomineralized superstructure and an elastic tissue filling in-between. In summary, this paper focuses on how scientists’ increasing ability to harness the innate computational capacity of living cells can help designers create self-constructing structures for architectural biofabrication. Through the discussions in this paper, we aim to initiate a shift in today’s biodesign practices toward a greater appreciation and adoption of bottom-up governance of living structures. We are confident that such a paradigm shift will allow for more efficient and sustainable biofabrication systems in the 4th industrial revolution and beyond.

IN VITRO SCREENING OF COLIPHAGE EFFICACY AGAINST MULTIDRUG DRUG RESISTANT CLINICAL ISOLATES OF ESCHERICHIA COLI

The incidence of multi-drug resistance (MDR) in bacteria has become a major problem in this era of modern medicine, making it necessary to look for other options that can be used to treat the infections caused by the MDR bacteria. Scientifically, one of the therapies that is now looking promising, is application of phage therapy. This research work was designed to determine the efficacy of coliphages isolated from sewage, against MDR clinical isolates of E. coli. Ten (10) clinical isolates were subjected to both microbiological conventional and PCR techniques for confirmation, thereafter, the confirmed isolates were tested against some 8 commonly used antibiotics using disk diffusion method to identify the MDR among them. The MDR isolates were subjected to various dilutions (1:10-1:108) of coliphage recovered from sewage, sampled from 5 different locations and isolated by using double agar overlay method. Out of the 10 isolates collected, 8(80%) were confirmed as E. coli, all (8/8: 100%) of which were MDR. However, 6(75%) out of the 8 MDR E. coli, were lysed by the coliphages-observed as cytopathic effect in form of clear zones (plaques) within the bacterial lawn and were recorded from all the dilutions (100%) tested and from all the locations (100%) sampled. Conclusively, infection by MDR E. coli may be life threatening, as it is highly prevalent (100% in our study). However, the coliphages were found to be abundant in sewage and the application of their therapy may provide a good alternative treatment against the MDR.

DETECTION OF BACTERIOPHAGES AGAINST ESKAPE GROUP OF NOSOCOMIAL PATHOGENS FROM GANGA RIVER WATER DURING COMMUNITY BATH AT VARIOUS RITUALS: SINCE 2013–2019

Introduction: Several species of bacterial contaminants are at the high level in river Ganga water but question arises that, why Ganga water is not spoiled even left for long time and answer is a presence of biological components including bacteriophage and bioactive component such as nanoparticles. Objective: In the present study our aim was to detect bacteriophages of resistant microbes such as ESKAPE group of nosocomial and S. Typhi. from different Ganga water samples collected on different rituals. Material & Methods: This study started since 2013 and completed in 2020. As per study design water sample from different places (Prayagraj, Mirzapur and Varanasi) and sites were collected. A total 210 strains (30 each) of Enterococcus faecium (E. faecium), Staphylococcus aureus (S. aureus), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumanii (A. baumannii), Pseudomonas aeruginosa (P. aeruginosa), Escherichia coli (E. coli) ( Called as ESKAPE group) and additionally S. Typhi were identified from the in 500 clinical samples. These identified strains were processed for their biochemical test microscopy and antibiotic sensitivity for its conformation. Confirmed ESKAPE and S. Typhi strains were used for lawn culture. The bacteriophages were isolated from the collected Ganga water samples by using the double layer agar assay method. Results and Discussion: Bacteriophages were observed in the form of plaques on the bacterial lawn culture. Among 210 strains (30 each) of E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, E. coli and S. Typhi total 52 phages were detected in the form of plaques on the bacterial lawn culture. Maximum no of phage sensitivity were identified with E. coli (13) then in S. aureus (11). Eight phages of ware specific to S. Typhi and seven were specific to P. aeruginosa and how ever in six phages are specific to K. pneumoniae and E. faecium. Minimum no of phage sensitivity were identified with A. baumanii (1). Conclusion: Our study concludes that Ganga water is a huge source of above detected bacteriophages among all possible natural sources with full of diversity. This is development of a phage bank, which will be useful for bacteriophage therapy in near future.

SENSITIVITY OF PURE CULTURES OF BRADYRHIZOBIUM JAPONICUM TO FUNGICIDES

Objective. Conduct screening of a wide variety of transgenic mutagenesis strains of Bradyrhizobium japonicum nodule bacteria by the sensitivity to Fever, Standak Top, Akanto Plus, Maxym XL, and Benorad fungicides, and obtain Tn5 mutants resistant to different normal rates of the above pesticides. Methods. Microbiological, statistical. Results. Under the conditions of laboratory experiments, Tn5 mutants of B. japonicum, resistant to the production and dual production normal rate of Fever, Standak Top, Akanto Plus, Maxym XL, and Benorad, were selected. It was shown that the active substances of the products with fungicidal activity Fever, Standak Top, Akanto Plus, Maxym XL do not have bactericidal effect on cell viability of the majority of Tn5 mutants obtained as a result of intergeneric conjugation between Escherichia coli S17-1 with different plasmid vectors and strains of B. japonicum 646 and 634b, and only in some cases reduce the intensity of their reproduction. It was established that Tn5 mutants of B. japonicum under study have different sensitivity to the influence of the normal rates of benomil-based Benorad recommended by the manufacturer and twice-increased. Nine Tn5 mutants were evaluated as low-sensitive to the production normal rate of Benorad, 11 were characterized by a higher sensitivity to fungicide, as evidenced by the zones of delayed growth of bacterial lawn around wells with the product over 15 mm. The influence of the double Benorad normal rate on transposon mutants was significantly stronger compared with other fungicides. Three Tn5 mutants, low-sensitive to the influence of the double rate of this product were selected, and delay of the reproduction of cells in these variants of the experiment was 14-15 mm around the wells. Conclusion. The use of bacterial fertilizers for soybeans based on Tn5 mutants of B. japonicum resistant to modern fungicides will help to reduce the consequences of chemical stress on the formation and functioning of symbiotic systems.

A cellular defense memory imprinted by early life toxic stress

Stress exposure early in life is implicated in various behavioural and somatic diseases. Experiences during the critical perinatal period form permanent, imprinted memories promoting adult survival. Although imprinting is widely recognized to dictate behaviour, whether it actuates specific transcriptional responses at the cellular level is unknown. Here we report that in response to early life stresses, Caenorhabditis elegans nematodes form an imprinted cellular defense memory. We show that exposing newly-born worms to toxic antimycin A and paraquat, respectively, stimulates the expression of toxin-specific cytoprotective reporters. Toxin exposure also induces avoidance of the toxin-containing bacterial lawn. In contrast, adult worms do not exhibit aversive behaviour towards stress-associated bacterial sensory cues. However, the mere re-encounter with the same cues reactivates the previously induced cytoprotective reporters. Learned adult defenses require memory formation during the L1 larval stage and do not appear to confer increased protection against the toxin. Thus, exposure of C. elegans to toxic stresses in the critical period elicits adaptive behavioural and cytoprotective responses, which do not form imprinted aversive behaviour, but imprint a cytoprotective memory. Our findings identify a novel form of imprinting and suggest that imprinted molecular defenses might underlie various pathophysiological alterations related to early life stress.