Non-coding RNA regulates phage sensitivity in Listeria monocytogenes

Non-coding RNAs (ncRNAs) have gained increasing attention as their diverse roles in virulence and environmental stress in Listeria monocytogenes have become clearer. The ncRNA rliB is an atypical member of the CRISPR family, conserved at the same genomic locus in all analyzed L. monocytogenes genomes and also in other Listeria species. In this study, rliB defective mutants (Lm3-22-ΔrliB) were constructed by homologous recombination. The growth cycle of Lm3-22-ΔrliB mutants was slower than that of wild-type Lm3-22. The sensitivity of Lm3-22-ΔrliB to the Listeria phage vB-LmoM-SH3-3 was significantly increased, and the efficiency of plaque formation was enhanced by 128 fold. Compared with wild type, the adhesion and invasion of Lm3-22-ΔrliB decreased significantly (9.3% and 1.33%, respectively). After 4 hours of infection, the proliferation of Lm3-22-ΔrliB in RAW264.7 cells also decreased significantly. Transcription level of invasion-related surface proteins showed that the internalin genes lmo0610 and lm0514, and the peptidoglycan binding protein gene lmo1799 in Lm3-22-ΔrliB were significantly increased. In addition, after interaction with phage, the transcription levels of inlA, lmo0610, lmo1799, lmo2085, and lmo0514 in Lm3-22-ΔrliB cells were significantly upregulated, while inlB was downregulated, compared with Lm3-22 control group with phage treatment. Therefore, rliB deletion effectively regulated the interaction between Listeria and phage, weaken its invasion ability, and provided a new theoretical basis for biocontrol of phage.

Identification of Novel Phage Resistance Mechanisms in Campylobacter jejuni by Comparative Genomics

Phages infecting Campylobacter jejuni are considered a promising intervention strategy at broiler farms, yet phage sensitivity of naturally occurring poultry isolates is not well studied. Here, we investigated phage sensitivity and identified resistance mechanisms of C. jejuni strains originating from Danish broilers belonging to the most prevalent MLST (ST) types. Determining plaque formation of 51 phages belonging to Fletchervirus or Firehammervirus showed that 21 out of 31 C. jejuni strains were susceptible to at least one phage. While C. jejuni ST-21 strains encoded the common phase variable O-methyl phosphoramidate (MeOPN) receptor of the Fletchervirus and were only infected by these phages, ST-45 strains did not encode this receptor and were exclusively infected by Firehammervirus phages. To identify internal phage resistance mechanism in ST-21 strains, we performed comparative genomics of two strains, CAMSA2002 sensitive to almost all Fletchervirus phages and CAMSA2038, resistant to all 51 phages. The strains encoded diverse clustered regularly interspaced short palindromic repeats (CRISPR) spacers but none matched the tested phages. Sequence divergence was also observed in a predicted SspE homolog and putative restriction modification systems including a methyl-specific McrBC endonuclease. Furthermore, when mcrB was deleted, CAMSA2038 became sensitive to 17 out of 43 phages, three being Firehammervirus phages that otherwise did not infect any ST-21 strains. Yet, 16 phages demonstrated significantly lower efficiencies of plating on the mcrB mutant suggesting additional resistance mechanism still restricting phage propagation in CAMSA2038. Thus, our work demonstrates that C. jejuni isolates originating from broilers may have acquired several resistance mechanisms to successfully prevent phage infection in their natural habitat.

A histidine kinase and a response regulator provide phage resistance to Marinomonas mediterranea via CRISPR-Cas regulation

CRISPR-Cas systems are used by many prokaryotes to defend against invading genetic elements. In many cases, more than one CRISPR-Cas system co-exist in the same cell. Marinomonas mediterranea MMB-1 possesses two CRISPR-Cas systems, of type I–F and III-B respectively, which collaborate in phage resistance raising questions on how their expression is regulated. This study shows that the expression of both systems is controlled by the histidine kinase PpoS and a response regulator, PpoR, identified and cloned in this study. These proteins show similarity to the global regulators BarA/UvrY. In addition, homologues to the sRNAs CsrB and CsrC and the gene coding for the post-transcriptional repressor CsrA have been also identified indicating the conservation of the elements of the BarA/UvrY regulatory cascade in M. mediterranea. RNA-Seq analyses have revealed that all these genetics elements are regulated by PpoS/R supporting their participation in the regulatory cascade. The regulation by PpoS and PpoR of the CRISPR-Cas systems plays a role in phage defense since mutants in these proteins show an increase in phage sensitivity.

Digital control of c-di-GMP in E. coli balances population-wide developmental transitions and phage sensitivity

Nucleotide-based signaling molecules (NSMs) are widespread in bacteria and eukaryotes, where they control important physiological and behavioral processes. In bacteria, NSM-based regulatory networks are highly complex, entailing large numbers of enzymes involved in the synthesis and degradation of active signaling molecules. How the converging input from multiple enzymes is transformed into robust and unambiguous cellular responses has remained unclear. Here we show that Escherichia coli converts dynamic changes of c-di-GMP into discrete binary signaling states, thereby generating heterogeneous populations with either high or low c-di-GMP. This is mediated by an ultrasensitive switch protein, PdeL, which senses the prevailing cellular concentration of the signaling molecule and couples this information to c-di-GMP degradation and transcription feedback boosting its own expression. We demonstrate that PdeL acts as a digital filter that facilitates precise developmental transitions, confers cellular memory, and generates functional heterogeneity in bacterial populations to evade phage predation. Based on our findings, we propose that bacteria apply ultrasensitive regulatory switches to convert dynamic changes of NSMs into binary signaling modes to allow robust decision-making and bet-hedging for improved overall population fitness.

Long-term persistence of crAss-like phage crAss001 is associated with phase variation in Bacteroides intestinalis

Background The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of lysogeny, these viruses demonstrate long-term persistence in the human gut microbiome, dominating the virome in some individuals. Results Here we show that rapid phase variation of alternate capsular polysaccharides in Bacteroides intestinalis cultures plays an important role in a dynamic equilibrium between phage sensitivity and resistance, allowing phage and bacteria to multiply in parallel. The data also suggests the role of a concomitant phage persistence mechanism associated with delayed lysis of infected cells, similar to carrier state infection. From an ecological and evolutionary standpoint, this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other “benign” forms of phage infection when the host is stably present at high abundance. Conclusion Long-term persistence of bacteriophage and host could result from mutually beneficial mechanisms driving bacterial strain-level diversity and phage survival in complex environments.

Long-term persistence of crAss-like phage crAss001 is associated with phase variation in Bacteroides intestinalis

The crAss-like phages are ubiquitous and highly abundant members of the human gut virome that infect commensal bacteria of the order Bacteroidales. Although incapable of classical lysogeny, these viruses demonstrate unexplained long-term persistence in the human gut microbiome, dominating the virome in some individuals. Here we demonstrate that rapid phase variation of alternate capsular polysaccharides plays an important role in dynamic equilibrium between phage sensitivity and resistance in B. intestinalis cultures, allowing phage and bacteria to multiply in parallel. The data also suggests the role of concomitant phage persistence mechanisms associated with delayed lysis of infected cells, such as carrier state infection. From an ecological and evolutionary standpoint this type of phage-host interaction is consistent with the Piggyback-the-Winner model, which suggests a preference towards lysogenic or other 'benign' forms of phage infection when the host is stably present at high abundance.

Bacteriophage Resistance Affects Flavobacterium columnare Virulence Partly via Mutations in Genes Related to Gliding Motility and Type IX Secretion System

Increasing problems with antibiotic resistance has directed interest towards phages as tools to treat bacterial infections in the aquaculture industry. However, phage resistance evolves rapidly in bacteria posing a challenge for successful phage therapy. To investigate phage resistance in the fish pathogenic bacterium Flavobacterium columnare, two phage-sensitive, virulent wild-type isolates, FCO-F2 and FCO-F9, were exposed to phages and subsequently analyzed for bacterial viability and colony morphology. Twenty-four phage-exposed isolates were further characterized for phage resistance, antibiotic susceptibility, motility, adhesion and biofilm formation on polystyrene surface, protease activity, whole genome sequencing and virulence against rainbow trout fry. Bacterial viability first decreased in the exposure cultures, subsequently increasing after 1-2 days. Simultaneously, the colony morphology of the phage-exposed isolates changed from original rhizoid to rough. The rough isolates arising in phage exposure were phage-resistant with low virulence, whereas rhizoid isolates maintained phage sensitivity, though reduced, and high virulence. Gliding motility and protease activity were also related to the phage sensitivity. Observed genetic mutations in phage-resistant isolates were mostly located in genes coding for type IX secretion system, a component of the flavobacterial gliding motility machinery. However, there were mutational differences between individual isolates, and not all phage-resistant isolates had genetic mutations. This indicates that development of phage resistance in F. columnare probably is a multifactorial process including both genetic mutations and changes in gene expression. Phage resistance may not, however, be a challenge for development of phage therapy against F. columnare infections, since phage resistance is associated with decrease in bacterial virulence.ImportancePhage resistance of infectious bacteria is a common phenomenon posing challenges for development of phage therapy. Along with growing World population and need for increased food production, constantly intensifying animal farming has to face increasing problems of infectious diseases. Columnaris disease, caused by F. columnare, is a worldwide threat for salmonid fry and juvenile farming. Without antibiotic treatments, infections can lead to 100% mortality in a fish stock. Phage therapy of columnaris disease would reduce a development of antibiotic-resistant bacteria and antibiotic loads by the aquaculture industry, but phage-resistant bacterial isolates may become a risk. However, phenotypic and genetic characterization of phage-resistant F. columnare isolates in this study revealed that they are less virulent than phage-sensitive isolates and thus not a challenge for phage therapy against columnaris disease. This is a valuable information for the fish farming industry globally when considering phage-based prevention and curing methods for F. columnare infections.

Characteristic and Potential Therapeutic Effect of Isolated MDR-Acinetobacter Baumannii Lytic Phage

Background: Acinetobacter baumannii is a major pathogen in the hospital, especially in Intensive Care Units (ICU) and the resistance to multiple drugs as a major contributor to hospital infection. Bacteriophages are viruses that attack bacteria and kill them that could be used for clinical treatment. The aim of the study is in evaluating the function of bacteriophage specificity of multi-drug resistant Acinetobacter baumannii, to be used as a useful method for treating of Acinetobacter Infections.Methods: Cross-sectional study during the year 2017, from patients admitted to the ICU, First, 48 isolates of Acinetobacter baumannii were identified by phenotypic method and amplified with blaOXA-51 gene. Then, the sensitivity of phages to pathogens namely ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp) evaluated. DNA of the phage was extracted using the Viral Nucleic Extraction Kit II (Geneaid, Taipei, Taiwan) according to the manufacturer's instructions. Then for protein analysis, PEG-precipitated purified phages were subjected directly to SDS-PAGE, and protein bands were visualized by coomassie Blue G-250 staining method. Finally for cell survival assay we investigated the toxicity of the isolated phage to Hela cells.Results: In the bacterial resistance pattern, the highest resistance belongs to ciprofloxacin. In optimal phage test, at dilution of 1 (MOI 1) it produced the best effect on bacteria in 30 minutes. Phage sensitivity to different hosts performed by double layer agar method, the phage was treated with ESKAPE bacteria and after 24 hours’ incubation at 37°C, only for Acinetobacter baumannii Plaque created. The genome analysis indicated that phage pIsf-AB2 has a double-stranded DNA genome. In bacterial control, all cells were killed by A. baumannii, and no live-cell was seen. The cells remained in control of the phage, and the phage did not affect the cells.Conclusion: Our findings support the potential application of the phage with potent endolysin activity against MDR A. baumannii and give useful information for its further study and use.

Characterization of Clinical MRSA Isolates from Northern Spain and Assessment of Their Susceptibility to Phage-Derived Antimicrobials

Methicillin-resistant Staphylococcus aureus (MRSA) is a prevalent nosocomial pathogen, causing a wide range of diseases. The increased frequency of MRSA isolates in hospitals and the emergence of vancomycin resistance have sparked the search for new control strategies. This study aimed to characterize sixty-seven MRSA isolates collected from both infected patients and asymptomatic carriers in a Spanish hospital. RAPD-PCR allowed the identification of six genetic patterns. We also investigated the presence of genes involved in producing adhesins, toxins and the capsule; the biofilm; and antimicrobial resistance. A notable percentage of the isolates carried virulence genes and showed medium-high ability to form biofilms. Next, we assessed the strains’ susceptibility to two phages (phiIPLA-C1C and phiIPLA-RODI) and one endolysin (LysRODI). All strains were resistant to phiIPLA-C1C, and most (70.2%) were susceptible to phiIPLA-RODI. Regarding LysRODI, all strains displayed susceptibility, although to varying degrees. There was a correlation between endolysin susceptibility and the random amplification of polymorphic DNA (RAPD) profile or the presence of some virulence genes (fnbA, eta, etb, PVL and czr), but that was not observed with biofilm-forming ability, strain origin or phage sensitivity. Taken together, these findings can help to explain the factors influencing endolysin effectiveness, which will contribute to the development of efficient therapies targeting MRSA infections.

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.