Rational Design of Profile HMMs for Sensitive and Specific Sequence Detection with Case Studies Applied to Viruses, Bacteriophages, and Casposons.

Profile hidden Markov models (HMMs) are a powerful way of modeling biological sequence diversity and constitute a very sensitive approach to detecting divergent sequences. Here, we report the development of protocols for the rational design of profile HMMs. These methods were implemented on TABAJARA, a program that can be used to either detect all biological sequences of a group or discriminate specific groups of sequences. By calculating position-specific information scores along a multiple sequence alignment, TABAJARA automatically identifies the most informative sequence motifs and uses them to construct profile HMMs. As a proof-of-principle, we applied TABAJARA to generate profile HMMs for the detection and classification of two viral groups presenting different evolutionary rates: bacteriophages of the Microviridae family and viruses of the Flavivirus genus. We obtained conserved models for the generic detection of any Microviridae or Flavivirus sequence, and profile HMMs that can specifically discriminate Microviridae subfamilies or Flavivirus species. In another application, we constructed Cas1 endonuclease-derived profile HMMs that can discriminate CRISPRs and casposons, two evolutionarily related transposable elements. We believe that the protocols described here, and implemented on TABAJARA, constitute a generic toolbox for generating profile HMMs for the highly sensitive and specific detection of sequence classes.

Polyamines and linear DNA mediate bacterial threat assessment of bacteriophage infection.

Monitoring the extracellular environment for danger signals is a critical aspect of cellular survival. However, the danger signals released by dying bacteria and the mechanisms bacteria use for threat assessment remain largely unexplored. Here, we show that lysis of Pseudomonas aeruginosa cells releases polyamines that are subsequently taken up by surviving cells via a mechanism that relies on Gac/Rsm signaling. While intracellular polyamines spike in surviving cells, the duration of this spike varies according to the infection status of the cell. In bacteriophage-infected cells, intracellular polyamines are maintained at high levels, which inhibits replication of the bacteriophage genome. Many bacteriophages package linear DNA genomes and linear DNA is sufficient to trigger intracellular polyamine accumulation, suggesting that linear DNA is sensed as a second danger signal. Collectively, these results demonstrate how polyamines released by dying cells together with linear DNA allow P. aeruginosa to make threat assessments of cellular injury.

Efficacy in Galleria mellonella Larvae and Application Potential Assessment of a New Bacteriophage BUCT700 Extensively Lyse Stenotrophomonas maltophilia.

In recent years, Stenotrophomonas maltophilia (S. maltophilia) has become an important pathogen of clinically acquired infections accompanied by high pathogenicity and high mortality. Moreover, infections caused by multidrug-resistant S. maltophilia have emerged as a serious challenge in clinical practice. Bacteriophages are considered a promising alternative for the treatment of S. maltophilia infections due to their unique antibacterial mechanism and superior bactericidal ability compared with traditional antibiotic agents. Here, we reported a new phage BUCT700 that has a double-stranded DNA genome of 43,214 bp with 70% GC content. A total of 55 ORFs and no virulence or antimicrobial resistance genes were annotated in the genome of phage BUCT700. Phage BUCT700 has a broad host range (28/43) and can lyse multiple ST types of clinical S. maltophilia (21/33). Furthermore, bacteriophage BUCT700 used the Type IV fimbrial biogenesis protein PilX as an adsorption receptor. In the stability test, phage BUCT700 showed excellent thermal stability (4 to 60°C) and pH tolerance (pH = 4 to 12). Moreover, phage BUCT700 was able to maintain a high titer during long-term storage. The adsorption curve and one-step growth curve showed that phage BUCT700 could rapidly adsorb to the surface of S. maltophilia and produce a significant number of phage virions. In vivo, BUCT700 significantly increased the survival rate of S. maltophilia-infected Galleria mellonella (G. mellonella) larvae from 0% to 100% within 72 h, especially in the prophylactic model. In conclusion, these findings indicate that phage BUCT700 has promising potential for clinical application either as a prophylactic or therapeutic agent. IMPORTANCE The risk of Stenotrophomonas maltophilia infections mediated by the medical devices is exacerbated with an increase in the number of ICU patients during the Corona Virus Disease 2019 (COVID-19) epidemic. Complications caused by S. maltophilia infections could complicate the state of an illness, greatly extending the length of hospitalization and increasing the financial burden. Phage therapy might be a potential and promising alternative for clinical treatment of multidrug-resistant bacterial infections. Here, we investigated the protective effects of phage BUCT700 as prophylactic and therapeutic agents in Galleria mellonella models of infection, respectively. This study demonstrates that phage therapy can provide protection in targeting S. maltophilia-related infection, especially as prophylaxis.

Identification of Phage Receptor-Binding Protein Sequences with Hidden Markov Models and an Extreme Gradient Boosting Classifier.

Receptor-binding proteins (RBPs) of bacteriophages initiate the infection of their corresponding bacterial host and act as the primary determinant for host specificity. The ever-increasing amount of sequence data enables the development of predictive models for the automated identification of RBP sequences. However, the development of such models is challenged by the inconsistent or missing annotation of many phage proteins. Recently developed tools have started to bridge this gap but are not specifically focused on RBP sequences, for which many different annotations are available. We have developed two parallel approaches to alleviate the complex identification of RBP sequences in phage genomic data. The first combines known RBP-related hidden Markov models (HMMs) from the Pfam database with custom-built HMMs to identify phage RBPs based on protein domains. The second approach consists of training an extreme gradient boosting classifier that can accurately discriminate between RBPs and other phage proteins. We explained how these complementary approaches can reinforce each other in identifying RBP sequences. In addition, we benchmarked our methods against the recently developed PhANNs tool. Our best performing model reached a precision-recall area-under-the-curve of 93.8% and outperformed PhANNs on an independent test set, reaching an F1-score of 84.0% compared to 69.8%.

Assessment of bacteriophage-encoded endolysin as a potent antimicrobial agent against antibiotic-resistant Salmonella Typhimurium.

This study was designed to evaluate the potential of using newly purified Salmonella phage-encoded endolysin LysPB32 as novel antibiotic alternative. The endolysin LysPB32 was characterized by analyzing pH and thermal stability, lytic spectrum, antimicrobial activity, and mutant frequency against Salmonella Typhimurium KCCM 40253 (STKCCM), S. Typhimurium ATCC 19585 (STATCC), S. Typhimurium CCARM 8009 (STCCARM), Klebsiella pneumoniae ATCC 23357 (KPATCC), K. pneumoniae CCARM 10237 (KPCCARM), Pseudomonas aeruginosa ATCC 27853 (PAATCC), Listeria monocytogenes ATCC 1911 (LMATCC), Staphylococcus aureus ATCC 25923 (SAATCC), and S. aureus CCARM 3080 (SACCARM). The molecular weight of LysPB32 is 17 kDa that was classified as N-acetyl-ß-d-muramidase. The optimum activity of LysPB32 against the outer membrane (OM) permeabilized STKCCM, STATCC, and STCCARM was observed at 37 °C and pH 6.5. LysPB32 had a broad spectrum of muralytic activity against antibiotic-sensitive STKCCM (41 mOD/min), STATCC (32 mOD/min), and SBKACC (25 mOD/min) and antibiotic-resistant STCCARM (35 mOD/min) and KPCCARM (31 mOD/min). The minimum inhibitory concentrations (MICs) of polymyxin B against STKCCM, STCCARM, and STATCC were decreased by 4-, 4-, and 8-folds, respectively, when treated with LysPB32. The combination of LysPB32 and polymyxin B effectively inhibited the growth of STKCCM, STCCARM, and STATCC after 24 h of incubation at 37 °C, showing 4.9-, 4.4-, and 3.3-log reductions, respectively. The mutant frequency was low in STKCCM, STCCARM, and STATCC treated with combination of LysPB32-polymyxin B system. The results suggest the LysPB32-polymyxin system can be a potential candidate for alternative therapeutic agent to control antibiotic-resistant pathogens.

Phage resistance mutation triggered by OmpC deficiency in Klebsiella pneumoniae induced limited fitness costs.

Outer membrane proteins (OMPs) play an important role in bacterial fitness costs. Derived from the interaction between Klebsiella pneumoniae K7 and phage GH-K3, K7RB is an outer membrane porin-deficient phage-resistant mutant strain triggered by ompC712 deletion, exhibits expression inhibition of OmpC, OmpN, KPN_02430 and OmpF, but its fitness costs and regulatory mechanism remains unknown. In this study, compared with K7, K7RB showed almost unaffected growth rate, slightly decreased virulence, and increased resistance to some antibiotics. Transcriptome analysis showed that the pathways of glycerolipid metabolism and nitrogen metabolism in K7RB were significantly inhibited, while the transcription of permeases belonging to ABC transporters tended to be active, nutrient uptakes such as citrate and phenylalanine were also enhanced. However, transcriptional up-regulation in K7RB was inhibited by overexpression of OmpC, OmpN, KPN_02430 and OmpF in general. Overexpression of OmpN, KPN_02430 and OmpF, respectively, restoring the sensitivity of strains to antibiotics to varying degrees, while OmpC overexpression aggravated the bacterial drug-resistance especially to ß-lactam antibiotics. Besides, unlike OmpC and OmpF, overexpression of OmpN and KPN_02430 reduced bacterial virulence. In brief, by revealing the limited fitness costs of phage-resistant mutant K. pneumoniae with porin-deficiency, our study providing a reference for the design and development of drugs to inhibit the ways of bacterial metabolic rewiring and to increase fitness costs.

PhageLeads: Rapid Assessment of Phage Therapeutic Suitability Using an Ensemble Machine Learning Approach.

The characterization of therapeutic phage genomes plays a crucial role in the success rate of phage therapies. There are three checkpoints that need to be examined for the selection of phage candidates, namely, the presence of temperate markers, antimicrobial resistance (AMR) genes, and virulence genes. However, currently, no single-step tools are available for this purpose. Hence, we have developed a tool capable of checking all three conditions required for the selection of suitable therapeutic phage candidates. This tool consists of an ensemble of machine-learning-based predictors for determining the presence of temperate markers (integrase, Cro/CI repressor, immunity repressor, DNA partitioning protein A, and antirepressor) along with the integration of the ABRicate tool to determine the presence of antibiotic resistance genes and virulence genes. Using the biological features of the temperate markers, we were able to predict the presence of the temperate markers with high MCC scores (>0.70), corresponding to the lifestyle of the phages with an accuracy of 96.5%. Additionally, the screening of 183 lytic phage genomes revealed that six phages were found to contain AMR or virulence genes, showing that not all lytic phages are suitable to be used for therapy. The suite of predictors, PhageLeads, along with the integrated ABRicate tool, can be accessed online for in silico selection of suitable therapeutic phage candidates from single genome or metagenomic contigs.

Comparative Assessment of Bacteriophage and Antibiotic Activity against Multidrug-Resistant Staphylococcus aureus Biofilms.

Problems connected with biofilm-related infections and antibiotic resistance necessitate the investigation and development of novel treatment strategies. Given their unique characteristics, one of the most promising alternatives to conventional antibiotics are bacteriophages. In the in vitro and in vivo larva model study, we demonstrate that phages vB_SauM-A, vB_SauM-C, and vB_SauM-D are effective antibiofilm agents. The exposure of biofilm to phages vB_SauM-A and vB_SauM-D led to 2-3 log reductions in the colony-forming unit number in most of the multidrug-resistant S. aureus strains. It was found that phage application reduced the formed biofilms independently of the used titer. Moreover, the study demonstrated that bacteriophages are more efficient in biofilm biomass removal and reduction in staphylococci count when compared to the antibiotics used. The scanning electron microscopy analysis results are in line with colony forming unit (CFU) counting but not entirely consistent with crystal violet (CV) staining. Additionally, phages vB_SauM-A, vB_SauM-C, and vB_SauM-D can significantly increase the survival rate and extend the survival time of Galleria mellonella larvae.

In Vitro Characterization and In Vivo Efficacy Assessment in Galleria mellonella Larvae of Newly Isolated Bacteriophages against Escherichia coli K1.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

Assessment of the microbiome during bacteriophage therapy in combination with systemic antibiotics to treat a case of staphylococcal device infection.

BACKGROUND: Infectious bacterial diseases exhibiting increasing resistance to antibiotics are a serious global health issue. Bacteriophage therapy is an anti-microbial alternative to treat patients with serious bacterial infections. However, the impacts to the host microbiome in response to clinical use of phage therapy are not well understood. RESULTS: Our paper demonstrates a largely unchanged microbiota profile during 4 weeks of phage therapy when added to systemic antibiotics in a single patient with Staphylococcus aureus device infection. Metabolomic analyses suggest potential indirect cascading ecological impacts to the host (skin) microbiome. We did not detect genomes of the three phages used to treat the patient in metagenomic samples taken from saliva, stool, and skin; however, phages were detected using endpoint-PCR in patient serum. CONCLUSION: Results from our proof-of-principal study supports the use of bacteriophages as a microbiome-sparing approach to treat bacterial infections. Video abstract.

Assessment of bacteriophage vB_Pd_PDCC-1 on bacterial dynamics during ontogenetic development of the longfin yellowtail (Seriola rivoliana).

The Seriola genus includes species of worldwide commercial importance due to its rapid growth and easy adaptability to confinement conditions. However, like other fish species, large mortalities occur during their early life stages, where the main problems are caused by opportunistic bacteria. Disease control strategies are thus urgently needed. The present study aimed to evaluate the efficacy of phage vB_Pd_PDCC-1 during the early development of longfin yellowtail (Seriola rivoliana), as well as its effect on microbial communities. This broad-host-range phage was added to the culture every 3 days starting from the egg-stage until 12 days after hatching (DAH) at a concentration of 1.41×1010 plaque-forming units (PFU) per mL and at a multiplicity of infection (MOI) of 1. The results showed positive effects (p<0.05) on egg hatching, survival, growth, and pigmentation area in treated larvae. Moreover, high-throughput sequencing analysis of 16S rRNA genes showed that phage administration did not produce significant changes (p>0.05) in the composition and structure of the associated microbiota. However, sequences affiliated to the Gammaproteobacteria class were displaced by those belonging to the Alphaproteobacteria class over time regardless of the treatment received. At the family level, there was a decrease in Rhodobacteraceae, Pseudoalteromonadaceae, and Flavobacteriaceae in both groups over time. To our best knowledge, this study represents the first attempt to evaluate the effect of a phage as a biological control agent during ontogenetic development of longfin yellowtail larvae. KEY POINTS: • Phages can be used against proliferation of Vibrio in fish cultures. • Seriola includes several important commercial fish species due to its rapid growth. • Phages do not cause significant changes in the associated microbiota.

Identification and classification of antiviral defence systems in bacteria and archaea with PADLOC reveals new system types.

To provide protection against viral infection and limit the uptake of mobile genetic elements, bacteria and archaea have evolved many diverse defence systems. The discovery and application of CRISPR-Cas adaptive immune systems has spurred recent interest in the identification and classification of new types of defence systems. Many new defence systems have recently been reported but there is a lack of accessible tools available to identify homologs of these systems in different genomes. Here, we report the Prokaryotic Antiviral Defence LOCator (PADLOC), a flexible and scalable open-source tool for defence system identification. With PADLOC, defence system genes are identified using HMM-based homologue searches, followed by validation of system completeness using gene presence/absence and synteny criteria specified by customisable system classifications. We show that PADLOC identifies defence systems with high accuracy and sensitivity. Our modular approach to organising the HMMs and system classifications allows additional defence systems to be easily integrated into the PADLOC database. To demonstrate application of PADLOC to biological questions, we used PADLOC to identify six new subtypes of known defence systems and a putative novel defence system comprised of a helicase, methylase and ATPase. PADLOC is available as a standalone package (https://github.com/padlocbio/padloc) and as a webserver (https://padloc.otago.ac.nz).

Phage gene expression and host responses lead to infection-dependent costs of CRISPR immunity.

CRISPR-Cas immune systems are widespread in bacteria and archaea, but not ubiquitous. Previous work has demonstrated that CRISPR immunity is associated with an infection-induced fitness cost, which may help explain the patchy distribution observed. However, the mechanistic basis of this cost has remained unclear. Using Pseudomonas aeruginosa PA14 and its phage DMS3vir as a model, we perform a 30-day evolution experiment under phage mediated selection. We demonstrate that although CRISPR is initially selected for, bacteria carrying mutations in the phage receptor rapidly invade the population following subsequent reinfections. We then test three potential mechanisms for the observed cost of CRISPR: (1) autoimmunity from the acquisition of self-targeting spacers, (2) immunopathology or energetic costs from increased cas gene expression and (3) toxicity caused by phage gene expression prior to CRISPR-mediated cleavage. We find that phages can express genes before the immune system clears the infection and that expression of these genes can have a negative effect on host fitness. While infection does not lead to increased expression of cas genes, it does cause differential expression of multiple other host processes that may further contribute to the cost of CRISPR immunity. In contrast, we found little support for infection-induced autoimmunological and immunopathological effects. Phage gene expression prior to cleavage of the genome by the CRISPR-Cas immune system is therefore the most parsimonious explanation for the observed phage-induced fitness cost.

Selection for Reducing Energy Cost of Protein Production Drives the GC Content and Amino Acid Composition Bias in Gene Transfer Agents.

Gene transfer agents (GTAs) are virus-like elements integrated into bacterial genomes, particularly, those of Alphaproteobacteria The GTAs can be induced under conditions of nutritional stress, incorporate random fragments of bacterial DNA into miniphage particles, lyse the host cells, and infect neighboring bacteria, thus enhancing horizontal gene transfer. We show that GTA genes evolve under conditions of pronounced positive selection for the reduction of the energy cost of protein production as shown by comparison of the amino acid compositions with those of both homologous viral genes and host genes. The energy saving in GTA genes is comparable to or even more pronounced than that in the genes encoding the most abundant, essential bacterial proteins. In cases in which viruses acquire genes from GTAs, the bias in amino acid composition disappears in the course of evolution, showing that reduction of the energy cost of protein production is an important factor of evolution of GTAs but not bacterial viruses. These findings strongly suggest that GTAs represent bacterial adaptations rather than selfish, virus-like elements. Because GTA production kills the host cell and does not propagate the GTA genome, it appears likely that the GTAs are retained in the course of evolution via kin or group selection. Therefore, we hypothesize that GTAs facilitate the survival of bacterial populations under energy-limiting conditions through the spread of metabolic and transport capabilities via horizontal gene transfer and increases in nutrient availability resulting from the altruistic suicide of GTA-producing cells.IMPORTANCE Kin selection and group selection remain controversial topics in evolutionary biology. We argue that these types of selection are likely to operate in bacterial populations by showing that bacterial gene transfer agents (GTAs), but not related viruses, evolve under conditions of positive selection for the reduction of the energy cost of GTA particle production. We hypothesize that GTAs are dedicated devices mediating the survival of bacteria under conditions of nutrient limitation. The benefits conferred by GTAs under nutritional stress conditions appear to include horizontal dissemination of genes that could provide bacteria with enhanced capabilities for nutrient utilization and increases of nutrient availability occurring through the lysis of GTA-producing bacteria.

Integration of phage and yeast display platforms: A reliable and cost effective approach for binning of peptides as displayed on-phage.

Hundreds of target specific peptides are routinely discovered by peptide display platforms. However, due to the high cost of peptide synthesis only a limited number of peptides are chemically made for further analysis. Here we describe an accurate and cost effective method to bin peptides on-phage based on binding region(s), without any requirement for peptide or protein synthesis. This approach, which integrates phage and yeast display platforms, requires display of target and its alanine variants on yeast. Flow cytometry was used to detect binding of peptides on-phage to the target on yeast. Once hits were identified, they were synthesized to confirm their binding region(s) by HDX (Hydrogen deuterium exchange) and crystallography. Moreover, we have successfully shown that this approach can be implemented as part of a panning process to deplete non-functional peptides. This technique can be applied to any target that can be successfully displayed on yeast; it narrows down the number of peptides requiring synthesis; and its utilization during selection results in enrichment of peptide population against defined binding regions on the target.

Cell-Free Bacteriophage Genome Synthesis Using Low-Cost Sequence-Verified Array-Synthesized Oligonucleotides.

Synthesizing engineered bacteriophages (phages) for human use has potential in various applications ranging from drug screening using a phage display to clinical use using phage therapy. However, the engineering of phages conventionally involves the use of an in vivo system that has low production efficiency because of high virulence against the host and low transformation efficiency. To circumvent these issues, de novo phage genome synthesis using chemically synthesized oligonucleotides (oligos) has increased the potential for engineering phages in a cell-free system. Here, we present a cell-free, low-cost, de novo gene synthesis technology called Sniper assembly for phage genome construction. With massively parallel sequencing of microarray-synthesized oligos, we generated and identified approximately 100 000 clonal DNA clusters in vitro and 5000 error-free ones in a cell-free environment. To demonstrate its practical application, we synthesized the Acinetobacter phage AP205 genome (4268 bp) using 65 sequence-verified DNA clones. Compared to previous reports, Sniper assembly lowered the genome synthesis cost ($0.0137/bp) by producing low-cost sequence-verified DNA.

Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion.

The intracellular pathogen Listeria monocytogenes is distinguished by its ability to invade and replicate within mammalian cells. Remarkably, of the 15 serovars within the genus, strains belonging to serovar 4b cause the majority of listeriosis clinical cases and outbreaks. The Listeria O-antigens are defined by subtle structural differences amongst the peptidoglycan-associated wall-teichoic acids (WTAs), and their specific glycosylation patterns. Here, we outline the genetic determinants required for WTA decoration in serovar 4b L. monocytogenes, and demonstrate the exact nature of the 4b-specific antigen. We show that challenge by bacteriophages selects for surviving clones that feature mutations in genes involved in teichoic acid glycosylation, leading to a loss of galactose from both wall teichoic acid and lipoteichoic acid molecules, and a switch from serovar 4b to 4d. Surprisingly, loss of this galactose decoration not only prevents phage adsorption, but leads to a complete loss of surface-associated Internalin B (InlB),the inability to form actin tails, and a virulence attenuation in vivo. We show that InlB specifically recognizes and attaches to galactosylated teichoic acid polymers, and is secreted upon loss of this modification, leading to a drastically reduced cellular invasiveness. Consequently, these phage-insensitive bacteria are unable to interact with cMet and gC1q-R host cell receptors, which normally trigger cellular uptake upon interaction with InlB. Collectively, we provide detailed mechanistic insight into the dual role of a surface antigen crucial for both phage adsorption and cellular invasiveness, demonstrating a trade-off between phage resistance and virulence in this opportunistic pathogen.

A simple, reproducible and cost-effective procedure to analyse gut phageome: from phage isolation to bioinformatic approach.

The microbiota of the human gut is a complex and rich community where bacteria and their viruses, the bacteriophages, are dominant. There are few studies on the phage community and no clear standard for isolating them, sequencing and analysing their genomes. Since this makes comparisons between studies difficult, we aimed at defining an easy, low-cost, and reproducible methodology. We analysed five different techniques to isolate phages from human adult faeces and developed an approach to analyse their genomes in order to quantify contamination and classify phage contigs in terms of taxonomy and lifestyle. We chose the polyethylene glycol concentration method to isolate phages because of its simplicity, low cost, reproducibility, and of the high number and diversity of phage sequences that we obtained. We also tested the reproducibility of this method with multiple displacement amplification (MDA) and showed that MDA severely decreases the phage genetic diversity of the samples and the reproducibility of the method. Lastly, we studied the influence of sequencing depth on the analysis of phage diversity and observed the beginning of a plateau for phage contigs at 20,000,000 reads. This work contributes to the development of methods for the isolation of phages in faeces and for their comparative analysis.

Assessment of the alteration in phage adsorption rates of antibiotic-resistant Salmonella typhimurium.

This study was designed to evaluate the phage-binding receptors on the surface of antibiotic-sensitive Salmonella typhimurium (ASST) and antibiotic-resistant S. typhimurium (ARST). The antibiotic susceptibilities of plasmid-cured ASST and ARST were evaluated against ampicillin, cephalothin, ciprofloxacin, kanamycin, penicillin, and tetracycline. The capsular polysaccharides (CPSs) and lipopolysaccharides (LPSs) were quantified using carbazole assay and HPLC, respectively. The amounts of CPSs and LPSs in ARST were decreased from 108 to 62 µg/ml and 284-111 ng/ml, respectively, after plasmid curing. The adsorption rates of P22, PBST10, and PBST13 to plasmid-uncured and plasmid-cured ASST and ARST were decreased after proteinase K and periodate treatments. The highest reduction in phage adsorption rate was observed for P22 to the plasmid-cured ARST treated with periodate (71%). The relative expression levels of btuB, fhuA, and rfaL were decreased by more than twofold in the plasmid-cured ASST, corresponding to the decrease in the adsorption rates of P22 and PBST10. The plasmid-cured ARST lost the ability to express the ß-lactamase gene, which was related to the loss of resistance to ampicillin, cephalothin, kanamycin, penicillin, and tetracycline. The results provide valuable insights into understanding the interaction between phage and antibiotic-resistant bacteria.

Cost and benefits of clustered regularly interspaced short palindromic repeats spacer acquisition.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-mediated immunity in bacteria allows bacterial populations to protect themselves against pathogens. However, it also exposes them to the dangers of auto-immunity by developing protection that targets its own genome. Using a simple model of the coupled dynamics of phage and bacterial populations, we explore how acquisition rates affect the probability of the bacterial colony going extinct. We find that the optimal strategy depends on the initial population sizes of both viruses and bacteria. Additionally, certain combinations of acquisition and dynamical rates and initial population sizes guarantee protection, owing to a dynamical balance between the evolving population sizes, without relying on acquisition of viral spacers. Outside this regime, the high cost of auto-immunity limits the acquisition rate. We discuss these optimal strategies that minimize the probability of the colony going extinct in terms of recent experiments. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.