Prophage Sequences Research Articles

Computational prediction of a phage cocktail active against multidrug-resistant bacteria

Background Antibiotic misuse and overuse have contributed to the emergence of multi-drug resistant (MDR) bacteria, posing a serious public health problem across the globe. Phage cocktails, which combine multiple phages, provide an efficient method to combat multidrug-resistant bacterial infections. This study integrated a computational pipeline to design a phage cocktail against the bacterial strains Acinetobacter baumannii AB0057, Klebsiella pneumoniae subsp. pneumoniae HS11286, and Pseudomonas aeruginosa UCBPP-PA14. Methods The whole genome sequences of selected multidrug-resistant bacteria were accessed. Prophage sequences were identified from them which could be expressed to produce viable lytic phages against MDR bacterial strains, thereby reducing the severity of infection. Prophages were annotated for open reading frames (ORFs), putative promoters, virulence factors, transcriptional terminators, ribosomal RNAs, and transfer RNAs. A dot plot was also generated to investigate similar phages and phylogenetic analysis was performed. Results A total of 11 prophages were predicted from the bacterial genomes. About 472 open reading frames were predicted along with 3 transfer RNAs. Additionally, the presence of 754 putative promoters and 281 transcription terminator sequences was also detected. Comparative genomic and phylogenetic analyses provided insight into the diversity, relatedness, and lytic potential of the phages. The final designed phage cocktail consisted of five selected prophages including Acinetobacter baumannii prophages (2759376-2809756) and (3311844-3364667), and Klebsiella pneumoniae prophages (1288317-1338719), (1778306-1808606), and (2280703-2325555). Conclusion The phage cocktail designed in this study might be useful against MDR Acinetobacter baumannii and Klebsiella pneumoniae infections, especially where conventional antibiotics fail. Sequence similarity analysis suggested that the phage cocktail may also be effective against other carbapenemase-producing K. pneumoniae strains.

Screening and in silico characterization of prophages in Helicobacter pylori clinical strains

The increase of antibiotic resistance calls for alternatives to control Helicobacter pylori, a Gram-negative bacterium associated with various gastric diseases. Bacteriophages (phages) can be highly effective in the treatment of pathogenic bacteria. Here, we develop a method to identify prophages in H. pylori genomes aiming at a future use in therapy. A PCR-based technique tested five primer pairs on 74 clinical H. pylori strains. With the PCR screening, 14 strains most likely to carry prophages were fully sequenced. After that, a more holistic approach was taken by studying the complete genome of the strains. The work allowed us to identify 12 intact prophage sequences, which were then characterized concerning their morphology, virulence, and antibiotic-resistance genes. To understand the variability of prophages, a phylogenetic analysis using the sequences of all H. pylori phages reported to date was performed. Using a PCR-based technique we increased the efficiency of identifying complete prophages to 54.1 %. Genes with homology to potential virulence factors were identified in some new prophages. Phylogenetic analysis revealed a close relationship among H. pylori-phages, although there are phages with different geographical origins. This study provides a deeper understanding of H. pylori-phages, providing valuable insights into their potential use in therapy.

Gene content, phage cycle regulation model and prophage inactivation disclosed by prophage genomics in the Helicobacter pylori Genome Project.

Prophages can have major clinical implications through their ability to change pathogenic bacterial traits. There is limited understanding of the prophage role in ecological, evolutionary, adaptive processes and pathogenicity of Helicobacter pylori, a widespread bacterium causally associated with gastric cancer. Inferring the exact prophage genomic location and completeness requires complete genomes. The international Helicobacter pylori Genome Project (HpGP) dataset comprises 1011 H. pylori complete clinical genomes enriched with epigenetic data. We thoroughly evaluated the H. pylori prophage genomic content in the HpGP dataset. We investigated population evolutionary dynamics through phylogenetic and pangenome analyses. Additionally, we identified genome rearrangements and assessed the impact of prophage presence on bacterial gene disruption and methylome. We found that 29.5% (298) of the HpGP genomes contain prophages, of which only 32.2% (96) were complete, minimizing the burden of prophage carriage. The prevalence of H. pylori prophage sequences was variable by geography and ancestry, but not by disease status of the human host. Prophage insertion occasionally results in gene disruption that can change the global bacterial epigenome. Gene function prediction allowed the development of the first model for lysogenic-lytic cycle regulation in H. pylori. We have disclosed new prophage inactivation mechanisms that appear to occur by genome rearrangement, merger with other mobile elements, and pseudogene accumulation. Our analysis provides a comprehensive framework for H. pylori prophage biological and genomics, offering insights into lysogeny regulation and bacterial adaptation to prophages.

Genomic characterization of prophage elements in Clostridium clostridioforme: an understudied component of the intestinal microbiome.

Genome sequencing of Clostridium clostridioforme strain LM41 revealed the presence of an atypically high proportion of mobile genetic elements for this species, with a particularly high abundance of prophages. Bioinformatic analysis of prophage sequences sought to characterize these elements and identify prophage-linked genes contributing to enhanced fitness of the host bacteria in the dysbiotic gut. Using PHASTER, PhageScope and manual curation, this work has identified 15 prophages: 4 predicted to be intact, 2 predicted to be defective and 9 which are unclassified. Quantitative PCR (qPCR) analysis revealed spontaneous release of four of the LM41 prophages (φ1, φ2, φ4 and φ10) into the culture supernatant, with virion-like particles visualized using transmission electron microscopy. The majority (12/14) of these particles had morphology akin to podoviruses, which is consistent with morphology predictions for φ1 and φ4. We observed diversity in the lysogeny mechanisms utilized by the prophages, with examples of the classical λ-like CI/Cro system, the ICEBs1 ImmR/ImmA-like system and the Mu-like C/Ner system. Classical morons, such as toxins or immune evasion factors, were not observed. We did, however, identify a variety of genes with roles in mediating restriction modification and genetic diversity, as well as some candidate genes with potential roles in host adaptation. Despite being the most abundant entities in the intestine, there is a dearth of information about phages associated with members of the microbiome. This work begins to shed light on the contribution of these elements to the lifestyle of C. clostridioforme LM41.

Comparative Genomic Analysis of Prophages in Human Vaginal Isolates of Streptococcus agalactiae.

Prophages, viral genomes integrated into bacterial genomes, are known to enhance bacterial colonization, adaptation, and ecological fitness, providing a better chance for pathogenic bacteria to disseminate and cause infection. Streptococcus agalactiae (Group B Streptococcus or GBS) is a common bacterium found colonizing the genitourinary tract of humans. However, GBS-colonized pregnant women are at risk of passing the organism to the neonate, where it can cause severe infections. GBS typically encode one or more prophages in their genomes, yet their role in pathogen fitness and virulence has not yet been described. Sequencing and bioinformatic analysis of the genomic content of GBS human isolates identified 42 complete prophages present in their genomes. Comparative genomic analyses of the prophage sequences revealed that the prophages could be classified into five distinct clusters based on their genomic content, indicating significant diversity in their genetic makeup. Prophage diversity was also identified across GBS capsule serotypes, sequence types (STs), and clonal clusters (CCs). Comprehensive genomic annotation revealed that all GBS strains encode paratox, a protein that prevents the uptake of DNA in Streptococcus, either on the chromosome, on the prophage, or both, and each prophage genome has at least one toxin-antitoxin system.

Genomic and taxonomic evaluation of 38 Treponema prophage sequences

BackgroundDespite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage.ResultsWe applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes.ConclusionsIn this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.

EVALUATION OF PROPHAGES AND ANTI MICROBIAL RESISTANCE PROFILE OF BACILLUS SUBTILIS USING IN SILICO APPROACH

The main objective of this study was to identify the prophages and antimicrobial resistance profile of Bacillus subtilis using computational methods. Prophages are potential therapeutic antimicrobial agents against antibiotic- resistant pathogens. The potential of prophage to induce antimicrobial resistance and increase pathogenicity in bacterial genomes resulted in renewed interest in the identification of prophage sequences and their associated antimicrobial resistant genes in bacterial genomes. Sequences of Bacillus subtilis retrieved from the sequence raw archive (SRA) of the National Centre for Biotechnology Information was submitted to bacterial viral bioinformatics resource centre for genome assembly. Prophage sequences were identified using the PHASTER server. The region length, region position and GC percentage of nucleotides of prophage sequences were identified. The antimicrobial resistance gene family, drug class, resistance mechanism and length of reference sequences were identified using the resistance gene identifier protocol of the comprehensive antimicrobial resistance database. It can be concluded that the Bacillus subtilis strain evaluated contained prophage sequences associated with antimicrobial resistance genes, there is, therefore, the need to assess the safety of bacteriophages before they are utilized as alternative to antibiotics.

A bioinformatic approach to identify confirmed and probable CRISPR-Cas systems in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex genomes.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex, or Acb complex, consists of six species: Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii, and Acinetobacter lactucae. A. baumannii is the most clinically significant of these species and is frequently related to healthcare-associated infections (HCAIs). Clustered regularly interspaced short palindromic repeat (CRISPR) arrays and associated genes (cas) constitute bacterial adaptive immune systems and function as variable genetic elements. This study aimed to conduct a genomic analysis of Acb complex genomes available in databases to describe and characterize CRISPR systems and cas genes. Acb complex genomes available in the NCBI and BV-BRC databases, the identification and characterization of CRISPR-Cas systems were performed using CRISPRCasFinder, CRISPRminer, and CRISPRDetect. Sequence types (STs) were determined using the Oxford scheme and ribosomal multilocus sequence typing (rMLST). Prophages were identified using PHASTER and Prophage Hunter. A total of 293 genomes representing six Acb species exhibited CRISPR-related sequences. These genomes originate from various sources, including clinical specimens, animals, medical devices, and environmental samples. Sequence typing identified 145 ribosomal multilocus sequence types (rSTs). CRISPR-Cas systems were confirmed in 26.3% of the genomes, classified as subtypes I-Fa, I-Fb and I-Fv. Probable CRISPR arrays and cas genes associated with CRISPR-Cas subtypes III-A, I-B, and III-B were also detected. Some of the CRISPR-Cas systems are associated with genomic regions related to Cap4 proteins, and toxin-antitoxin systems. Moreover, prophage sequences were prevalent in 68.9% of the genomes. Analysis revealed a connection between these prophages and CRISPR-Cas systems, indicating an ongoing arms race between the bacteria and their bacteriophages. Furthermore, proteins associated with anti-CRISPR systems, such as AcrF11 and AcrF7, were identified in the A. baumannii and A. pittii genomes. This study elucidates CRISPR-Cas systems and defense mechanisms within the Acb complex, highlighting their diverse distribution and interactions with prophages and other genetic elements. This study also provides valuable insights into the evolution and adaptation of these microorganisms in various environments and clinical settings.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Complete genome assemblies of several Xylella fastidiosa subspecies multiplex strains reveals high phage content and novel plasmids

The Gram-negative bacterium Xylella fastidiosa (Xf) was originally found in the Americas, but has now been identified in more than 20 countries across America, Asia, and Europe. This plant pathogen is currently listed as a priority pest in Europe due to its socio-economic and ecological impacts. Within the three Xf subspecies fastidiosa, multiplex and pauca, subsp. multiplex displays a notably wider range of host plants than the other two subspecies. Comparative genomics may allow determination of how Xf subsp. multiplex adapts to new and diverse hosts and environments, so it is important that more genomes of this subspecies are defined. Twelve complete closed genomes sequences of Xf subsp. multiplex were obtained using a hybrid assembly approach combining Illumina and Oxford Nanopore technologies. The combined use of Canu and Unicycler assemblers enabled identification and closure of several plasmid sequences with high similarity to other plasmids described in strains of Xf subsp. fastidiosa and subsp. pauca. The analysis also revealed prophage sequences and contigs outside the chromosomes, annotated as phages. These new genomes, in conjunction with those existing in GenBank, will facilitate exploration of the evolutionary dynamics of Xf subsp. multiplex, its host adaptation mechanisms, and the potential emergence of novel strains of this important plant pathogen.

Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

Whole genome analysis of Pseudomonas mandelii SW-3 and the insights into low-temperature adaptation.

Pseudomonas mandelii SW-3, isolated from the Napahai plateau wetland, can survive in cold environments. The mechanisms underlying the survival of bacteria in low temperatures and high altitudes are not yet fully understood. In this study, the whole genome of SW-3 was sequenced to identify the genomic features that may contribute to survival in cold environments. The results showed that the genome size of strain SW-3 was 6,538,059bp with a GC content of 59%. A total of 67 tRNAs, a 34,110bp prophage sequence, and a large number of metabolic genes were found. Based on 16S rRNA gene phylogeny and average nucleotide identity analysis among P. mandelii, SW-3 was identified as a strain belonging to P. mandelii. In addition, we clarified the mechanisms by which SW-3 survived in a cold environment, providing a basis for further investigation of host-phage interaction. P. mandelii SW-3 showed stress resistance mechanisms, including glycogen and trehalose metabolic pathways, and antisense transcriptional silencing. Furthermore, cold shock proteins and glucose 6-phosphate dehydrogenase may play pivotal roles in facilitating adaptation to cold environmental conditions. The genome-wide analysis provided us with a deeper understanding of the cold-adapted bacterium.

1896. Analysis of Virulence Factors and Antibiotic Resistance Genes in Mycobacterium abscessus Prophage Sequences genomes and Relationships with Clinical Patient Prognoses

Abstract Background M. abscessus is known for its multiple resistance to antibiotics, and diseases caused by M. abscessus have limited methods to treat with. Little is known for prophages carried by M. abscessus, nor their role played in diseases. Methods Strains identified as M. abscessus from patients from December 2017 to August 2021 were included. Prophages, virulence factors and antibiotic resistance genes in genomes were analyzed. The correlation between prophages, virulence factors and antibiotic resistance genes contained, and clinical patient prognoses were analyzed. The sequence similarity between prophages and known phages were analyzed. Results A total of 145 prophage sequences were detected in 56 M. abscessus strains. The average number of intact prophages was 0.7 ± 0.6. The average number and gene proportion of virulence factors and drug resistance genes in prophages were higher than those in known mycobacteriophages. Differences of average prophage carried, virulence factors and drug-resistant genes, and their proportions between different prognoses were not statistically significant, except for virulence factors. The average similarity of prophage sequences in single patient was 99.86%; which was significantly higher of the average matching rate of 89.27% in different patients and the total similarity of 78.28% between prophages and known phages. No clear positive correlation between tested common virulence factors or virulence factors and clinical patient prognoses was observed. Conclusion M. abscessus commonly carries prophages, and they have higher contents of virulence factors and drug resistance genes than known phages. The prophage detected in patients with good prognosis carried more virulence factors, and its meaning was unclear. The composition of the prophage sequences in the strains isolated from the same patient is stable, with occasional base mutation or deletion. Prophages carried in strains of different patients were highly diverse, and shared low similarity with known mycobacterium phages. Disclosures All Authors: No reported disclosures

Genomic characterization and comparative genomic analysis of pathogenic Vibrio isolated from aquaculture-grown white-leg shrimp (Penaeus vannamei) in Guangdong and Jiangsu, China

The proliferation of opportunistic pathogens Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio owensii have impeded the rapid development of aquaculture farming. The study aimed to analyze the whole genome of pathogenic Vibrio strains associated with hepatopancreatic necrosis in Penaeus vannamei. The analysis included genomic characterization, virulence genes profiling, antibiotic resistance genes (ARGs) identification, antimicrobial resistance phenotypes, clustered regularly interspaced short palindromic repeats (CRISPR) and prophage prediction, multi-locus sequence typing, and pan-genome analysis of Vibrio isolates from Guangdong and Jiangsu provinces, China. Sequence analysis of the specimens revealed 29 V. parahaemolyticus isolates, three V. alginolyticus isolates, and one V. owensii isolate. Their genomes are 4.9–5.9 Mbp in size, with a GC content of 44.4–45.4%. Each Vibrio isolate harbored >120 virulence genes. Out of the 33 isolates, 13 (39%) demonstrated multidrug-resistant (MDR). Notably, at least 14 isolates exhibited heightened resistance to sulfamethoxazole, erythromycin, and amoxicillin, with a cumulative detection of 20 ARGs. Nevertheless, no significant correlation was found between the composition of ARGs and the observed antimicrobial resistance phenotypes. All Vibrio isolates differed in the presence of prophage and CRISPR sequences; and two of these prophages harbored virulence genes but not ARGs. The study identified 17 distinct sequence types (STs) in V. parahaemolyticus isolates, primarily ST415 (n = 5), while two Vibrio isolates were classified as ST3. Using the 117 V. parahaemolyticus strains available in the National Center for Biotechnology Information database as a reference, pan-genomic analysis and phylogenetic analysis of the core genome revealed that clinical isolates of the same STs clustered into the same clade, irrespective of isolation date and geographic origins. In summary, these data enhance our understanding of the molecular characteristics and epidemiological risk of Vibrio strains in southern China, especially regarding shrimp aquaculture.

Comparative genomic analysis of strong biofilm-forming Klebsiella pneumoniae isolates uncovers novel ISEcp1-mediated chromosomal integration of a full plasmid-like sequence

Background The goal of the current study was to elucidate the genomic background of biofilm formation in Klebsiella pneumoniae. Methods Clinical isolates were screened for biofilm formation using the crystal violet assay. Antimicrobial resistance (AMR) profiles were assessed by disk diffusion and broth microdilution tests. Biofilm formation was correlated to virulence and resistance genes screened by PCR. Draft genomes of three isolates that form strong biofilm were generated by Illumina sequencing. Results Only the siderophore-coding gene iutA was significantly associated with more pronounced biofilm formation. ST1399-KL43-O1/O2v1 and ST11-KL15-O4 were assigned to the multidrug-resistant strain K21 and the extensively drug-resistant strain K237, respectively. ST1999-KL38-O12 was assigned to K57. Correlated with CRISPR/Cas distribution, more plasmid replicons and prophage sequences were identified in K21 and K237 compared to K57. The acquired AMR genes (bla OXA-48, rmtF, aac(6′)-Ib and qnrB) and (bla NDM-1, bla CTX-M, aph(3′)-VI, qnrS, and aac(6′)-Ib-cr) were found in K237 and K21, respectively. The latter showed a novel ISEcp1-mediated chromosomal integration of replicon type IncM1 plasmid-like structure harboring bla CTX-M-14 and aph(3′)-VI that uniquely interrupted rcsC. The plasmid-mediated heavy metal resistance genes merACDEPRT and arsABCDR were spotted in K21, which also exclusively carried the acquired virulence genes mrkABCDF and the hypervirulence-associated genes iucABCD-iutA, and rmpA/A2. Pangenome analysis revealed NTUH-K2044 accessory genes most frequently shared with K21. Conclusions While less virulent to Galleria mellonella than ST1999 (K57), the strong biofilm former, multidrug-resistant, NDM-producer K. pneumoniae K21 (ST1399-KL43-O1/O2v1) carries a novel chromosomally integrated plasmid-like structure and hypervirulence-associated genes and represents a serious threat to countries in the area.

The heterogenous and diverse population of prophages in Mycobacterium genomes.

Bacterial genomes commonly carry one or more integrated prophages, although prophage prevalence varies greatly in different bacterial genera and species. We have used the recently developed DEPhT software to discover prophages present in sequenced Mycobacterium genomes. Prophages are unevenly distributed among mycobacteria; Mycobacterium tuberculosis strains are devoid of intact prophages, whereas 75% of Mycobacterium abscessus-chelonae strains carry at least one prophage, and some have up to nine. Newly discovered prophage sequences are generally distinct from those of lytic phages isolated using Mycobacterium smegmatis as a host, although assembly of over 3,800 phage and prophage genomes into clusters based on shared sequence similarity yields seven clusters containing both phage and prophage genomes. M. abscessus prophages use a usually large repertoire of attB sites for prophage integration, and new integration-proficient vectors demonstrate the functionality of integration systems identified in silico and provide tools useful for advancing M. abscessus genetics. Genomic analysis reveals additional accessory and likely mobile parts of Mycobacterium genomes including defective prophages, prophage-like regions, and satellite elements proposed to be candidate phage-inducible chromosomal islands (PICIs). M. abscessus prophages and PICIs are unusual in that they often encode phage-encoded ESX-secreted toxin (PEST) systems that are located adjacent to the phage integration functions and are lysogenically expressed and likely provide a fitness advantage to their host. IMPORTANCE Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages.

Novel endolysin LysMP for control of Limosilactobacillus fermentum contamination in small-scale corn mash fermentation

BackgroundTraditional bioethanol fermentation industries are not operated under strict sterile conditions and are prone to microbial contamination. Lactic acid bacteria (LAB) are often pervasive in fermentation tanks, competing for nutrients and producing inhibitory acids that have a negative impact on ethanol-producing yeast, resulting in decreased yields and stuck fermentations. Antibiotics are frequently used to combat contamination, but antibiotic stewardship has resulted in a shift to alternative antimicrobials.ResultsWe demonstrate that endolysin LysMP, a bacteriophage-encoded peptidoglycan hydrolase, is an effective method for controlling growth of LAB. The LysMP gene was synthesized based on the prophage sequence in the genome of Limosilactobacillus fermentum KGL7. Analysis of the recombinant enzyme expressed in E. coli and purified by immobilized metal chelate affinity chromatography (IMAC) showed an optimal lysis activity against various LAB species at pH 6, with stability from pH 4 to 8 and from 20 to 40 °C up to 48 h. Moreover, it retains more than 80% of its activity at 10% ethanol (v/v) for up to 48 h. When LysMP was added at 250 µg/mL to yeast corn mash fermentations containing L. fermentum, it reduced bacterial load by at least 4-log fold compared to the untreated controls and prevented stuck fermentation. In comparison, untreated controls with contamination increased from an initial bacterial load of 1.50 × 107 CFU/mL to 2.25 × 109 CFU/mL and 1.89 × 109 CFU/mL after 24 h and 48 h, respectively. Glucose in the treated samples was fully utilized, while untreated controls with contamination had more than 4% (w/v) remaining at 48 h. Furthermore, there was at least a fivefold reduction in lactic acid (0.085 M untreated contamination controls compared to 0.016 M treated), and a fourfold reduction in acetic acid (0.027 M untreated contamination controls vs. 0.007 M treated), when LysMP was used to treat contaminated corn mash fermentations. Most importantly, final ethanol yields increased from 6.3% (w/v) in untreated contamination samples to 9.3% (w/v) in treated contamination samples, an approximate 50% increase to levels comparable to uncontaminated controls 9.3% (w/v).ConclusionLysMP could be a good alternative to replace antibiotics for mitigation of LAB contamination in biofuel refineries.

Distribution, inducibility, and characteristics of Latilactobacillus curvatus temperate phages.

Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture. Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy. Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype. Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.

An extended catalog of integrated prophages in the infant and adult fecal microbiome shows high prevalence of lysogeny.

The acquisition and gradual maturation of gut microbial communities during early childhood is central to an individual's healthy development. Bacteriophages have the potential to shape the gut bacterial communities. However, the complex ecological interactions between phages and their bacterial host are still poorly characterized. In this study, we investigated the abundance and diversity of integrated prophages in infant and adult gut bacteria by detecting integrated prophages in metagenome assembled genomes (MAGs) of commensal bacteria. Our study included 88 infants sampled at 3 weeks, 3 months, 6 months, and 12 months (n = 323 total samples), and their parents around delivery time (n = 138 total samples). Fecal DNA was extracted and characterized by using shotgun metagenomic sequencing, and a collection of prokaryotic MAGs was generated. The MAG collection was screened for the presence of integrated bacteriophage sequences, allowing their taxonomic and functional characterization. A large collection of 6,186 MAGs from infant and adult gut microbiota was obtained and screened for integrated prophages, allowing the identification of 7,165 prophage sequences longer than 10 kb. Strikingly, more than 70% of the near-complete MAGs were identified as lysogens. The prevalence of prophages in MAGs varied across bacterial families, with a lower prevalence observed among Coriobacteriaceae, Eggerthellaceae, Veillonellaceae and Burkholderiaceae, while a very high prevalence of lysogen MAGs were observed in Oscillospiraceae, Enterococcaceae, and Enterobacteriaceae. Interestingly for several bacterial families such as Bifidobacteriaceae and Bacteroidaceae, the prevalence of prophages in MAGs was higher in early infant time point (3 weeks and 3 months) than in later sampling points (6 and 12 months) and in adults. The prophage sequences were clustered into 5,616 species-like vOTUs, 77% of which were novel. Finally, we explored the functional repertoire of the potential auxiliary metabolic genes carried by these prophages, encoding functions involved in carbohydrate metabolism and degradation, amino acid metabolism and carbon metabolism. Our study provides an enhanced understanding of the diversity and prevalence of lysogens in infant and adult gut microbiota and suggests a complex interplay between prophages and their bacterial hosts.

Complete De Novo Assembly of Wolbachia Endosymbiont of Frankliniella intonsa.

As an endosymbiont, Wolbachia exerts significant effects on the host, including on reproduction, immunity, and metabolism. However, the study of Wolbachia in Thysanopteran insects, such as flower thrips Frankliniella intonsa, remains limited. Here, we assembled a gap-free looped genome assembly of Wolbachia strain wFI in a length of 1,463,884 bp (GC content 33.80%), using Nanopore long reads and Illumina short reads. The annotation of wFI identified a total of 1838 protein-coding genes (including 85 pseudogenes), 3 ribosomal RNAs (rRNAs), 35 transfer RNAs (tRNAs), and 1 transfer-messenger RNA (tmRNA). Beyond this basic description, we identified mobile genetic elements, such as prophage and insertion sequences (ISs), which make up 17% of the entire wFI genome, as well as genes involved in riboflavin and biotin synthesis and metabolism. This research lays the foundation for understanding the nutritional mutualism between Wolbachia and flower thrips. It also serves as a valuable resource for future studies delving into the intricate interactions between Wolbachia and its host.