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ACS Infectious Diseases Jun 2024Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly...
Endolysins, peptidoglycan hydrolases derived from bacteriophages (phages), are being developed as a promising alternative to conventional antibiotics. To obtain highly active endolysins, a diverse library of these endolysins is vital. We propose here microbial single-cell genome sequencing as an efficient tool to discover dozens of previously unknown endolysins, owing to its culture-independent sequencing method. As a proof of concept, we analyzed and recovered endolysin genes within prophage regions of single-amplified genomes in human skin microbiome samples. We constructed a library of chimeric endolysins by shuffling domains of the natural endolysins and performed high-throughput screening against . One of the lead endolysins, bbst1027, exhibited desirable antimicrobial properties, such as rapid bactericidal activity, no detectable resistance development, and in vivo efficacy. We foresee that this endolysin discovery pipeline is in principle applicable to any bacterial target and boost the development of novel antimicrobial agents.
PubMed: 38906534
DOI: 10.1021/acsinfecdis.4c00039 -
Advanced Science (Weinheim,... Jun 2024The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential...
The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.
PubMed: 38899839
DOI: 10.1002/advs.202404049 -
BioRxiv : the Preprint Server For... Jun 2024Antimicrobial resistance (AMR) poses a critical threat to hospital infections particularly in the context of hospital-acquired infections (HAIs). This study leverages...
UNLABELLED
Antimicrobial resistance (AMR) poses a critical threat to hospital infections particularly in the context of hospital-acquired infections (HAIs). This study leverages genomic tools to predict AMR and identify resistance markers in clinical bacterial samples associated with HAIs. Using comprehensive genomic and phenotypic analyses, we evaluated the genetic profiles of Pseudomonas aeruginosa and Staphylococcus aureus to uncover resistance mechanisms. Our results demonstrate that genomic tools, such as CARD-RGI and the Solu platform, can accurately identify resistance genes and predict AMR phenotypes in nosocomial pathogens. These findings underscore the potential of integrating genomic approaches into clinical practice to enhance the management of resistant infections in hospital settings and inform the development of novel antimicrobial strategies.
IMPORTANCE
This study investigates the impact of prophages on antibiotic resistance in two clinically significant bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. Understanding how prophages influence resistance mechanisms in these pathogens is crucial, as Pseudomonas aeruginosa is known for its role in chronic infections in cystic fibrosis patients, while Staphylococcus aureus, including MRSA strains, is a leading cause of hospital-acquired infections. By exploring the relationship between prophage presence and resistance, this research provides insights that could inform the development of more effective treatment strategies and enhance our ability to combat antibiotic-resistant infections, ultimately improving patient outcomes and public health.
PubMed: 38895396
DOI: 10.1101/2024.06.02.595912 -
MBio Jun 2024Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in are integrated into...
Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the gene coding for the lambda phage repressor using single-molecule fluorescence hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in .
PubMed: 38888367
DOI: 10.1128/mbio.02078-23 -
BMC Microbiology Jun 2024Bergeyella porcorum is a newly identified bacterium that has an ambiguous relationship with pneumonia in pigs. However, few studies have adequately characterized this...
BACKGROUND
Bergeyella porcorum is a newly identified bacterium that has an ambiguous relationship with pneumonia in pigs. However, few studies have adequately characterized this species.
RESULTS
In this study, we analyzed the morphological, physiological, and genomic characteristics of the newly identified B. porcorum sp. nov. strain QD2021 isolated from pigs. The complete genome sequence of the B. porcorum QD2021 strain consists of a single circular chromosome (2,271,736 bp, 38.51% G + C content), which encodes 2,578 genes. One plasmid with a size of 70,040 bp was detected. A total of 121 scattered repeat sequences, 319 tandem repeat sequences, 4 genomic islands, 5 prophages, 3 CRISPR sequences, and 51 ncRNAs were predicted. The coding genes of the B. porcorum genome were successfully annotated across eight databases (NR, GO, KEGG, COG, TCDB, Pfam, Swiss-Prot and CAZy) and four pathogenicity-related databases (PHI, CARD, VFDB and ARDB). In addition, a comparative genome analysis was performed to explore the evolutionary relationships of B. porcorum QD2021.
CONCLUSIONS
To our knowledge, this is the first study to provide fundamental phenotypic and whole-genome sequences for B. porcorum. Our results extensively expand the current knowledge and could serve as a valuable genomic resource for future research on B. porcorum.
Topics: Animals; China; Genome, Bacterial; Swine; Base Composition; Whole Genome Sequencing; Phylogeny; Flavobacteriaceae; Swine Diseases; DNA, Bacterial; Genomic Islands; Plasmids; Flavobacteriaceae Infections; Sequence Analysis, DNA; Molecular Sequence Annotation
PubMed: 38886642
DOI: 10.1186/s12866-024-03366-6 -
Nucleic Acids Research Jun 2024Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We...
Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuity or directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.
PubMed: 38884209
DOI: 10.1093/nar/gkae489 -
Current Microbiology Jun 2024Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic...
Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZD) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZD enterococci carried plasmid rep genes (2-3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43-86). These findings strongly suggest the transmission of LZD-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZD enterococci in all ecological niches and body parts to direct appropriate control strategies.
Topics: Animals; Phylogeny; Linezolid; Swine; Drug Resistance, Bacterial; Genome, Bacterial; Dogs; Anti-Bacterial Agents; Enterococcus; Gram-Positive Bacterial Infections; Humans; Whole Genome Sequencing; Spain; Polymorphism, Single Nucleotide; Microbial Sensitivity Tests; Bacterial Proteins; Genomics; Plasmids
PubMed: 38877167
DOI: 10.1007/s00284-024-03737-2 -
Journal of Bacteriology Jun 2024Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and...
UNLABELLED
Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, has a poorly transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is expressed at higher levels in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins, and permeabilization activity is concentration-dependent. Time-lapse microscopy reveals that competent cells producing ComI are first permeabilized and then die. Based on these observations, we propose a new model for the mechanism of ComI in which response to competence activation leads to selective elimination of the competent subpopulation.
IMPORTANCE
Natural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of into the "competent" state, such that cells with the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.
PubMed: 38874341
DOI: 10.1128/jb.00413-23 -
Cell Host & Microbe Jun 2024Stress-induced prophages commonly "jump ship" by inducing lysis via the host SOS response. In a recent work, Uppalapati et al. reports an alternate, stress-selective...
Stress-induced prophages commonly "jump ship" by inducing lysis via the host SOS response. In a recent work, Uppalapati et al. reports an alternate, stress-selective strategy. Instead of promoting lysis, the Salmonella Gifsy-1 prophage arrests growth specifically when the SOS response coincides with oxidative stress.
Topics: Oxidative Stress; Prophages; SOS Response, Genetics; Salmonella Phages; Salmonella
PubMed: 38870894
DOI: 10.1016/j.chom.2024.05.010 -
Microbiology Resource Announcements Jun 2024We have revealed the genomic sequence of strain Hakim RU_CBWP isolated from pond surface water. Our assembled genome covers 3.787 Mb with 45.5629× coverage, showcasing...
We have revealed the genomic sequence of strain Hakim RU_CBWP isolated from pond surface water. Our assembled genome covers 3.787 Mb with 45.5629× coverage, showcasing an average GC content of 38.60%. This genome contains two CRISPR arrays, 17 prophages, 22 antibiotic resistance genes, and 20 virulence factor genes.
PubMed: 38864657
DOI: 10.1128/mra.00440-24