Unveiling Prasinovirus diversity and host specificity through targeted enrichment in the South China Sea.

Unicellular green picophytoplankton from the Mamiellales order are pervasive in marine ecosystems and susceptible to infections by prasinoviruses, large double-stranded DNA viruses within the Nucleocytoviricota phylum. We developed a double-stranded DNA virus enrichment and shotgun sequencing method, and successfully assembled 80 prasinovirus genomes from 43 samples in the South China Sea. Our research delivered the first direct estimation of 94% accuracy in correlating genome similarity to host range. Stirkingly, our analyses uncovered unexpected host-switching across diverse algal lineages, challenging the existing paradigms of host-virus co-speciation and revealing the dynamic nature of viral evolution. We also detected six instances of horizontal gene transfer between prasinoviruses and their hosts, including a novel alternative oxidase. Additionally, diversifying selection on a major capsid protein suggests an ongoing co-evolutionary arms race. These insights not only expand our understanding of prasinovirus genomic diversity but also highlight the intricate evolutionary mechanisms driving their ecological success and shaping broader virus-host interactions in marine environments.

A nanoluciferase-encoded bacteriophage illuminates viral infection dynamics of Pseudomonas aeruginosa cells.

Bacteriophages (phages) are increasingly considered for both treatment and early detection of bacterial pathogens given their specificity and rapid infection kinetics. Here, we exploit an engineered phage expressing nanoluciferase to detect signals associated with Pseudomonas aeruginosa lysis spanning single cells to populations. Using several P. aeruginosa strains we found that the latent period, burst size, fraction of infected cells, and efficiency of plating inferred from fluorescent light intensity signals were consistent with inferences from conventional population assays. Notably, imaging-based traits were obtained in minutes to hours in contrast to the use of overnight plaques, which opens the possibility to study infection dynamics in spatial and/or temporal contexts where plaque development is infeasible. These findings support the use of engineered phages to study infection kinetics of virus-cell interactions in complex environments and potentially accelerate the determination of viral host range in therapeutically relevant contexts.

Potential of training of anti-Staphylococcus aureus therapeutic phages against Staphylococcus epidermidis multidrug-resistant isolates is restricted by inter- and intra-sequence type specificity.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus, while a limited number of studies investigated phage activity against S. epidermidis. We studied the potential of phage training to extend the host range of two types of anti-S. aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S. epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti-S. aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.ImportanceS. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

Pangenomic landscapes shape performances of a synthetic genetic circuit across Stutzerimonas species.

Engineering identical genetic circuits into different species typically results in large differences in performance due to the unique cellular environmental context of each host, a phenomenon known as the "chassis-effect" or "context-dependency". A better understanding of how genomic and physiological contexts underpin the chassis-effect will improve biodesign strategies across diverse microorganisms. Here, we combined a pangenomic-based gene expression analysis with quantitative measurements of performance from an engineered genetic inverter device to uncover how genome structure and function relate to the observed chassis-effect across six closely related Stutzerimonas hosts. Our results reveal that genome architecture underpins divergent responses between our chosen non-model bacterial hosts to the engineered device. Specifically, differential expression of the core genome, gene clusters shared between all hosts, was found to be the main source of significant concordance to the observed differential genetic device performance, whereas specialty genes from respective accessory genomes were not significant. A data-driven investigation revealed that genes involved in denitrification and components of trans-membrane transporter proteins were among the most differentially expressed gene clusters between hosts in response to the genetic device. Our results show that the chassis-effect can be traced along differences among the most conserved genome-encoded functions and that these differences create a unique biodesign space among closely related species.IMPORTANCEContemporary synthetic biology endeavors often default to a handful of model organisms to host their engineered systems. Model organisms such as Escherichia coli serve as attractive hosts due to their tractability but do not necessarily provide the ideal environment to optimize performance. As more novel microbes are domesticated for use as biotechnology platforms, synthetic biologists are urged to explore the chassis-design space to optimize their systems and deliver on the promises of synthetic biology. The consequences of the chassis-effect will therefore only become more relevant as the field of biodesign grows. In our work, we demonstrate that the performance of a genetic device is highly dependent on the host environment it operates within, promoting the notion that the chassis can be considered a design variable to tune circuit function. Importantly, our results unveil that the chassis-effect can be traced along similarities in genome architecture, specifically the shared core genome. Our study advocates for the exploration of the chassis-design space and is a step forward to empowering synthetic biologists with knowledge for more efficient exploration of the chassis-design space to enable the next generation of broad-host-range synthetic biology.

Characterization of a novel phage against multidrug-resistant Klebsiella pneumoniae.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) poses a significant challenge in global healthcare, underscoring the urgency for innovative therapeutic approaches. Phage therapy emerges as a promising strategy amidst rising antibiotic resistance, emphasizing the crucial need to identify and characterize effective phage resources for clinical use. In this study, we introduce a novel lytic phage, RCIP0100, distinguished by its classification into the Chaoyangvirus genus and Fjlabviridae family based on International Committee on Taxonomy of Viruses (ICTV) criteria due to low genetic similarity to known phage families. Our findings demonstrate that RCIP0100 exhibits broad lytic activity against 15 out of 27 tested MDR-KP strains, including diverse profiles such as carbapenem-resistant K. pneumoniae (CR-KP). This positions phage RCIP0100 as a promising candidate for phage therapy. Strains resistant to RCIP0100 also showed increased susceptibility to various antibiotics, implying the potential for synergistic use of RCIP0100 and antibiotics as a strategic countermeasure against MDR-KP.

A novel lytic phage infecting MDR Salmonella enterica and its application as effective food biocontrol.

Salmonella enterica is a foodborne pathogen associated with both typhoid and non-typhoid illness in humans and animals. This problem is further exacerbated by the emergence of antibiotic-resistant strains of Salmonella enterica. Therefore, to meet public health and safety, there is a need for an alternative strategy to tackle antibiotic-resistant bacteria. Bacteriophages or (bacterial viruses), due to their specificity, self-dosing, and antibiofilm activity, serve as a better approach to fighting against drug-resistant bacteria. In the current study, a broad-host range lytic phage phiSalP219 was isolated against multidrug-resistant Salmonella enterica serotypes Paratyphi from a pond water sample. Salmonella phage phiSalP219 was able to lyse 28/30 tested strains of Salmonella enterica. Salmonella phage phiSalP219 exhibits activity in acidic environments (pH3) and high temperatures (70°C). Electron microscopy and genome analysis revealed that phage phiSalP219 is a member of class Caudoviricetes. The genome of Salmonella phage phiSalP219 is 146Kb in size with 44.5% GC content. A total of 250 Coding Sequence (CDS) and 25 tRNAs were predicted in its genome. Predicted open reading frames (ORFs) were divided into five groups based on their annotation results: (1) nucleotide metabolism, (2) DNA replication and transcription, (3) structural proteins, (4) lysis protein, and (5) other proteins. The absence of lysogeny-related genes in their genome indicates that Salmonella phage phiSalP219 is lytic in nature. Phage phiSalP219 was also found to be microbiologically safe (due to the absence of toxin or virulence-related genes) in the control of Salmonella enterica serovar Typhimurium infections in the ready-to-eat meat and also able to eradicate biofilm formed by the same bacterium on the borosilicate glass surface.

Electrophysiological Responses of Trissolcus japonicus, T. basalis, and T. oenone (Hymenoptera: Scelionidae) to Volatile Compounds Associated with New Zealand Stink Bugs (Hemiptera: Pentatomidae).

Parasitoid biological control agents rely heavily on olfaction to locate their hosts. Chemical cues associated with hosts and non-hosts are known to influence the expression of host preferences and host-specificity. A better understanding of how and why parasitoids attack some species and not others, based on volatile organic compounds associated with potential hosts, can provide key information on the parasitoid's host preferences, which could be applied to pre-release risk assessments for classical biological control agents. Electrophysiological techniques such as electroantennography (EAG) and GC-EAD (gas chromatography coupled with electroantennographic detection) are widely used to identify bioactive semiochemicals. But the application of these techniques to understanding how chemical ecological cues mediate parasitoid host specificity has not been as thoroughly explored. We conducted GC-EAD and EAG studies to identify olfactory-active compounds associated with adult females of nine stink bug species from Aotearoa/New Zealand on the antennae of three closely related parasitoid species: Trissolcus japonicus Ashmead, a pre-emptively (= proactively) approved biocontrol agent against brown marmorated stink bug; T. basalis (Wollaston), a biocontrol agent introduced against Nezara viridula L. in 1949; and T. oenone Johnson, a native Australasian pentatomid parasitoid. Eight compounds associated with stink bugs elicited antennal responses from all three parasitoids, and we were able to identify seven of these. (E)-2-hexenal, (E)-4-oxo-2-hexenal, (E)-2-octenal and (E)-2-decenal generally elicited stronger responses in the three parasitoids, while n-tridecane, n-dodecane, and (E)-2-decenyl acetate elicited weaker responses. We discuss how and why the results from electrophysiological experiments can be applied to non-target risk assessments within biological control programmes.

Targeted phage hunting to specific Klebsiella pneumoniae clinical isolates is an efficient antibiotic resistance and infection control strategy.

Klebsiella pneumoniae is one of the most threatening multi-drug-resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to the phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerases. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular-type strains and determining the ability to lyse relevant clinical isolates. A combination of 12 phages capable of infecting 55% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Thirty-one percent of isolates were susceptible to the phage cocktail. However, our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.IMPORTANCEThe emergence of resistant bacteria is a serious global health problem. In the absence of effective treatments, phages are a personalized and effective therapeutic alternative. However, little is still known about phage-host interactions, which are key to implementing effective strategies. Here, we focus on the study of Klebsiella pneumoniae, a highly pathogenic encapsulated bacterium. The complexity and variability of the capsule, where in most cases phage receptors are found, make it difficult for phage-based treatments. Here, we isolated a large collection of Klebsiella phages against all the reference strains and in a cohort of clinical isolates. Our results suggest that clinical isolates represent a challenge, especially high-risk clones. Thus, we propose targeted phage hunting as an effective strategy to implement phage-derived therapies. Our results are a step forward for new phage-based strategies to control K. pneumoniae infections, highlighting the importance of understanding phage-host interactions to design personalized treatments against Klebsiella spp.

Can genomic signatures guide the selection of host-specific agents for weed biological control?

Biological control of weeds involves deliberate introduction of host-specific natural enemies into invaded range to reduce the negative impacts of invasive species. Assessing the specificity is a crucial step, as introduction of generalist natural enemies into a new territory may pose risks to the recipient communities. A mechanistic understanding of host use can provide valuable insights for the selection of specialist natural enemies, bolster confidence in non-target risk assessment and potentially accelerate the host specificity testing process in biological control. We conducted a comprehensive analysis of studies on the genomics of host specialization with a view to examine if genomic signatures can help predict host specificity in insects. Focusing on phytophagous Lepidoptera, Coleoptera and Diptera, we compared chemosensory receptors and enzymes between "specialist" (insects with narrow host range) and "generalist" (insects with wide host range) insects. The availability of genomic data for biological control agents (natural enemies of weeds) is limited thus our analyses utilized data from pest insects and model organisms for which genomic data are available. Our findings revealed that specialists generally exhibit a lower number of chemosensory receptors and enzymes compared with their generalist counterparts. This pattern was more prominent in Coleoptera and Diptera relative to Lepidoptera. This information can be used to reject agents with large gene repertoires to potentially accelerate the risk assessment process. Similarly, confirming smaller gene repertoires in specialists could further strengthen the risk evaluation. Despite the distinctive signatures between specialists and generalists, challenges such as finite genomic data for biological control agents, ad hoc comparisons, and fewer comparative studies among congeners limit our ability to use genomic signatures to predict host specificity. A few studies have empirically compared phylogenetically closely related species, enhancing the resolution and the predictive power of genomics signatures thus suggesting the need for more targeted studies comparing congeneric specialists and generalists.

Diversity and biology of Spirometra tapeworms (Cestoda: Diphyllobothriidea), zoonotic parasites of wildlife: A review.

Tapeworms of the genus Spirometra Faust, Campbell et Kellogg, 1929 have long been known as intestinal parasites of carnivores and their larvae (spargana) have been found in various vertebrates. Nevertheless, their species diversity, host associations and geographical distribution remain poorly understood. Molecular data clearly confirm the validity of the genus, which has been synonymised by several authors with Diphyllobothrium Cobbold, 1858. Despite morphological similarities between the species of the two genera, they are not closely related and also differ in their life cycle. The present review provides a list of the species recognised as valid and additional genotypes that may represent other species, with a basic characterisation of each taxon and comments on their validity, the probable range of definitive and intermediate hosts, and their distribution. The existing taxonomic problems and the insufficient knowledge of the host specificity and distribution of Spirometra tapeworms can only be solved by combining molecular and morphological data, i.e. by comparing genetically characterised specimens with corresponding morphological vouchers (hologenophores). Further targeted sampling and surveys are required to clarify the distribution and host associations.

Construction and Characterization of a High-Capacity Replication-Competent Murine Cytomegalovirus Vector for Gene Delivery.

We investigated the basic characteristics of a new murine cytomegalovirus (MCMV) vector platform. Using BAC technology, we engineered replication-competent recombinant MCMVs with deletions of up to 26% of the wild-type genome. To this end, we targeted five gene blocks (m01-m17, m106-m109, m129-m141, m144-m158, and m159-m170). BACs featuring deletions from 18% to 26% of the wild-type genome exhibited delayed virus reconstitution, while smaller deletions (up to 16%) demonstrated reconstitution kinetics similar to those of the wild type. Utilizing an innovative methodology, we introduced large genomic DNA segments, up to 35 kbp, along with reporter genes into a newly designed vector with a potential cloning capacity of 46 kbp (Q4). Surprisingly, the insertion of diverse foreign DNAs alleviated the delayed plaque formation phenotype of Q4, and these large inserts remained stable through serial in vitro passages. With reporter-gene-expressing recombinant MCMVs, we successfully transduced not only mouse cell lines but also non-rodent mammalian cells, including those of human, monkey, bovine, and bat origin. Remarkably, even non-mammalian cell lines derived from chickens exhibited successful transduction.

Cordyceps cateniannulata: An endophyte of coffee, a parasite of coffee leaf rust and a pathogen of coffee pests.

Here, we report on a Cordyceps species entering into a multi-trophic, multi-kingdom association. Cordyceps cateniannulata, isolated from the stem of wild Coffea arabica in Ethiopia, is shown to function as an endophyte, a mycoparasite and an entomopathogen. A detailed polyphasic taxonomic study, including a multilocus phylogenetic analysis, confirmed its identity. An emended description of C. cateniannulata is provided herein. Previously, this species was known as a pathogen of various insect hosts in both the Old and New World. The endophytic status of C. cateniannulata was confirmed by re-isolating it from inoculated coffee plants. Inoculation studies have further shown that C. cateniannulata is a mycoparasite of Hemileia vastatrix, as well as an entomopathogen of major coffee pests; infecting and killing Hypothenemus hampei and Leucoptera coffeella. This is the first record of C. cateniannulata from Africa, as well as an endophyte and a mycoparasite. The implications for its use as a biocontrol agent are discussed.

IbpAB small heat shock proteins are not host factors for bacteriophage ϕX174 replication.

Bacteriophage ϕX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ϕX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ϕX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ϕX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ϕX174 replication; moreover, their absence has no discernible effect on ϕX174 fecundity. These results suggest IbpA/B upregulation is a response to ϕX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.

H19 influenza A virus exhibits species-specific MHC class II receptor usage.

Avian influenza A virus (IAV) surveillance in Northern California, USA, revealed unique IAV hemagglutinin (HA) genome sequences in cloacal swabs from lesser scaups. We found two closely related HA sequences in the same duck species in 2010 and 2013. Phylogenetic analyses suggest that both sequences belong to the recently discovered H19 subtype, which thus far has remained uncharacterized. We demonstrate that H19 does not bind the canonical IAV receptor sialic acid (Sia). Instead, H19 binds to the major histocompatibility complex class II (MHC class II), which facilitates viral entry. Unlike the broad MHC class II specificity of H17 and H18 from bat IAV, H19 exhibits a species-specific MHC class II usage that suggests a limited host range and zoonotic potential. Using cell lines overexpressing MHC class II, we rescued recombinant H19 IAV. We solved the H19 crystal structure and identified residues within the putative Sia receptor binding site (RBS) that impede Sia-dependent entry.

Effect of Domain Manipulation in the Staphylococcal Phage Endolysin, Endo88, on Lytic Efficiency and Host Range.

The bacteriophage endolysin Endo88 targeting Staphylococcus aureus PS88 consists of the CHAP and Amidase-2 enzymatic domains and one SH3b targeting domain. In this study, the effects of domain manipulations on Endo88 functionality were determined. Three truncated mutants of Endo88 (CHAP, CHAPAmidase and CHAPSH3) and two chimeras (CHAPAmidase-Cpl7Cpl7 and Endo88-Cpl7Cpl7) containing the Cpl7Cpl7 targeting domains of the streptococcal LambdaSa2-ECC endolysin were cloned in E. coli (pET28a), expressed, and then purified. Lytic efficiency and host range were assessed through plate lysis assays and turbidity reduction assays. Endo88 required all domains for maximum functionality, with activity detected against Staphylococcus aureus PS88 (host strain), S. aureus Mu50 (VISA), CoNS (Staphylococcus epidermidis and Staphylococcus hominis), and Enterococcus faecalis. The truncated constructs maintained the original host range but with reduced lytic efficiency. The Amidase-2 and SH3b domains are interdependent in maximizing functionality. The chimera constructs demonstrated reduced functionality, without activity against Streptococcus agalactiae in both assays. This study provides insights into domain function in a staphylococcal endolysin, which could enable the development of prospective engineered antimicrobials against multidrug-resistant pathogens.

Identification of common human infectious and potentially zoonotic novel genotypes of Enterocytozoon bieneusi in cavernicolous bats in Thailand.

Enterocytozoon bieneusi is a common cause of human microsporidiosis and can infect a variety of animal hosts worldwide. In Thailand, previous studies have shown that this parasite is common in domestic animals. However, information on the prevalence and genotypes of this parasite in other synanthropic wildlife, including bats, remains limited. Several pathogens have been previously detected in bats, suggesting that bats may serve as a reservoir for this parasite. In this study, a total of 105 bat guano samples were collected from six different sites throughout Thailand. Of these, 16 from Chonburi (eastern), Ratchaburi (western), and Chiang Rai (northern) provinces tested positive for E. bieneusi, representing an overall prevalence of 15.2%. Based on ITS1 sequence analysis, 12 genotypes were identified, including two known genotypes (D and type IV) frequently detected in humans and ten novel potentially zoonotic genotypes (TBAT01-TBAT10), all belonging to zoonotic group 1. Lyle's flying fox (Pteropus lylei), commonly found in Southeast Asia, was identified as the host in one sample that was also positive for E. bieneusi. Network analysis of E. bieneusi sequences detected in this study and those previously reported in Thailand also revealed intraspecific divergence and recent population expansion, possibly due to adaptive evolution associated with host range expansion. Our data revealed, for the first time, multiple E. bieneusi genotypes of zoonotic significance circulating in Thai bats and demonstrated that bat guano fertilizer may be a vehicle for disease transmission.

Experimental Evolution Studies in Φ6 Cystovirus.

Experimental evolution studies, in which biological populations are evolved in a specific environment over time, can address questions about the nature of spontaneous mutations, responses to selection, and the origins and maintenance of novel traits. Here, we review more than 30 years of experimental evolution studies using the bacteriophage (phage) Φ6 cystovirus. Similar to many lab-studied bacteriophages, Φ6 has a high mutation rate, large population size, fast generation time, and can be genetically engineered or cryogenically frozen, which facilitates its rapid evolution in the laboratory and the subsequent characterization of the effects of its mutations. Moreover, its segmented RNA genome, outer membrane, and capacity for multiple phages to coinfect a single host cell make Φ6 a good non-pathogenic model for investigating the evolution of RNA viruses that infect humans. We describe experiments that used Φ6 to address the fitness effects of spontaneous mutations, the consequences of evolution in the presence of coinfection, the evolution of host ranges, and mechanisms and consequences of the evolution of thermostability. We highlight open areas of inquiry where further experimentation on Φ6 could inform predictions for pathogenic viruses.

Does Phage Therapy Need a Pan-Phage?

The emergence of multidrug-resistant bacteria is undoubtedly one of the most serious global health threats. One response to this threat that has been gaining momentum over the past decade is 'phage therapy'. According to this, lytic bacteriophages are used for the treatment of bacterial infections, either alone or in combination with antimicrobial agents. However, to ensure the efficacy and broad applicability of phage therapy, several challenges must be overcome. These challenges encompass the development of methods and strategies for the host range manipulation and bypass of the resistance mechanisms developed by pathogenic bacteria, as has been the case since the advent of antibiotics. As our knowledge and understanding of the interactions between phages and their hosts evolves, the key issue is to define the host range for each application. In this article, we discuss the factors that affect host range and how this determines the classification of phages into different categories of action. For each host range group, recent representative examples are provided, together with suggestions on how the different groups can be used to combat certain types of bacterial infections. The available methodologies for host range expansion, either through sequential adaptation to a new pathogen or through genetic engineering techniques, are also reviewed.

Data-Driven Engineering of Phages with Tunable Capsule Tropism for Klebsiella pneumoniae.

Klebsiella pneumoniae, a major clinical pathogen known for causing severe infections, is attracting heightened attention due to its escalating antibiotic resistance. Phages are emerging as a promising alternative to antibiotics; however, their specificity to particular hosts often restricts their use. In this study, a collection of 114 phages is obtained and subjected to analysis against 238 clinical K. pneumoniae strains, revealing a spectrum of lytic behaviors. A correlation between putative tail protein clusters and lysis patterns leads to the discovery of six receptor-binding protein (RBP) clusters that determine host capsule tropism. Significantly, RBPs with cross-capsular lysis capabilities are identified. The newly-identified RBPs provide a toolbox for customizing phages to target diverse capsular types. Building on the toolbox, the engineered phages with altered RBPs successfully shifted and broadened their host capsule tropism, setting the stage for tunable phage that offer a precise and flexible solution to combat K. pneumoniae infections.

Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria.

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.