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AbstractFew measures explicitly assess general youth sexual knowledge in the United States, The Acquired Sexual Knowledge Questionnaire (ASKQ) was designed to be a practical and useful measure of sexual health knowledge for youth (Grades 7-12) participating in sexuality education courses in schools. This article outlines the construction, development, and evaluation of the ASKQ on a sample of 244 middle and high school students who participated in a United Way approved eight-week sexual health education program. The questionnaire was found to have good internal consistency (α = .80) and will be a useful tool in measuring sexual health knowledge. [ABSTRACT FROM AUTHOR]

This study examined the association between a dental implant and changes in adjacent teeth over time. Electronic health records of 1818 patients who received a dental implant were retrospectively evaluated over 14 years (2005–2019) in a university setting. The status of the adjacent tooth and vertical and horizontal distance from the implant platform to adjacent teeth were determined using digital intraoral radiographs taken at baseline and the last follow-up visit (1–14 years, median 4 years). In total, 1085 dental implants were evaluated. There were 234 instances of a change in the adjacent tooth. Decay was observed in 83 (7.6%) of adjacent teeth; the mean time to development was 4 years (range 1–14 years). Approximately 9% of adjacent teeth received direct restorations, 4.8% received indirect restorations, 1% received endodontic root canal treatment, and 5.6% were extracted. The mean horizontal distance between the implant platform and the adjacent teeth was 3.56 mm; the mean vertical distance from the contact point to the alveolar crest on the tooth side was 6.2 mm at the first time of the reported decay on X ray. These distances did not significantly influence the occurrence of caries. The prevalence of interproximal contact loss was higher on the mesial of the implant crown at 63% compared with 20% on the distal side. This large retrospective analysis identified that teeth adjacent to a dental implant were at risk of decay and changes in their condition. In addition, the implant-to-tooth distance and inadequate emergence profile may contribute as caries risk factors in addition to hygiene and a high sugar level diet. These findings appear essential for clinicians when making treatment decisions and discussing outcomes with patients. [ABSTRACT FROM AUTHOR]

There is a complex interplay between viral infection and host innate immune response regarding disease severity and outcomes. Neutrophil hyperactivation, including excessive release of neutrophil extracellular traps (NETs), is linked to exacerbated disease in acute COVID-19, notably in hospitalized patients. Delineating protective versus detrimental neutrophil responses is essential to developing targeted COVID-19 therapies and relies on high-quality translational animal models. In this study, we utilize a previously established feline model for COVID-19 to investigate neutrophil dysfunction in which experimentally infected cats develop clinical disease that mimics acute COVID-19. Specific pathogen-free cats were inoculated with SARS-CoV-2 (B.1.617.2; Delta variant) (n = 24) or vehicle (n = 6). Plasma, bronchoalveolar lavage fluid, and lung tissues were collected at various time points over 12 days post-inoculation. Systematic and temporal evaluation of the kinetics of neutrophil activation was conducted by measuring markers of activation including myeloperoxidase (MPO), neutrophil elastase (NE), and citrullinated histone H3 (citH3) in SARS-CoV-2-infected cats at 4 and 12 days post-inoculation (dpi) and compared to vehicle-inoculated controls. Cytokine profiling supported elevated innate inflammatory responses with specific upregulation of neutrophil activation and NET formation-related markers, namely IL-8, IL-18, CXCL1, and SDF-1, in infected cats. An increase in MPO-DNA complexes and cell-free dsDNA in infected cats compared to vehicle-inoculated was noted and supported by histopathologic severity in respiratory tissues. Immunofluorescence analyses further supported correlation of NET markers with tissue damage, especially 4 dpi. Differential gene expression analyses indicated an upregulation of genes associated with innate immune and neutrophil activation pathways. Transcripts involved in activation and NETosis pathways were upregulated by 4 dpi and downregulated by 12 dpi, suggesting peak activation of neutrophils and NET-associated markers in the early acute stages of infection. Correlation analyses conducted between NET-specific markers and clinical scores as well as histopathologic scores support association between neutrophil activation and disease severity during SARS-CoV-2 infection in this model. Overall, this study emphasizes the effect of neutrophil activation and NET release in SARS-CoV-2 infection in a feline model, prompting further investigation into therapeutic strategies aimed at mitigating excessive innate inflammatory responses in COVID-19. [ABSTRACT FROM AUTHOR]

Objectives: The underlying mechanisms of burning mouth syndrome (BMS) remain unclear leading to challenges and unsatisfactory management. Current treatments focus primarily on symptom relief, with few consistently achieving a 50% reduction in pain. This review aims to explore animal models of BMS to gain a better understanding of the underlying mechanisms and to discuss potential and existing knowledge gaps. Methods: A comprehensive review of PubMed®, Google Scholar, and Scopus was performed to assess advances and significant gaps of existing rodent models that mimic BMS‐related symptoms. Results: Rodent models of BMS involve reproduction of dry‐tongue, chorda tympani transection, or overexpression of artemin protein. Existing preclinical models tend to highlight one specific etiopathogenesis and often overlook sex‐ and hormone‐specific factors. Conclusion: Combining aspects from various BMS models could prove beneficial in developing comprehensive experimental designs and outcomes encompassing the multifaceted nature of BMS. [ABSTRACT FROM AUTHOR]

Cytauxzoonosis, a highly fatal tick-borne disease in domestic cats caused by Cytauxzoon felis, poses diagnostic and therapeutic challenges due to the inability to culture the parasite in vitro. This study aimed to artificially replicate C. felis infection and characterize in vitro replication kinetics. Concanavalin A-activated feline embryonal macrophages (Fcwf-4) were plated at 3–5 × 105 cells/mL and incubated with C. felis-positive blood samples from either a (1) chronically infected bobcat (Lynx rufus), (2) chronically infected domestic cat, or (3) acutely infected domestic cat with clinical signs of cytauxzoonosis. Temporal changes in parasite load were quantified by droplet digital PCR (ddPCR), and the inhibition of infection/replication was assessed using atovaquone, imidocarb dipropionate (ID), artemisinin, ponazuril, and neutralizing antibodies. Tick cell lines AAE2 and ISE6 were also tested for infection. In vitro inoculation with chronic infection led to transient replication, while acute infection resulted in sustained replication beyond 10 days post-inoculation. Atovaquone, ID, and artemisinin inhibited replication, and neutralizing antibodies prevented infection. The inoculation of tick cells in vitro indicated infection; however, parasite replication was not observed. The results of this study established an in vitro model for studying infection dynamics, assessing therapy efficacy, and testing vaccination strategies in cytauxzoonosis-infected cats. [ABSTRACT FROM AUTHOR]

People living with human immunodeficiency virus (PLWH) are a significant population globally. Research delineating our understanding of coinfections in PLWH is critical to care for those navigating infection with other pathogens. The recent COVID-19 pandemic underscored the urgent need for studying the effects of SARS-CoV-2 infections in therapy-controlled and uncontrolled immunodeficiency viral infections. This study established the utility of a feline model for the in vivo study of coinfections. Domestic cats are naturally infected with SARS-CoV-2 and Feline Immunodeficiency Virus, a lentivirus molecularly and pathogenically similar to HIV. In this study, comparisons are made between FIV-positive and FIV-negative cats inoculated with SARS-CoV-2 (B.1.617.2.) in an experimental setting. Of the FIV+ cats, three received Zidovudine (AZT) therapy in the weeks leading up to SARS-CoV-2 inoculation, and two did not. SARS-CoV-2 viral RNA was quantified, histopathologic comparisons of respiratory tissues were made, and T-cell populations were analyzed for immune phenotype shifts between groups. CD4+ T lymphocyte responses varied, with FIV+-untreated cats having the poorest CD4+ response to SARS-CoV-2 infection. While all cats had significant pulmonary inflammation, key histopathologic features of the disease differed between groups. Additionally, viral genomic analysis was performed, and results were analyzed for the presence of emerging, absent, amplified, or reduced mutations in SARS-CoV-2 viral RNA after passage through the feline model. Positive selection is noted, especially in FIV+ cats untreated with AZT, and mutations with potential relevance were identified; one FIV+-untreated cat had persistent, increasing SARS-CoV-2 RNA in plasma five days post-infection. These findings and others support the utility of the feline model for studying coinfection in people with HIV and highlight the importance of antiretroviral therapy in clearing SARS-CoV-2 coinfections to minimize transmission and emergence of mutations that may have deleterious effects. [ABSTRACT FROM AUTHOR]

For a semigroup S$S$ whose universal right congruence is finitely generated (or, equivalently, a semigroup satisfying the homological finiteness property of being type right‐FP1$FP_1$), the right diameter of S$S$ is a parameter that expresses how 'far apart' elements of S$S$ can be from each other, in a certain sense. To be more precise, for each finite generating set U$U$ for the universal right congruence on S$S$, we have a metric space (S,dU)$(S,d_U)$ where dU(a,b)$d_U(a,b)$ is the minimum length of derivations for (a,b)$(a,b)$ as a consequence of pairs in U$U$; the right diameter of S$S$ with respect to U$U$ is the diameter of this metric space. The right diameter of S$S$ is then the minimum of the set of all right diameters with respect to finite generating sets. We develop a theoretical framework for establishing whether a semigroup of transformations or partitions on an arbitrary infinite set X$X$ has a finitely generated universal right/left congruence, and, if it does, determining its right/left diameter. We apply this to prove results such as the following. Each of the monoids of all binary relations on X$X$, of all partial transformations on X$X$, and of all full transformations on X$X$, as well as the partition and partial Brauer monoids on X$X$, have right diameter 1 and left diameter 1. The symmetric inverse monoid on X$X$ has right diameter 2 and left diameter 2. The monoid of all injective mappings on X$X$ has right diameter 4, and its minimal ideal (called the Baer–Levi semigroup on X$X$) has right diameter 3, but neither of these two semigroups has a finitely generated universal left congruence. On the other hand, the semigroup of all surjective mappings on X$X$ has left diameter 4, and its minimal ideal has left diameter 2, but neither of these semigroups has a finitely generated universal right congruence. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been especially devastating to patients with comorbidities, including metabolic and cardiovascular diseases. Elevated blood glucose during SARS-CoV-2 infection increased mortality of patients with COVID-19, although the mechanisms are not well understood. It has been previously demonstrated that glucose transport and utilization is a crucial pathway for other highly infectious RNA viruses. Thus, we hypothesized that SARS-CoV-2 infection could lead to alterations in cellular and whole body glucose metabolism. Specific pathogen-free domestic cats were intratracheally inoculated with USA-WA1/2020 (wild-type) SARS-CoV-2 or vehicle-inoculated, then euthanized at 4- and 8-days postinoculation (dpi). Blood glucose and cortisol concentrations were elevated at 4 and 8 dpi. Blood ketones, insulin, and angiotensin II concentrations remained unchanged throughout the experimental timeline. SARS-CoV-2 RNA was detected in the lung and heart, without changes in angiotensin-converting enzyme 2 (ACE2) RNA expression. In the lung, SARS-CoV-2 infection increased glucose transporter 1 (GLUT1) protein levels at 4 and 8 dpi, whereas GLUT4 level was only upregulated at 8 dpi. In the heart, GLUT-1 and -4 protein levels remained unchanged. Furthermore, GLUT1 level was upregulated in the skeletal muscle at 8 dpi, and AMPK was activated in the hearts of infected cats. SARS-CoV-2 infection increased blood glucose concentration and pulmonary GLUT protein levels. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming primarily in the lung to support viral replication. Furthermore, this translational feline model mimicked human COVID-19 and could be used to explore novel therapeutic targets to treat metabolic disease during SARS-CoV-2 infection. NEW & NOTEWORTHY Our study on a feline model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, mirroring human COVID-19, revealed alterations in whole body and cellular glucose metabolism. Infected cats developed mild hyperglycemia, increased protein levels of glucose transporters in the lung, and AMPK activation in the heart. These findings suggest that SARS-CoV-2 infection induces metabolic reprogramming in the cardiorespiratory system to support viral replication. Understanding these mechanisms could lead to novel antiviral therapeutic strategies. [ABSTRACT FROM AUTHOR]

In the era of personalized/precision health care, additional effort is being expended to understand the biology and molecular mechanisms of disease processes. How these mechanisms are affected by individual genetics, environmental exposures, and behavioral choices will encompass an expanding role in the future of optimally preventing and treating diseases. Considering saliva as an important biological fluid for analysis to inform oral disease detection/description continues to expand. This review provides an overview of saliva as a diagnostic fluid and the features of various biomarkers that have been reported. We emphasize the use of salivary biomarkers in periodontitis and transport the reader through extant literature, gaps in knowledge, and a structured approach toward validating and determine the utility of biomarkers in periodontitis. A summation of the findings support the likelihood that a panel of biomarkers including both host molecules and specific microorganisms will be required to most effectively identify risk for early transition to disease, ongoing disease activity, progression, and likelihood of response to standard periodontal therapy. The goals would be to develop predictive algorithms that serve as adjunctive diagnostic tools which provide the clinician and patient important information for making informed clinical decisions. [ABSTRACT FROM AUTHOR]

Case series summary: This case series describes six cases involving seven cats naturally infected with Cytauxzoon felis in Indiana, USA. Medical records were retrospectively reviewed and all available information on signalment, history, clinical and diagnostic findings, treatment, outcome and pathology was reported. Cats infected with C felis were domestic shorthairs, were aged between 2 and 9 years and all but one of the cats were male. The seven infected cats originated from five counties in southwestern Indiana. Six of seven cats were found to have acute cytauxzoonosis based on clinical signs, gross pathologic lesions, observation of C felis in tissues and/or detection of C felis DNA. One cat was identified as a subclinical survivor cat with no known clinical history of cytauxzoonosis. Relevance and novel information: The reported cases are the first confirmed reports of acute and chronic cytauxzoonosis in cats from Indiana and document an expansion in the range of C felis. Veterinary practitioners in Indiana should consider infection with C felis as a differential diagnosis for cats that present with fever, inappetence, lethargy, depression, dehydration, dyspnea, hemolytic crisis, anorexia or icterus. Administration of approved acaricides to cats currently offers the best protection and control against C felis infection. [ABSTRACT FROM AUTHOR]

Because Null Hypothesis Significance Tests (NHST) can be misleading particularly when sample sizes are large, reporting effect sizes is recommended. Effect sizes such as Cohen's d, however, are abstract statistics and not relevant to decision-making. This article introduced an alternative formulation of Cohen's d based on a "minimal important percent difference" (MIPD) between groups that has an intuitive, practical meaning. Cohen's d was calculated based on detecting MIPDs of 5% and 10% differences in means. Power analyses were used to determine the number of responses needed to detect differences in means. Power-probability sample size requirements were examined for an 80% and a 50% chance of accepting a difference of MIPD at p <.05. We proposed a four-step process to show how both effect size and NHST jointly contribute to decision-making. To demonstrate the process, data were obtained for 6 years of annual waterfowl surveys in Illinois (N = 17,265, average response rate = 41%). [ABSTRACT FROM AUTHOR]

Silicic caldera volcanoes present major volcanic and seismic hazards but also host dynamic hydrothermal and groundwater systems and a rich but largely unexplored subsurface biosphere. Many of these volcanoes are hosted in rift settings. The intricate connections and feedbacks among magmatism, rifting, hydrothermal processes, and the biosphere in these complex systems remain poorly understood, necessitating subsurface joint observations that are only enabled by scientific drilling. The CALDERA (Connections Among Life, geo-Dynamics and Eruptions in a Rifting Arc caldera) project workshop funded by the International Continental Scientific Drilling Program (ICDP) gathered multi-disciplinary international experts in January 2023 to advance planning of a scientific drilling project within one of these dynamic, rift-hosted calderas, the Okataina Volcanic Centre (OVC), Aotearoa New Zealand. The OVC's high eruption rate, frequent unrest events and earthquake swarms, location in a densely faulted rapidly extending rift, abundant groundwater–geothermal fluid circulations, and diverse surface hot spring microbiota make it an ideal location for exploring a connected geo-hydro-biosphere via scientific drilling and developing a test bed for novel volcano monitoring approaches. Drilling configurations with at least two boreholes (∼ 200 and ∼ 1000–1500 m deep) were favoured to achieve the multi-disciplinary objectives of the CALDERA project. Decadal monitoring including biosphere activity and composition has the potential to evaluate the response of the hydro-bio system to volcano-tectonic activity. In addition to the OVC caldera-scale datasets already available, site surveys will be conducted to select the best drilling locations. The CALDERA project at the OVC would provide, for the first time, an understanding of volcanic–tectonic–hydrological–biological connections in a caldera–rift system and a baseline for global comparisons with other volcanoes, rifts, and hydrothermal systems. CALDERA would serve as an unprecedented model system to understand how and how quickly the subsurface biosphere responds to geologic activities. Discoveries will improve assessment of volcanic and seismic hazards, guide the sustainable management and/or conservation of groundwater and geothermal resources and microbial ecosystems, and provide a forum for interweaving mātauranga Māori and Western knowledge systems. [ABSTRACT FROM AUTHOR]

Iatrogenic vascular air embolism is a relatively infrequent event but is associated with significant morbidity and mortality. These emboli can arise in many clinical settings such as neurosurgery, cardiac surgery, and liver transplantation, but more recently, endoscopy, hemodialysis, thoracentesis, tissue biopsy, angiography, and central and peripheral venous access and removal have overtaken surgery and trauma as significant causes of vascular air embolism. The true incidence may be greater since many of these air emboli are asymptomatic and frequently go undiagnosed or unreported. Due to the rarity of vascular air embolism and because of the many manifestations, diagnoses can be difficult and require immediate therapeutic intervention. An iatrogenic air embolism can result in both venous and arterial emboli whose anatomic locations dictate the clinical course. Most clinically significant iatrogenic air emboli are caused by arterial obstruction of small vessels because the pulmonary gas exchange filters the more frequent, smaller volume bubbles that gain access to the venous circulation. However, there is a subset of patients with venous air emboli caused by larger volumes of air who present withmore protean manifestations. There have been significant gains in the understanding of the interactions of fluid dynamics, hemostasis, and inflammation caused by air emboli due to in vitro and in vivo studies on flow dynamics of bubbles in small vessels. Intensive research regarding the thromboinflammatory changes at the level of the endothelium has been described recently. The obstruction of vessels by air emboli causes immediate pathoanatomic and immunologic and thromboinflammatory responses at the level of the endothelium. In this review, we describe those immunologic and thromboinflammatory responses at the level of the endothelium as well as evaluate traditional and novel forms of therapy for this rare and often unrecognized clinical condition. [ABSTRACT FROM AUTHOR]

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage–host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage. IMPORTANCE Directed evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies. [ABSTRACT FROM AUTHOR]

Objective Background Methods Results Conclusion To examine the characteristics of the salivary microbiome in Type 2 diabetes mellitus (T2DM) patients with or without periodontitis.Periodontitis has been identified as clear sequelae of T2DM. This chronic oral disease also impacts the management of the clinical features of diabetes. The oral microbiome characteristics in T2DM with and without periodontitis, as well as the response of this oral microbiome to nonsurgical therapy have not been well described. Knowledge of key oral biological features could help address the observed poorer clinical presentation of T2DM patients.The oral microbiome in saliva of adult cohorts periodontally healthy/non‐diabetic (non‐periodontitis; NP; n = 31), T2DM without periodontitis (DWoP; n = 32), and T2DM with periodontitis (DWP; n = 29) were characterized by microbial molecular analysis using V3–V4 sequencing and Luminex or ELISA techniques for salivary host analytes.Phyla distribution showed DWP with significantly lower levels of Firmicutes and Actinobacteria and higher levels of Fusobacteria and Spirochetes compared to the healthier groups. Approximately 10% of the detected microbial species showed significant differences in frequency and level of colonization among the DWP, DWoP, and NP samples. A subset of bacteria were significantly correlated with clinical disease features, as well as a specific repertoire of salivary analytes, in particular matrix metalloproteinase (MMP)8/MMP9, interleukin‐1ß, B‐cell activating factor, and resistin differed between the groups and were related to specific taxa. Principal component analysis that identified a majority of the DWP subjects microbiome was unique based upon an array of 27 taxa out of up to 255 detected in the saliva samples.T2DM patients with periodontitis show unique oral microbiome and salivary analyte composition compared to diabetics or non‐diabetic persons without periodontitis. Specific members of the oral microbiome relate directly to the clinical disease features and/or salivary biomolecules in T2DM individuals. [ABSTRACT FROM AUTHOR]

Bacteriophages (phages) that are intended to be used to treat bacterial infections are often improved using genetic engineering or experimental evolution. A protocol called "Appelmans" utilizes evolution in microtiter plates to promote the evolution of phages that can infect nonpermissive hosts. We tested a modification of the Appelmans protocol using the honey bee pathogen, Paenibacillus larvae . Three phages evolved together on four P. larvae strains following the standard Appelmans protocol and a modified version to ensure high phage diversity throughout ten rounds of passaging. The host range of 360 plaques were characterized and six new phage lysis patterns were identified. These new phage lysis patterns included plaque formation on previously nonpermissive, phage-resistant isolates that were used to identify phage types. The modified protocol did not drastically change the rate or number of new phage types observed but did prevent the phage population from being dominated by one phage that tended to rapidly raise in frequency. These findings showed how a minor modification of the Appelmans protocol influenced the development of phages for phage therapy. The method also provided improved phages for the treatment of bacterial infections in honey bees.

Feline immunodeficiency virus (FIV) is the domestic cat analogue of HIV infection in humans. Both viruses induce oral disease in untreated individuals, with clinical signs that include gingivitis and periodontal lesions. Oral disease manifestations in HIV patients are abated by highly effective combination antiretroviral therapy (cART), though certain oral manifestations persist despite therapy. Microorganisms associated with oral cavity opportunistic infections in patients with HIV cause similar pathologies in cats. To further develop this model, we evaluated characteristics of feline oral health and oral microbiome during experimental FIV infection over an 8-month period following cART. Using 16S metagenomics sequencing, we evaluated gingival bacterial communities at four timepoints in uninfected and FIV-infected cats treated with cART or placebo. Comprehensive oral examinations were also conducted by a veterinary dental specialist over the experimental period. Gingival inflammation was higher in FIV-infected cats treated with placebo compared to cART-treated cats and controls at study endpoint. Oral microbiome alpha diversity increased in all groups, while beta diversity differed among treatment groups, documenting a significant effect of cART therapy on microbiome community composition. This finding has not previously been reported and indicates cART ameliorates immunodeficiency virus-associated oral disease via preservation of oral mucosal microbiota. Further, this study illustrates the value of the FIV animal model for investigations of mechanistic associations and therapeutic interventions for HIV oral manifestations., Competing Interests: Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.

Honey bee ( Apis mellifera ) larvae are susceptible to the bacterial pathogen Paenibacillus larvae , which causes severe damage to bee colonies. Antibiotic treatment requires veterinary supervision in the United States, is not used in many parts of the world, perpetuates problems associated with antibiotic resistance, and can necessitate residual testing in bee products. There is interest in using bacteriophages to treat infected colonies (bacteriophage therapy) and several trials are promising. Nevertheless, the safety of using biological agents in the environment must be scrutinized. In this study we analyzed the ability of P. larvae to evolve resistance to several different bacteriophages. We found that bacteriophage resistance is rapidly developed in culture but often results in growth defects. Mutations in the bacteriophage-resistant isolates are concentrated in genes encoding potential surface receptors. Testing one of these isolates in bee larvae, we found it to have reduced virulence compared to the parental P. larvae strain. We also found that bacteriophages are likely able to counteract resistance evolution. This work suggests that while bacteriophage-resistance may arise, its impact will likely be mitigated by reduced pathogenicity and secondary bacteriophage mutations that overcome resistance., Competing Interests: COMPETING INTEREST STATEMENT The authors declare that they have no known competing financial interests or personal relationships that could influence the research reported in this paper.

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods is to propagate phage on multiple bacterial hosts, pool the lysate, and repeat the propagation process until phage(s) can form plaques on the target host(s). In theory, this propagation process produces a phage lysate that contains input phages and their evolved phage progeny. However, in practice, this phage lysate can also include prophages originating from bacterial hosts. Here we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage-host system, in which we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing analysis revealed that this observed host-range expansion was due to a Casadabanvirus prophage that originated from one of the Appelmans hosts. Host-range analysis of the prophage showed that it could infect five of eight bacterial hosts initially used, allowing it to proliferate and persist through the end of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment. This work highlights the impact of prophages in directed evolution experiments and the importance of incorporating sequencing data in analyses to verify output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine presence of prophage., Importance: Directed evolution is a common strategy for evolving phages to expand host range, often targeting pathogenic strains of bacteria. In this study we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophage are active players in directed evolution and can contribute to observation of host-range expansion. Since prophage are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving experimental design of future directed evolution studies.