Long-term aging survivors of human immunodeficiency virus or acquired immunodeficiency syndrome (HIV/AIDS) were surveyed. Some did or did not have companion dogs or cats while experiencing both the AIDS and COVID-19 (COVID) pandemics. While antiretroviral treatments have reduced AIDS fatalities, survivors still suffer challenges with disabilities and finances. The surveyed 147 HIV/AIDS survivors reported experiencing more frequent stigma, aloneness, and sadness/grief during the AIDS pandemic than during COVID. During AIDS, sadness was greater among those with cats than those without cats. During COVID, older respondents unexpectedly were sad less often than younger ones; dog owners less often felt alone and isolated than those without dogs. Support during AIDS retrospectively was better for older respondents, and better for young gays than young straight ones. During COVID, support was better for men than women. Men with dogs felt more supported than those without; women with pets felt least supported. Compared to dog owners, cat owners more often felt isolated and less often felt supported. Few dog or cat owners received support from family members in either pandemic; but during the AIDS pandemic, dog owners had family support more than cat owners.Long-term HIV/AIDS survivors responded online concerning their experiences during the AIDS and COVID pandemics. Recruited from web-based organizations for AIDS survivors, 147 answered questions on: frequency of experiencing stigma, isolation, aloneness, or grief/sadness; pet ownership; and sources of human support during each pandemic. Conditional inference trees were run to identify relevant demographic factors. Post-hoc comparisons were conducted to compare dog owners and cat owners. AIDS survivors reported more frequent feelings of stigma, aloneness, and sadness/grief during the AIDS pandemic than during COVID. Cat owners’ sadness/grief during AIDS was greater than non-owners. During COVID, older respondents unexpectedly were less often sad/grieving than younger ones; dog owners less often felt alone and isolated than non-dog owners. Support during the AIDS pandemic retrospectively was rated better for older respondents; young gays’ support was greater than young straights. During COVID, support was better for men than women. Contrastingly, women with pets felt less support than those without; men with dogs felt more support than those without. Cat owners more often felt isolated and unsupported during COVID than dog owners. Few dog or cat owners received support from family members in either pandemic; during AIDS, family support was better for owners of dogs than cats. This paper describes the effects of the COVID-19 (COVID) pandemic and the retrospective effects of the HIV/AIDS (AIDS) pandemic on the lives of long-term survivors of the AIDS pandemic, some of whom had companion animals. These survivors, who are 30% more likely to be infected with COVID than the general population, are defined as people who developed HIV before 1996 [The first cases of what would later become known as AIDS were reported in the United States in 1981. More than 1.2 million people still live with AIDS in the United States . Life exThe term “AIDS survivor syndrome” was coined in 2012 to describe the psychological results of the HIV epidemic, including anxiety, depression, irritability, lack of future orientation, low self-esteem, substance abuse, social withdrawal, isolation, and survivor’s guilt . SymptomMany older survivors with AIDS now live with multiple physical complications from aging and using prolonged active retroviral therapy. These older adults exhibit higher rates of illnesses, including cardiovascular, liver and kidney diseases; cancers; frailty; and osteoporosis ,14. HIV Isolation was characteristic for most people with AIDS prior to the retroviral treatment and has been essential for the health of our entire society during the COVID pandemic. People find varied ways of coping with isolation or loneliness. Expansion of electronic communication methods that were employed for teaching, such as Zoom, also provided solutions for loneliness. Some persons focused on the companionship of pets, perhaps also gaining additional social support from people that initially was facilitated by friends’ and neighbors’ interest in their pets, wanting to talk about them or even assist with their care.Animal companionship generally involves dogs or cats. While people can gain support from their pets, resulting in feeling less lonely, the pets require some care, especially dogs, and this can be challenging for people with AIDS who have disabilities and limited income. Vulnerable populations may especially benefit from being caregivers to animals, as nurturing others can enhance self-esteem and a sense of resilience . CaregivPets played an important role in the lives of men with AIDS. By the early 1990s, an estimated 45% of Americans with HIV or AIDS had a pet . Having Young and old people, including some in distress, may seek the comfort of companionship. During the society wide COVID pandemic, news stories reported that the number of people adopting pets from shelters increased ,28. WithHow did survivors rate their experienced stigma, aloneness, isolation, sadness/grief, and human support during each of the pandemics?Did age or gender play a role in survivors’ reported experiences?What companion animals did survivors have during each pandemic, and were the animals related to the aloneness, isolation, sadness/grief, and human support that survivors reported?Were there contrasts in experiences among owners of dogs, owners of cats, and non-owners?In this study, we sought to explore the experiences of long-term survivors with AIDS who have faced serious challenges with their health and wellbeing. We focused on the following questions:A bespoke questionnaire was developed to address the questions relevant to AIDS survivors’ experiences during the AIDS and COVID pandemics. Advised that the survey needed to be short, AIDS survivors, academic professionals, and AIDS service providers teamed up to create the survey, including only 30 questions. In addition to a few demographic items, the questions were created to address the research questions of interest. Members of the team vetted the survey questions among some contacts who were AIDS survivors.The web-based survey was posted from 1 May until 15 July 2021. The survey was designed to take 10–15 min to complete. A convenience sample was obtained by distributing the survey through Facebook groups serving long-term AIDS survivors and through a targeted listserv specific for AIDS survivors.Survey participants initially viewed an introduction page inviting them to participate in a survey for long-term survivors of the AIDS pandemic. Participants were required to be at least 18 years of age. Questions addressed their sources of support and experiences with stigma, aloneness, isolation, sadness/grief, and social support during both the early AIDS pandemic (1981–1997) and the current COVID pandemic (2019–2021). A total of 150 survey responses were collected. Of those responses, 147 surveys  provided sufficient information on whether they had any animal companions or not, and this group was used for analyses. The socio-demographic information gathered included: sex at birth, gender identity, sexual orientation, age, and location, summarized in p-value below 0.0001, indicating that the variables are not normal (on their own). Then, statistical results were generated using R, version 3.6.1. The formal comparisons  were all done using Wilcoxon signed rank tests. For these analyses, data were prepared by sorting dog owners who had a dog during the specified pandemic, or not, and similarly with cats. A conditional inference tree [Responses were either dichotomous or measured on 5-point Likert scales. As such, normality was unlikely. To assure the lack of normality, Wilk-Shapiro tests were run on each of the Likert scales used in the paper , and they were all significant with a nce tree  was run Descriptive statistics with Chi-square and Fisher tests were used to compare reported experiences during each pandemic of people who had dogs with those who had cats.Analyses were conducted to assess participants’ overall comparisons of their experiences during the two pandemics. Next, conditional inference trees analyzed for the demographic factors associated with the variables of primary concern: isolation, aloneness, sadness/grief, and sources of support during each pandemic. Finally, data from people who had chosen to keep dogs or cats during each pandemic were compared with each other.p < 0.0001). The experience of stigma during the AIDS pandemic was rated as significantly more extreme by the respondents who were more than 60 years of age (p = 0.004). See Figure 1 TREE.These analyses compared respondents’ recollections of their experiences during the long-term AIDS pandemic with their recent experiences during the shorter COVID pandemic. In terms of experiencing stigma related to having AIDS, 111 respondents rated their experiences during the AIDS pandemic as worse than during the COVID pandemic, 20 rated the two pandemics as similar, and only one said COVID was worse: a highly significant result (p = 0.00012). Respondents’ frequency of feeling isolated was similar during the two pandemics (p = ns), both pandemics involving isolation. Respondents reported having felt sadness and grief more frequently during the AIDS pandemic than during the COVID pandemic, a highly statistically significant result (p < 0.0001). Respondents reported the quality of their human support network as better during the COVID pandemic than during the AIDS pandemic (p = 0.0016).Using only paired scores, long-term survivors gave higher scores for their frequency of feeling alone during AIDS as compared with during COVID . See Figure 2 TREE. During the COVID pandemic, sex played a role; male respondents felt alone less frequently than females (p = 0.048), and the men with dogs felt alone even less frequently than those without dogs (p < 0.033). See Figure 3 TREE.Age played a role in recollections of feelings of aloneness during the AIDS pandemic. Respondents older than 70 years retrospectively reported feeling alone less often during the AIDS pandemic than younger respondents (p = 0.003). See Figure 4 TREE. During COVID, those with a dog felt isolated less frequently than those without a dog (p = 0.043). See Figure 5 TREE. While the group medians for dog owners and non-owners of dogs were the same, the groups differed significantly; the means provide additional information.Similar to feelings of aloneness, age played a role in retrospective feelings of isolation during the AIDS pandemic, with these respondents older than 70 years of age reporting feeling isolated less frequently during AIDS than those younger (p = 0.03). See Figure 6 TREE. Age played a role during the COVID pandemic, with those 40 years of age or younger being sad more often than older respondents (p = 0.002). Those aged over 40 years of age through age 70 years reported having sadness and grief more frequently than those over 70 years of age (p = 0.041). See Figure 7 TREE.Those who had a cat during the AIDS pandemic retrospectively reported feeling sadness and grief more frequently than those who did not have a cat at that time (p = 0.019). See Figure 8 TREE. Among these respondents over 60 years of age, those older than 70 years reported significantly better human support than the younger group (p = 0.043). During COVID, men experienced more adequate human support than women (p = 0.007). See Figure 9 TREE. Men who had dogs experienced significantly greater human support than those without dogs (p = 0.015).Age also played a role in recollections of human support that respondents recalled receiving during the AIDS pandemic. These respondents over 60 years of age retrospectively gave better assessments of their human support during the AIDS pandemic than the younger respondents. Among these younger respondents, gays reported having better support during the AIDS pandemic than straights (p = 0.0007). Among these cat owners, about 26% described themselves retrospectively as usually very stressed in these ways during the AIDS pandemic, as compared with only 9% of these dog owners.During AIDS but not COVID, a strong majority of cat owners frequently or usually felt alone and sad. People who had ever had a cat described themselves as more stressed during the AIDS pandemic in a composite score of loneliness, isolation, and sadness than those long-term survivors who had ever had a dog (p = 0.0261); this was similar during the AIDS pandemic (p = 0.0151). Cumulative high scores on isolation, being lonely, and being sad were higher for those having only a cat during the COVID pandemic, as compared with those having only a dog (p < 0.0001). The smaller groups having only either a cat or dog during the AIDS pandemic did not show this significant difference between cat and dog owners.During the COVID pandemic, more than 1/3 of cat owners usually or frequently felt isolated. People who had ever owned a cat felt more isolated than those who had ever had a dog (p = 0.0670) was significant during the COVID pandemic (p = 0.0036). These cat owners significantly more often than the dog owners reported two or more stressors associated with having their cats (p < 0.0096). One major stressor for cat owners was transporting their cat to the veterinarians, reported more often by these cat owners than by the dog owners (p < 0.015).Similarly, more long-term AIDS survivors who were cat owners felt generally unsupported as compared with dog owners, with the cat owners more often specifying they had no support: this insignificant tendency during the AIDS pandemic (p < 0.0005); during COVID this was only an insignificant tendency (p = 0.0751). Interestingly, during COVID, some dog owners said they received no support at all from their dog, whereas only one cat owner gave this answer about their cat; there was an insignificant tendency toward some dog owners having less support than cat owners from their pets (p = 0.0636).Long-term survivors of AIDS who were dog owners found their dogs to have a socializing effect with other people during the AIDS pandemic significantly more than cat owners (p = 0.0448). During COVID, there was an insignificant tendency for more long-term survivors with dogs to receive support from a partner or spouse than those with cats (p = 0.0657). Most cat owners reported having no human support during COVID. During COVID, analyses of survivors with cats having support from a friend or a spouse as compared with those with dogs having support from a friend or spouse showed that those with cats had less support (p = 0.0302); this was not the case during AIDS.Only a minority of long-term survivors with dogs or cats listed family members as being a source of support during AIDS or COVID, but during AIDS significantly more survivors with dogs than cats had support from family members  but considered themselves deeply involved in the AIDS community and were included in the analyses.In responding to questions about the two pandemics, survivors were retrospectively considering their memories of the decades-long AIDS pandemic, 20–40 years in their experiences, as well as just 1–2 years of the COVID pandemic at the time of this survey. Naturally the AIDS experience was more extensive and it was to be expected that respondents would rate the AIDS pandemic as worse than the COVID pandemic.Older male respondents, despite describing great adversity during the AIDS pandemic, showed current coping that was even further enhanced for those who had chosen to have a dog. The healthier scores of older men regarding their experiences of stigma, aloneness, isolation, and human support were unexpected and differ from other studies of long-term survivors with AIDS. The fact that these respondents belonged to organizations that networked them with other long-term survivors speaks to the importance of community organizing and communication and how it offers meaningful support. The contrasting responses from owners of cats versus dogs suggest that programs providing support in keeping pets to people with special needs could tailor their approaches more specifically for those with cats and dogs: for cat owners to reduce their isolation; for dog owners, assisting with physical care of the dog, such as in walking the dog if the person has mobility challenges.


Background: The dramatic increase in intravitreal injections (IVTs) has been accompanied by a greater need for safer procedures. The ongoing debate about topical antibiotic prophylaxis after IVTs emphasizes the importance of large-scale studies. We aimed to study the role of topical antibiotic prophylaxis in reducing the risk of acute endophthalmitis after IVTs. Methods: Population-based cohort study, in France, from 2009 to 2018, including all French patients receiving IVTs of corticosteroids or anti-VEGF agents. Results: A total of 5,291,420 IVTs were performed on 605,434 patients. The rate of topical antibiotic prophylaxis after IVTs progressively decreased during the study period, with a sharp drop in 2014 (from 84.6% in 2009 to 27.4% in 2018). Acute endophthalmitis occurred in 1274 cases (incidence rate = 0.0241%). Although antibiotic prophylaxis did not alter the risk of endophthalmitis (p = 0.06), univariate analysis showed an increased risk after fluoroquinolone and aminoglycoside prophylaxis. This increased risk was not found in multivariate analysis. However, we observed an increased risk related to the use of fixed combinations of fluoroquinolones and aminoglycosides with corticosteroids . Conclusion: These results are consistent with the literature. Endophthalmitis rates after IVTs did not decrease with topical antibiotic prophylaxis. The use of a combination of antibiotics and corticosteroids doubles the risk of endophthalmitis and should be avoided. Avoiding antibiotic prophylaxis would reduce the costs and the potential risks of antibiotic resistance. Intravitreal injections (IVTs) have dramatically improved the visual prognosis of patients suffering from various ocular conditions, such as age-related macular degeneration, macular edema linked to diabetes, and retinal vein occlusion. Consequently, a rapidly growing number of patients have been treated with anti-VEGF  agents or corticosteroids. In France, more than 500,000 injections are administered to 150,000 patients annually ,3. GivenCaisse Nationale de l’Assurance Maladie, CNAM), the SNDS includes data from the Assurance Maladie , hospital data , and databases on deaths and disability, collecting data for the whole country, i.e., 66 million inhabitants. The high quality of this database has previously been evaluated and used in several epidemiological studies [https://clinicaltrials.gov/ct2/show/NCT03635268 (accessed on 5 September 2022)).This study is part of the French Epidemiology and Safety (EPISAFE) collaborative program . This pr studies ,10,11,12Purulent endophthalmitis and Other endophthalmitis, respectively; 10th edition of the International Classification of Diseases) within 42 days of the injection index date [®; Novartis Pharma SAS, Basel, Switzerland), bevacizumab , aflibercept , pegaptanib , triamcinolone acetonide , and dexamethasone implants . To study the impact of the date of injection on the occurrence of endophthalmitis in multivariate analysis, we considered the periods before and after 2014, in relation to the univariate results of lower IRRs after 2014 and to the authorization of prefilled syringes of ranibizumab as well as the recommendations of the French Society of Ophthalmology in 2014 regarding the lack of benefit of antibiotic prophylaxis after IVT [The dataset for this study included all adult patients in the database who received at least one IVT from 1 January 2007 to 31 December 2018. Data were not included if a lookback period or follow-up of 42 days was unavailable or if the patient died within the 42-day follow-up period. Procedures performed before 2009 were used to verify the uniqueness of cause, medical history, and medicine consumption, since the exact dates of hospitalization were unavailable for 2007 and 2008 see . As a redex date . Cases odex date . Informafter IVT .p < 0.05 (two-tailed tests). Data processing and statistical analyses were performed using the SAS statistical analysis software package .According to the results of a Kolmogorov–Smirnov normality test , most ofFrom 2009 to 2018, a total of 5,291,420 IVTs were performed and analyzed in the present study, after excluding IVTs with insufficient lookback or lacking 42 days of follow-up, as well as IVTs concomitant with ocular surgery . Continuous variables are displayed as medians and interquartile ranges. Categorical variables are displayed as numbers and percentages.IVTs were performed on 605,434 patients aged 78.0 years at treatment initiation , and most of them were women (58.8%). Most IVTs were anti-VEGF injections, accounting for 91.0% of all procedures; 4.1% were corticosteroids, and 4.9% were not identified in the database. The most frequently injected agent was ranibizumab , followed by aflibercept (27.5%) .The years 2009 and 2018 were truncated to allow for the required 42 days of lookback and follow-up.p < 0.001). The prevalence of IVTs performed with antibiotic prophylaxis decreased during the study period , followed by aminoglycosides (21.8%) and fluoroquinolones (20.3%). Combination medications with antibiotics and corticosteroids were administered in 5.4% of the IVTs. Corticosteroid IVTs were most likely performed with topical antibiotic prophylaxis  during the study period. The incidence of endophthalmitis following anti-VEGF and corticosteroid injections was 0.0197% and 0.0699%, respectively. n = 189, 222, and 439, respectively). No cases or few cases of endophthalmitis were observed under less frequently prescribed antibiotic prophylaxes such as phenicols, polypeptides, fusidic acid, or rifamycin . In contrast, a higher number of endophthalmitis cases was observed for more frequently prescribed antibiotic prophylaxes such as fluoroquinolones, aminoglycosides, and macrolides . No statistically significant differences were found for sex and diabetes when considering IVTs with or without endophthalmitis. Patients with endophthalmitis were significantly younger than those without it (78 years (70–84) vs. 79 (71–85), p < 0.001). Endophthalmitis occurred earlier with antibiotic prophylaxis than without: 6.6 days (±7.8) vs. 7.8 (±8.6), respectively (p = 0.01). In univariate analysis, acute post-IVT endophthalmitis was more likely to occur in younger patients, with corticosteroid IVTs, with non-prefilled anti-VEGF syringes (vs. prefilled ranibizumab), and at the beginning of the study period, with a decreasing IRR over the years . Other antibiotics did not decrease or increase the risk of post-IVT endophthalmitis. A major finding was that IVT with a topical combination of antibiotics and corticosteroids was associated with a higher risk of endophthalmitis versus no prophylaxis , independent of the therapeutic class injected. Other variables positively associated with a higher occurrence of endophthalmitis included corticosteroid IVTs, with a significant risk after dexamethasone and triamcinolone injection . Variables associated with a lower likelihood of endophthalmitis were older age, prefilled vs. non-prefilled ranibizumab, and period after 2014 (except 2018) (p < 0.001).There was no association when the role of antibiotic prophylaxis in the occurrence of endophthalmitis was examined in univariate analysis, regardless of the type of antibiotic, while antibiotic prophylaxis by aminoglycosides or fluoroquinolones yielded a greater risk of endophthalmitis than no prophylaxis  or with a combination of topical aminoglycoside and corticosteroids  . The incIn this study examining all IVTs performed in France over a period of almost 10 years, we observed a low post-injection endophthalmitis rate of 0.0241% (1/4153 injections). This rate is consistent with other reports, ranging from 0.02% to 0.08% ,18,19,20p = 0.02) and multivariate (p = 0.001) analyses [There is a long-running debate on the effectiveness of antibiotic prophylaxis after IVTs. None of the previously published data were sufficiently powered to reach a robust conclusion. Moreover, published studies suggested that patients with endophthalmitis were at higher risk of bacterial resistance following antibiotic prophylaxis ,24, due analyses . In 2016analyses , while Manalyses . As a reanalyses ,31, incranalyses .The French guidelines were amended in 2014, resulting in a significant reduction in post-IVT antibiotic prophylaxis, as shown in our study see , withoutWe recognize some limitations in this study, which are partly inherent to medical-administrative database studies.First, concerning the event under study, acute post-IVT endophthalmitis was defined by the record of a hospital stay for endophthalmitis. In France, the practice is to admit patients with endophthalmitis to hospital. In addition, the consumption of care  was verified in those cases. Finally, although we could not obtain laboratory confirmation of the diagnosis, it is well documented that this piece of information is missing in a substantial number of cases . MoreoveSecond, we could not determine precisely whether the topical antibiotic prophylaxis was given before or after the IVTs, or its duration. However, this did not appear to modify the risk of endophthalmitis ,39.Third, the potential role of patient adherence to their drops once prescriptions were delivered may have to be taken into consideration.Fourth, we had no information on the asepsis protocol. However, as reported by Dossarps et al., there is a unique asepsis protocol that is used exclusively in France ,40.Fifth, we could not adjust our data to systemic or local infectious risk factors such as immunosuppression, except for diabetic status or conjunctiva at risk.Sixth, we limited our main outcome measures to infectious events occurring in the 42 days following the procedure, as defined by the Endophthalmitis Vitrectomy Study Group (EVS) . HoweverSeventh, our findings cannot be fully extended to other countries; French guidelines for IVTs are somewhat different from American guidelines ,41. In FThe strength of this study is the collection of all IVTs registered in a single administrative database over 10 years in our country.The subgroup size was large enough to detect a statistically significant difference between exposure groups;In a quasi-exhaustive population;Without exclusion criteria as found in RCTs or selected population studies .In conclusion, this study found that antibiotic prophylaxis for IVTs does not lower the risk of endophthalmitis and could be detrimental through the selection of resistant germs in the conjunctival flora, thereby leading to more aggressive endophthalmitis cases, as reported in the literature. In addition, using a combination of antibiotics with corticosteroids doubles the risk of endophthalmitis and should be avoided. Patients receiving anti-VEGF or corticosteroid IVTs should therefore not be given antibiotic prophylaxis.


Pseudomonas aeruginosa. All AMPs exhibited antimicrobial activity against all strains, with minimum inhibitory concentrations (MICs) ranging from 4–512 µg/mL. Time-kill kinetics of all AMPs at 3× MIC values against strains PAO1 and LES431 showed complete kill within 10 min to 4 h, although P15s was not bactericidal against PAO1. All AMPs significantly inhibited biofilm formation by strains PAO1 and LES431, and induction of resistance assays showed no decrease in activity against these strains. AMP cytotoxicity against human lung cells was also minimal. In terms of mechanism of action, the AMPs showed affinity towards PAO1 and LES431 bacterial membrane lipids, efficiently permeabilising the P. aeruginosa membrane. Transcriptome and metabolome analysis revealed increased catalytic activity at the cell membrane and promotion of β-oxidation of fatty acids. Finally, tests performed with the Galleria mellonella infection model showed that Lynronne 1 and 2 were efficacious in vivo, with a 100% survival rate following treatment at 32 mg/kg and 128 mg/kg, respectively. This study illustrates the therapeutic potential of microbiome-derived AMPs against P. aeruginosa infections.Microbiomes are rife for biotechnological exploitation, particularly the rumen microbiome, due to their complexicity and diversity. In this study, antimicrobial peptides (AMPs) from the rumen microbiome  were assessed for their therapeutic potential against seven clinical strains of  However, like most microbiomes, competitive behaviour is displayed by rumen microbes when conditions require resilience for survival. Production of novel antimicrobials by the rumen microbes has been recently demonstrated, and these may well aid competitive prowess6. Many of these antimicrobials are classified as antimicrobial peptides (AMPs) and have also been shown to be efficient against a range of pathogenic bacteria, illustrating their potential medical application alongside their role in maintaining the function of the rumen microbiome3. Indeed, a review by O’Neill in 2016 outlined six possible strategies requiring research in order to treat multidrug-resistant bacterial infections, which included AMPs7.Microbiomes offer a largely untapped resource of novel bioactives for biotechnological exploitation due to their complexity and diversity. The rumen is a prime example, whereby bacteria, fungi, protozoa and phage interact symbiotically to harvest energy from ingested feed8. There are a growing number of AMPs with a broad spectrum of biological activity, showing great promise for potential biomedical applications, since they can regulate pro-inflammatory reactions, stimulate cell proliferation, promote wound healing by modulating the cell migration, and more9. Their antimicrobial mechanisms are unique and when used in combination with traditional antibiotics, cationic AMPs may broaden their spectrum and therapeutic effects10. Cationic AMPs present naturally in a wide variety of organisms and constitute a major component of the innate immune system11 and are continually being discovered and if developed strategically, could advance the treatment of drug-resistant infections.AMPs are normally cationic and constitute a structurally diverse group of molecules, composed of short peptide sequences, which are effective against potentially pathogenic bacteria, whilst being capable of modulating innate defences and the inflammatory processEnterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species) as being of most concern as they display vast MDR and are responsible for most nosocomial and wound infections worldwide12. ESKAPE pathogens possess a range of antimicrobial resistance mechanisms, including enzymatic inactivation, biofilm formation, changing cell permeability and modification of drug targets13. Indeed, microbial biofilms can render many antibiotic treatments and components of the host immune system ineffective14. Furthermore, complex biofilm formation by P. aeruginosa has been implicated as a major contributor to delayed wound healing in chronic wounds15 and for over 40 years, chronic P. aeruginosa infection in cystic fibrosis patients has been considered a biofilm-oriented infection14. Consequently, very few novel antibiotics are available to treat P. aeruginosa infections and developing novel strategies to combat biofilm infections is of utmost importance in order to curtail the considerable healthcare costs and reduce patient morbidity16.Indeed, strategic development of novel antimicrobials has never been as important as multidrug-resistance (MDR) is increasing and consequently decreases our ability to treat MDR bacterial infections, with the World Health Organisation (WHO) predicting that by 2050 death associated with MDR bacteria will be greater than deaths due to cancer. The WHO have also identified the ESKAPE pathogens  and other clinically relevant bacterial pathogens, including P. aeruginosa strains2. The activities of Lynronne 1–3 against methicillin-resistant Staphylococcus aureus (MRSA) were extensively characterised while only the antimicrobial susceptibility of pathogens to P15s was performed in the study by Oyama et al.2. Structural modelling showed that Lynronne 1–3 adopt an α-helical conformation of an amphipathic nature, with a net positive charge and hydrophobicity ratio of ≥40%2. In this study, we characterised the 3D structure of P15s. We also investigated the mechanism of action of all four AMPs, Lynronne 1–3 and P15s against clinical strains of P. aeruginosa. Some pre-clinical data for the topical application of Lynronne 1–3 to a mouse model of MRSA wound infection is already available, however, efficacy in a PAO1 Galleria mellonella infection model was investigated here. This study provides the fundamental pre-clinical data required to determine the viability of these AMPs as future alternative therapeutics.In this study, we determined the in vitro and in vivo efficacy of four rumen microbiome-derived AMPs Lynronne 1 (19 AAs: LPRRNRWSKIWKKVVTVFS-NH2), Lynronne 2 (20 AAs: HLRRINKLLTRIGLYRHAFG-NH2), Lynronne 3 (20 AAs: NRFTARFRRTPWRLCLQFRQ-NH2) and P15s (20 AAs: KFVRLKIYCRDKNKGRGISF-NH2) against 2. In that study, Lynronne 1, Lynronne 2 and Lynronne 3 were shown to be effective against numerous bacterial pathogens, most notably against MRSA. For this study, susceptibility of multidrug-resistant clinical isolates of P. aeruginosa isolates to Lynronne 1, 2, 3 and the less characterised P15s is shown as minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) data in Table P. aeruginosa strains were selected for their diverse AMR profiles and geographical locations, additional metadata for the strains is shown in Table A large number of novel AMPs from the rumen metagenome were identified and characterised in the foundational study by Oyama et al.Subsequent MBC values were determined by the presence of viable bacteria following a 24-h incubation on Muller Hinton agar Table . MBC det2 found that Lynronne 1–3 had a net positive charge of +6, +5 and +6, respectively, with a hydrophobicity ratio of ≥40%, whereas P15s is positively charged (+6) with a hydrophobicity ratio of 35% and an arginine–lysine (R + K) ratio of 35%  counting after exposure of bacteria to 3× MIC of the AMPs in cation-adjusted Mueller Hinton broth. Antimicrobial concentrations for in vitro time-kill assays vary in the literature, 3× MIC was chosen in this study to align with the previously published AMP data2. The data showed a rapid time-dependent kill of both strains by Lynronne 1 and 2 with ≥3 log CFU/mL reduction within the first 2 h for PAO1  biofilms were investigated. For this assay, we utilised a 96-well plate biofilm model2. At 3× MIC concentration, the AMPs were capable of significantly decreasing PAO1    Fig.  and LES424) Fig.  biofilm 431 Fig. .19. To identify promising therapeutic AMP candidates, mechanisms of resistance must be assessed to determine whether specific mutations or resistance genes have already been acquired by the target pathogen20. No resistant mutants of P. aeruginosa strains PAO1 and LES431 strains were obtained following serial passage of cultures in the presence of sub-MIC concentrations of Lynronne 1, 2 and P15s over a 25-day period 2.Resistance to cationic AMPs is an emerging phenomenon and compromises their efficacy as potential therapeutic agents2. In this study, cytotoxicity of the AMPs was evaluated against fibroblastic (IMR90 cells) lung cells  on cell viability for the AMPs Lynronne 1, 2 and P15s are as follows  at 4× MIC values were capable of permeabilising PAO1  for each of the microbiome-derived peptides Table  revealedUsing TEM, we observed changes in PAO1 bacterial cell morphology following treatment with Lynronne 1 and P15s Fig. . Due to 2. The interaction of peptides with lipid extracts from the target P. aeruginosa strains PAO1 and LES431 431 Fig.  was meas431 Fig. .Fig. 4PeWhen assessing the AMP lipid monolayer interactions with PAO1 and LES431, Lynronne 2 was the most efficacious showing affinity to the bacterial lipid extracts. Despite lower cytotoxicity to mammalian cell lines BEAS-2B and IMR90, P15s showed an affinity to PC (the mean lipid of the outer leaflet of the human cell membrane) in AMP lipid monolayer interaction studies. Despite the rapid kill of LES431 by P15s demonstrated in time-kill kinetics, the peptide had a lower critical pressure of insertion when tested against LES431 pure lipid extracts. P15s showed higher critical pressure of insertion against PAO1 pure lipid extracts, but a considerably slower reduction in CFU/mL in time-kill kinetics, indicating that the mechanism of action against PAO1 and LES431 is likely distinct.21.All AMPs interacted with the lipids with varied selectivity Table . LynronnP. aeruginosa strains PAO1 and LES431 were subjected to treatment with Lynronne 1, Lynronne 2 and P15s, to further understand the transcriptome-level effect of the peptides in comparison with untreated cells. Treatment with the AMPs at MIC concentration for 60 min was chosen to ensure sufficient RNA yield and quality from viable cells. From this assay, 24 samples were sequenced  yielding ~26 million reads for PAO1 and ~31 million reads for LES431 (36.54 GB of data combined) with an average read length before trimming of 126 for both strains. These sequences were then mapped to the reference genomes (NCBI: txid208964 for PAO1 and txid1408272 for LES431). Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA) with one-way analysis of variance (ANOVA) were performed upon the aligned reads to show the control vs treatment effects on PAO1 and LES431 22. Disruption of arginine metabolism suggests that Lynronne 1 and 2 have reduced the protective capacity of PAO1 to adjust cytoplasmic pH in response to hostile extracellular changes in acidity. For P15s, arginine metabolism was less significantly affected, however, expression of the arginine-ornithine antiporter (arcD) responsible for the transport exchange of ornithine and arginine at the bacterial membrane was significantly downregulated. The AMPs also significantly affected genes associated with PAO1 respiratory chain. cbb3 oxidases enable P. aeruginosa to grow in low-oxygen environments (such as biofilms), and they are more highly expressed in P. aeruginosa under anoxic conditions23. Therefore, reduced expression of cbb3 oxidases ccoN2 and ccoP2 in the presence of the AMPs could be an indication of reduced aggregation and potential anti-biofilm peptide activity. NO-reductase has been shown to protect pathogenic bacteria against NO, a toxic free radical produced by host macrophages24. Increased expression of NO-reductase subunits norB and norC by PAO1 in the presence of Lynronne 1, 2 and P15s, indicates potential respiratory inhibition and stress response to the accumulation of NO.Both ion Fig. . Compari22. This highlights a potential link between the ‘omic datasets as genes for arc operons involved in the ADI pathway were shown to be affected by the AMPs in the RNA-seq analysis. Following an assessment of the metabolomics data for PAO1 and LES431 combined, it is evident that both strains are distinct metabolomically and therefore must be considered independently , it was possible to see distinct clusters for the different antimicrobial treatments for both PAO1 and LES431 Fig. . SuggestG. mellonella  was determined as ~1.17 × 104 CFU/larvae . The effect of the peptides in protecting the larvae from a PAO1 infection was then evaluated using ~1.17 × 104 or ~1.32 × 103 CFU/larvae PAO1. For the larvae group infected with 104 cells; the death rate was 100 % at 24 h . For statistical analysis, a simple pairwise comparison was performed to compare the effect of an AMP between antimicrobial dosages and the control group. Mixing of bacteria and specified AMP prior to injection was kept to less than one minute, therefore, would not affect significance as time-kill kinetics data for all AMPs showed complete kill >50 min for PAO1. No melanisation or death was observed in the larvae inoculated with PBS. Additionally, larvae treated with Lynronne 1 and Lynronne 2 showed no melanisation after 96 h , could be attributed to a higher proportion of outer membrane-associated genes and a smaller proportion of cytoplasmic genes26. When compared with other strains such as C3719, higher porin activity of AES-1R could be responsible for the increased MIC value.MIC and MBC values obtained in this study are in line and even better than those found with other AMPs against 27. As previously stated by Oyama et al., Lynronne 1 and Lynronne 2 have a net positive charge (+6 and +5) with a hydrophobicity ratio (≥40%), greater than was seen for P15s (35%)2. Minor differences in peptide hydrophobicity and charge distribution can have a considerable impact on the penetration and disruption of the bacterial membranes capabilities and determine the level of damage to host mammalian membranes28.Throughout this study, it is evident that the β-sheet peptide P15s acts distinctly from the α-helical Lynronne 1 and Lynronne 2 peptides, and the root of these efficacy differences are likely attributable to their structural adoption. Despite similar antimicrobial activity potentials, it is known that cationic AMPs adopting α-helical, or β-sheet amphipathic structures can differ greatly in their ability to bind and disrupt cell membranes, with β-sheet conformations often proving less lyticP. aeruginosa PAO1 and LES431 isolates with ≥3 log CFU/mL reduction within the first 60 min and 10 min of treatment, respectively. P15s showed similar bactericidal activity on LES431 cultures, but not on PAO1 cultures. These data suggest that P15s may utilise an alternative mechanism of action to Lynronne 1 and Lynronne 2. We also speculate that the difference in activity of the AMPs between PAO1 and LES431 may be due to their distinct lipopolysaccharide (LPS) expression profiles. Reduced AMP efficacy against PAO1 could be expected, as LES431 has shown to exhibit weaker motility, swarming and biofilm formation capabilities29. In turn, this may result in lower bactericidal effects against higher growth density strains, such as PAO1. The reference compounds, levofloxacin and polymyxin B, showed reduced bactericidal killing against LES431 when compared to PAO1. Against less susceptible strains such as PAO1, Lynronne 1, 2 and P15s may not need to be delivered mono-therapeutically and could be utilised in combination with other conventional compounds to deliver synergistic effects25.Lynronne 1 and Lynronne 2 exhibited rapid bactericidal killing against P. aeruginosa is notoriously difficult to treat. Amongst other factors, P. aeruginosa can rapidly grow within a biofilm and reduce the effectiveness of host defences or antimicrobial treatment30. These biofilms are a major problem in the clinic and often present in susceptible hosts with burn or chronic wounds, chronic obstructive pulmonary disorder (COPD), cystic fibrosis, implanted biomaterials and water supplies31. The AMPs were able to prevent biofilm formation in both strains PAO1 and LES431, however, they were not capable of significantly dislodging pre-established biofilms. Therefore, rapid action against the maximum number of bacterial cells before they enter and progress into the biofilm phenotype would be more optimal32. AMPs with inhibition of biofilm formation properties should not be overlooked and could have unique applications, for example, in the prevention of urinary catheter-associated infections by using antimicrobial peptide coating34. Alternatively, combining these antimicrobial peptides with conventional antibiotics that are known to target distinct physiological subpopulations of P. aeruginosa could also improve biofilm eradication35. In addition, it may be that a combination of Lynronne 1, Lynronne 2 or P15s alongside physical treatment of biofilms, such as photodynamic inactivation could induce biofilm detachment, therefore increasing antimicrobial efficacy against biofilms that are pre-established36.Once established, 19. However, resistance to AMP-like compounds is often a result of selective pressure, rather than the acquisition of dedicated resistant genes via horizontal gene transfer20. Therefore, a gradual increase in resistance or ‘MIC-creep’ following repeated exposure to AMP-based drugs is to be expected. Furthermore, pharmacodynamic studies have shown that in vivo the probability of resistance evolution is lower in AMPs when compared to conventional antibiotics37.Importantly, serial-passage mutagenesis assays presented no resistant mutants against the rumen microbiome-derived AMPs tested in this study. The fast-acting nature of the compounds likely reduced the resistance acquisition potential of PAO1 and LES431, even at sub-lethal concentrations. The absence of resistant mutants suggests that Lynronne 1, Lynronne 2 and P15s may have multiple cellular targets and highlights their promise as potential therapeutic agents. Resistance to cationic AMPs has been noted in the literature as an emerging obstacle that compromises AMPs efficacyP. aeruginosa can cause severe lung infections in patients with cystic fibrosis, due to the clinical sample origin and the premise of these AMPs, therefore it was appropriate to test the cytotoxicity of the peptides in human lung cells. Despite higher activity against PAO1 and LES431 in susceptibility testing, Lynronne 1 appears to be cytotoxic at lower concentrations (~200 µg/mL) to the human lung cell lines IMR90 and BEAS-2B. Alternative mechanisms of action and differences in hydrophobicity may explain the higher toxicity of Lynronne 1 and 2 to human cells. Peptide hydrophobicity and charge distribution have been shown to play a key role in the penetration and disruption of lipid membranes, therefore, determining toxicity of cationic antimicrobial peptides to mammalian cells28. To improve efficacy, the stability of Lynronne 1 could be improved with synthesis enhancements. Partial modification of an AMP, such as D-amino acid substitution, has been shown to reduce cytotoxicity38. P15s was revealed to be the least cytotoxic to both IMR90 and BEAS-2B, indicating that the compound could be employed at higher concentrations. Once again, P15s exhibits unique characteristics when compared to Lynronne 1 and Lynronne 2, providing evidence to support a distinct mechanism of action and a model to further investigate any mechanistic differences.39. Although the cellular membrane composition of target and non-target cells are distinct, both natural and synthetic AMPs often exert unwanted collateral toxicity to mammalian cells41. Lynronne 1 exhibited the lowest TI of the tested AMPs, suggesting that the cytotoxicity of the peptide to BEAS-2B and IMR90 cells could outweigh the benefits of increased antimicrobial activity against PAO1 and LES431. The higher TI for Lynronne 2 and P15s in comparison to Lynronne 1, indicates that without modification, Lynronne 2 and P15s would be the safest candidates for development in therapeutic applications with regards to toxicity.Due to their innate cellular selectivity, maintaining a sufficiently large Therapeutic Index (TI) for AMPs over non-target host cells is challenging42. In this study, TEM provided visual confirmation of the proposed differences in the mechanism of action between Lynronne 1 and P15s. The cell morphology of the PAO1 cells treated with Lynronne 1 showed considerable disruption of cellular integrity when compared to P15s, confirming the cell-disruption mechanism of action.Using TEM to observe alterations in bacterial membrane integrity can play an important role in clarifying cell death mechanisms at lethal AMP concentrationsP. aeruginosa may present several independent expression changes in the presence of antimicrobials not associated with mechanism of action. Annotation of the PAO1 transcriptome revealed that the most variable genes are likely associated with stress response, including catalytic activity and transmembrane activities. Like other known cationic antimicrobial peptide treatments, disruption of the bacterial cell membrane is a common mechanism of action42. Further analysis of significant gene expressions using GO Enrichment Analysis uncovered information about the effect of the peptide upon molecular function. Different strategies have been developed by pathogenic bacteria to target arginine and the ADI pathway for self-preservation22. Arginine metabolism via the ADI pathway enables microorganisms to adapt to host defences and hostile environmental niches43. The ADI pathway requires multiple enzymes encoded by arc operon genes, including arcA, arcB and arcC that hydrolyse arginine to ornithine producing by-products of ATP, CO2 and ammonia44. Production of ammonia and ATP protects the bacteria by enabling the maintenance of cytoplasmic pH homoeostasis44. Arginine metabolic functions and the ADI pathway have been shown to be crucial for bacterial survival in changing environmental conditions44 and significant disruption of this pathway by Lynronne 1 and Lynronne 2 would likely inhibit the adaptive stress response of PAO1. The ADI pathway is reliant upon the transport of arginine in and ornithine out of the cell, and this is regulated by the arginine-ornithine antiporter (arcD) at the bacterial cell membrane22. From the enrichment data, it appears that the presence of P15s may be distinct from Lynronne 1 and 2 as it does not reduce arcA, arcB and arcC expression as significantly. However, it appears to more indirectly impact arginine metabolism by significantly reducing the expression of arcD. P. aeruginosa has five terminal oxidases that includes two cbb3-type cytochrome c oxidases essential for aerobic respiration and uses these oxidases under different growth conditions via isoform combinations of CcoN, CcoO, and CcoP subunits45. Transcriptomic assessment revealed significantly higher expression of genes such as ccoN2 and ccoP2 associated with respiratory chain in untreated PAO1 when compared to all the peptide treatments, most notably with P15s. Reduced expression of ccoN2 and ccoP2 could reflect antimicrobial peptide membrane disruption of PAO1 and the organism’s maintenance of electrochemical potential and ATP production, as these genes are localised within the cytoplasmic membrane and outer membrane vesicle. Downregulation of genes associated with the respiratory chain could also be a sign of a reduced capacity to adapt under low-oxygen conditions, such as biofilms45. Alternative respiratory chain-associated genes, norB and norC encode cytochrome subunits of NO-reductase, and this active enzyme catalyses the reduction of NO to N2046. NO-reductase functions as a respiratory enzyme that conserves anaerobic energy and detoxification of exogenous NO46. P. aeruginosa deficient in NO-reductase has been shown to have reduced survival against nitrosative attack by the host immune system46. Increased expression of NO-reductase most significantly by P15s but also with Lynronne 1 and Lynronne 2, suggests that in the presence of the AMPs there may be an increased accumulation of the toxic NO intermediate within PAO1. In addition, downregulation of norB and norC genes could further indicate membrane interaction, as these genes are localised within the cytoplasmic membrane. As expected, many of the top-variable genes highlighted for LES431 have not yet been characterised and information on their function was not available. From transcriptomic analysis of PAO1 and previous bactericidal assays, following 1 h of treatment with the peptides, we hypothesise that significant death is likely to have already occurred. Therefore, it is expected that genes associated with stress response and membrane-associated pathways would be affected in both PAO1 and LES431.Transcriptomics and metabolomics were used to further assess the mechanism of action of Lynronne 1, 2 and P15s against strains PAO1 and LES43 following 1 h of exposure. It must be noted that inference of mechanism of action from ‘omic analysis is complex, and G. mellonella were used for in vivo assessment of efficacy, as it is highlighted as an appropriate model system for identifying the mammalian virulence factors of P. aeruginosa47 and can reveal insights into antimicrobial pharmacokinetics and therapeutic effects48. From the data in this study, it is evident that Lynronne 1 and Lynronne 2 were efficacious against PAO1 in vivo, with 100% survival rate of larvae. In contrast, despite proving to be much less cytotoxic in previous assays, treating the larvae with P15s at 128 mg/kg resulted in both visual melanisation and death within 24 h. This may be due to the relatively slower killing of P15s, as observed in the time-kill assays, allowing PAO1 to form uninhibited bacterial aggregations that are more difficult to prevent from growing. Despite this, it is important to note that the survival of G. mellonella was improved when treated with P15s at higher concentrations (384 mg/kg).P. aeruginosa infections.Due to their demonstrated efficacy in primary in vivo assessment, it is possible that Lynronne 1 could undergo partial modifications to reduce its cytotoxicity. Lynronne 2 and P15s could also be partially modified and applied at considerably higher concentrations than Lynronne 1 without cytotoxic effects. In summary, this study provides the fundamental pre-clinical data required to determine the viability of these AMPs as future alternative therapeutics, particularly for the treatment of 49. PyMOL v1.7.6 software programme was used to visualise the results .The 3D conformation modelling of peptide 15 S was carried out using the de novo structural prediction method PEP-FOLD50 in cation-adjusted Mueller Hinton broth (MHB) (Cation-adjusted), Tryptic Soy broth (TSB), Brain-Heart Infusion (BHI) broth following the International Organization for Standardisation 20776-1 standard for MIC testing with a final bacterial inoculum concentration of 5 × 105 CFU/mL51. Peptides and comparator antibiotics were dissolved in sterile distilled water and added to sterile U-bottom polypropylene 96-well microplates at desired concentrations. MIC was defined as the lowest concentration of peptide or antibiotic required to inhibit the visible growth of bacteria after 18–24 h of incubation at 37 °C. Immediately following, a Minimum Bactericidal Concentration (MBC) was determined. Each individual MIC plate-well was sub-sampled and 5 μl plated onto MH agar , then incubated overnight at 37 °C. Any visible colony growth was recorded the following day to determine MBC value i.e where no growth was seen.MICs were determined using a modified broth microdilution methodP. aeruginosa strains PAO1 and LES431 was performed52. Exponential-phase cultures of PAO1 and LES431 were grown in MHB (1 × 108–10 CFU/mL), and treated with peptides  at concentrations three times their MIC values. Samples were serially diluted in phosphate-buffered saline and plated onto MHA. Following incubation at 37 °C for 18 to 24 h, the number of colonies was recorded52. Experiments were performed in triplicates, and CFU/mL was calculated at different time points after overnight incubation.Assessment of the killing times of peptides and the comparator antibiotic (Levofloxacin) against 2. For prevention of biofilm attachment, overnight cultures of both P. aeruginosa PAO1 and LES431 were grown and resuspended in MHB. Individually, peptides at 3× MIC were added to the resuspended cultures, then added to 96-well tissue culture plates and placed in a static incubator at 37 °C for a further 24 h. Following incubation, planktonic cells were washed from the biofilms using a minimum of three PBS wash steps. Biofilms were fixed with methanol for 20 min, stained with 0.4% (w/v) crystal violet solution for 20 min and re-solubilised with 33% (v/v) acetic acid. A microplate spectrophotometer was used to measure the re-solubilised biofilms at 570 nm, due to differences in starting OD for biofilm replicates, biofilm activity was plotted as a percentage of reduction in OD5700nm. Statistical significance was calculated using a single-factor ANOVA.To evaluate the efficacy of peptides Lynronne 1, 2, 3 and P15s in the prevention and disruption of pre-established biofilms, a 96-well plate method was adoptedP. aeruginosa PAO1 and LES431 were diluted and added to 96-well tissue culture plates without treatment and placed in a static incubator at 37 °C for 24 h. Planktonic cells were removed by three PBS washes and then fresh MH with antimicrobial compounds at 3× MIC were added. This was followed by a further 24 h of static incubation at 37 °C. Planktonic cells were removed once more with PBS washes and biofilm fixing, staining, re-solubilising and optical density reading steps remained the same.To measure disruption of pre-established biofilms, the assay was modified to add an additional incubation. Overnight cultures of P. aeruginosa strains PAO1 and LES431 were continuously subjected to Lynronne 1, 2, 3 and P15s over a 25-day period to expose potential AMP resistance. As described in the MIC protocol above, for the first passage microdilution susceptibility testing in broth was performed utilising the standard doubling-dilution series using cation-adjusted Muller Hinton. MIC was determined following incubation of the cultures for 24 h. Wells containing the highest concentration of AMPs enabling growth were then diluted 1:1000 in MHB and utilised as inoculum for the subsequent MIC assay. This process was repeated over 25 days.Cultures of l-glutamine and 1% antibiotics . Cells were routinely grown onto 25 cm2 flasks maintained in a 5% CO2 incubator at 37 °C. Cells grown on 25-cm2 flasks were detached using trypsin-EDTA solution (Thermofisher) and seeded into 96-well cell culture plates (Greiner Bio-one) at ~104 cells per well  and were incubated at 37 °C, 5% CO2 incubator until they reached confluence (~48–72 h post-seeding). Wells were then aspirated and increasing concentrations of peptides were added to the cells, followed by incubation for 48 h at 37 °C in a 5% CO2 incubator. Subsequently, the wells were aspirated, and cell viability was determined using a resazurin based in vitro toxicity assay kit (Sigma-Aldrich) following the manufacturer’s instructions. The resazurin stock solution was diluted at 1:100 in sterile PBS containing calcium and magnesium  and emptied wells were filled with 100 μl of the resazurin diluted solution. After 4 h incubation at 37 °C, fluorescence intensity was measured using a microplate reader (excitation wavelength of 530 nm/emission wavelength of 590 nm). The fluorescence values were normalised by the controls and expressed as a percentage of cell viability. The IC50 values  were calculated using GraphPad® Prism 7 software. Non-linear regression parameter was utilised for curve, pre-set as dose response to determine inhibition vs normalised response.Cytotoxic activity of the rumen-derived peptides against BEAS-2B (ATCC CRL-9609) and IMR90 cells (ATCC CCL-186) was determined. BEAS-2B and IMR90 cell lines were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% foetal calf serum (FCS), 1% 50 values for each cell line (BEAS-2B or IMR90) with the given MIC value for each organism (PAO1 or LES431). Standard error values (+/−) follow the TI, and MIC values were placed in brackets as reference.The AMP Therapeutic Index (TI) was calculated by dividing the ICThe effects of Lynronne 1 and P15s on the PAO1 bacterial cell morphology were investigated using transmission electron microscopy (TEM). Bacterial cultures in mid-log phase were treated with Lynronne 1 and P15s independently (at 3× MIC for 1 h) and then fixed with 2.5% (v/v) glutaraldehyde. Cells were post-fixed with 1% osmium tetroxide (w/v), stained with 2% (w/v) uranyl acetate and Reynold’s lead citrate and assessed using a JEOL JEM1010 transmission electron microscope  at 80 kV. TEM was performed by Alan Cookson, IBERS Aberystwyth University Advanced Microscopy and Bio-Imaging Laboratory.P. aeruginosa strains, then resuspended in chloroform and stored under nitrogen conditions at −20 °C. Pure bacterial and eukaryotic lipids, PE, PG, PC, cardiolipin, LTA and LPS  were also resuspended in chloroform (1 mg/mL) and stored under the same conditions. Phosphatidylglycerol (PG), cardiolipin, Phosphatidylglycerol (PC) and phosphatidylethanolamine (PE). Phosphatidylglycerol (PG) and cardiolipin represent lipids abundant on the bacterial membrane surface. Phosphatidylglycerol (PC), represents the main lipid present on the surface of eukaryotic membranes while phosphatidylethanolamine (PE) is present in both the inner and outer membrane leaflet of bacterial and eukaryotic membranes. Interaction of the peptides with total Pseudomonas lipid extracts or pure lipids was evaluated through the measurement of their critical pressure of insertion53. Critical pressure of insertion was determined by changing the initial pressure of lipid monolayer (from 10 and 30 mN/m) and measuring the variation of pressure caused by the injection of peptide . Briefly, lipids were added until the desired initial surface pressure was reached. After an incubation period of 5–10 min required to allow for evaporation of solvent and stabilisation of initial surface pressure, peptides were injected sub-phase into the PBS  using a 10 µl Hamilton syringe. Variations of surface pressure caused by peptide’s insertion into lipid monolayer were continuously measured using a full automated microtensiometer  until equilibrium was reached . All experiments were carried out in a controlled atmosphere at 20 °C ± 1 °C and data were analysed using the Filmware 2.5 programme . Variation of surface pressure was plotted as a function of initial surface pressure and critical pressure of insertion was calculated as the theoretical value of initial pressure of lipid monolayer not permissive to peptide insertion, i.e., a variation of pressure equals to 0 mN/m. The accuracy of the system under our experimental conditions was determined to be ±0.25 mN/m for surface pressure measurements.Propidium iodide assays were used to assess membrane permeabilisation using CTAB (300 µM) as control. Peptide–lipid interaction was measured utilising a reconstituted lipid monolayer . Folch extraction was used to obtain complete lipid extracts from overnight cultures of 54. Overnight bacterial cultures were inoculated into MHB using a direct colony suspension method and incubated at 37 °C (200 rpm) to reach a mid-log growth phase (approx. OD600 0.35–0.5). Following this, a 2 × 106 CFU/mL aliquot was taken from the culture and diluted in MHB. The cultured strains were then treated with Lynronne 1, Lynronne 2, and P15s at their respective MIC concentration (n = 3). Treatment time was restricted to 1 h in order to maintain RNA quality and yield; all untreated cultures were used as a control.Lynronne 1, Lynronne 2 and P15s against LES431 and PAO1 were investigated by transcriptomic analysis following pre-established methodsg for 10 min. To the formed pellet, Proteinase K (QIAGEN) and a Bacterial Lysis Mix (QIAGEN) consisting of Mutanolysin and Lysozyme was added. Tris-EDTA buffer could then be added, vortexed and incubated at room temperature for a further 10 min. An RNeasy Plus Mini Kit (QIAGEN) was then utilised to perform the RNA extraction process on the treated cells, following the manufacturer’s guidelines. A Qubit™ fluorometer with a broad range RNA kit  was used to quantify the total RNA samples according to the manufacturer’s instructions.Following treatment, all suspensions were washed twice and resuspended in a PBS and RNAprotect Bacteria reagent  mixture (1:2). This suspension was promptly vortexed and incubated at room temperature (20 °C ± 1 °C) for 5 min, before centrifugation at 5000×® TruSeq® Stranded mRNA Sample Preparation Kit following the Illumina protocol (poly-A enrichment step was omitted). Libraries were then quantified using EpochTM (BIOTEK) plate spectrophotometer prior to pooling, and the final library pool quantified via QubitTM. The size of amplicons was determined via agarose gel electrophoresis, then library pool was diluted to 10 nM based on DNA concentration and average amplicon size. This stock pool was then diluted to 8 pM for sequencing in 2 × 125 bp format on an Illumina HiSeq 2500 platform.Total RNA was subjected to ribosomal RNA depletion using the MICROBExpressTM kit (Thermo Fisher) as per the manufacturer’s guidelines. Four hundred ng of enriched mRNA was then used to prepare dual-indexed sequencing libraries with the IlluminaBacterial incubation remained consistent with all previous assays (200 rpm at 37 °C overnight). For each isolate, six biological replicates were individually treated with the relevant peptide, except the control group which remained untreated. Following subsequent incubation, all samples were collected during the mid-exponential growth phase. An aliquot of bacterial culture was taken at predetermined time points; 1 h following treatment with a test compound. Liquid N2 was used to rapidly freeze the samples, halting any further cellular metabolism. These samples were then stored at −80 °C until all sampling was completed.55. Upon thawing, all samples were centrifuged at 10 °C, 1800 × g. Individually, the samples were then washed with a saline solution (0.85% NaCl) and centrifuged again under the same conditions. The supernatant was discarded, and the pellet resuspended once more in a saline solution. All samples were adjusted to an OD600 of 1 before the addition of a 200 μL extraction buffer, chloroform/methanol/water (1:3:1) solution. Four rapid freeze-thaw cycles with liquid nitrogen and periodic vortexing were used to achieve sufficient extraction. All samples were then centrifuged for a final time  and transferred to new microcentrifuge tubes. Fifty microliters of this final mixture was transferred into a HPLC vial containing a 0.2 mL flat-bottom micro insert required for flow infusion electrospray ion high-resolution mass spectrometry (FIE-HRMS) analysis.Extraction of metabolites from the samples was undertaken using a method developed by Baptista et al.−1 flow of methanol and water (70:30). Ion intensities were recorded with an m/z between 50 and 1000 for 3.5 min at a resolution of 100,000 (m/z 200), with a ppm mass accuracy level of 3 (±1).Metabolite fingerprinting via flow injection electrospray high-resolution mass spectrometry (FIE-HRMS) was performed using an Exactive HCD mass analyser. The system is equipped with an Accela UHPLC system (Thermo-Scientific) which can generate metabolite fingerprints in both positive and negative ionisation modes, within a single run. Samples were injected into a 100 µl min56 and FastQC/MultiQC (v0.11.9/v1.11). For both strains, all Illumina-specific sequences and adapter regions were cut . Kallisto 57 was used as a mapping tool to the reference genomes, and differential expression analysis was undertaken using DESEQ258 with no threshold . Numerical data were exported from the read-counting programme and entered into R for statistical analysis. The Geneontology.org online resource was used to determine the gene designations for the significant genes identified in DESEQ2, Padj of <= 0.05 and a logfold change of >=2 was used to make the volcano plots, ShinyGO 0.76 was used for GO Enrichment Analysis figures.RNA sequences as ‘fastq’ files exported from the Miseq/Hiseq were then paired, trimmed and quality checked accordingly using Trimmomatic (v0.38)m/z). Before further statistical analysis, all data from the intensity matrix was log-transformed. The main statistical analysis platform used to generate PCA and Heatmap figures for the metabolomics dataset was MetaboAnalyst 4.059, this software also supported integrative analysis between the transcriptomic and metabolomics datasets.For metabolomics, the manufacturer’s recommendations were used to set the ESI source parameters, and all raw files were converted to CDF-files, to be mass aligned and centroided in MATLAB  maintaining the highest mass accuracy. For each ion mode, the mass spectra around the apex of the infusion peak were combined into a single intensity matrix  larvae with medium weight 275 mg each were randomly selected both for cytotoxicity and efficacy testing in duplicate . Larvae with previous melanisation of the cuticle were not used in the experiments. For cytotoxicity testing, peptides Lynronne 1, Lynronne 2, and P15s were injected in larvae’s last proleg at the MIC concentration, namely 32 mg/kg of larvae body weight (LBW) for Lynronne 1 and 128 mg/kg LBW for Lynronne 2 and P15s; and 3× MIC, namely 96 mg/kg LBW for Lynronne 1 and 384 mg/kg LBW Lynronne 2 and P15s as determined in the MIC assay. The larvae were maintained at 37 °C in the dark. The survival phenotypic aspects as activity, melanisation, cocoon production (extent of silk production) and survival were monitored every 24 h for 96 h. The control group corresponded to larvae that received injection with water (peptides toxicity assays) or PBS .50 and LD of P. aeruginosa PAO1 in G. mellonella, an inoculum of 10 μl in PBS 1× (101 to 105 CFU/larvae) was injected into the larvae. CFU > 104 killed all the larvae before 24 h after inoculation. After the injections, the larvae were maintained at 37 °C in the dark. The LD50 and LD were determined by linear regression. To evaluate the efficacy of peptides in G. mellonella infected with P. aeruginosa 104 and 103 CFU/larvae was used and the bacteria inoculum and the peptides solutions were mixed and immediately inoculated in the larvae (<1 min). Larvae injected with PBS and bacteria were used as negative and positive controls, respectively. The larvae were maintained at 37 °C in the dark, and their survival was monitored and analysed as above. The Kaplan–Meier method was used to plot the survival curves. Differences in survival were calculated using the log-rank test using the software R, version 2.13.0.To determine the LDSupplementary Material


Since the first case of HIV infection was reported in China in 1985, the incidence and mortality of AIDS have been increasing rapidly, which has caused serious damage to the life and health of people in China and all over the world. Therefore, it is of great significance to study the technique for predicting AIDS morbidity and mortality. The purpose of this research is to explore the applicability of the mean generation function model (MGFM) in the early warning of AIDS morbidity and mortality, to predict its prevalence trend, to enrich the prediction techniques and methods of AIDS research and to provide suggestions for AIDS transmission control.In this research, the MGFM was applied to predict the incidence and mortality of AIDS in China. AIDS incidence and mortality data in China from 2008 to 2019 were used to construct the prediction model.The MGFM can predict the annual incidence and mortality of AIDS. The model constructed in this research predicted that the incidence and mortality of AIDS in China will continue to increase from 2020 to 2023.The mean birth function model was an effective method to monitor and predict the changing trend of AIDS incidence and mortality in China.The online version contains supplementary material available at 10.1186/s12911-022-01825-6. Global progress report on HIV, viral hepatitis and sexually transmitted infections, 2021 shows that approximately 1.5 million people were newly infected with HIV in 2020, and 680,000 people died from HIV-related diseases. By 2020, there were 37.7 million HIV-infected people in the world [Human immunodeficiency virus (HIV) is a virus that attacks the human immune system. HIV destroys CD4 cells and weakens people's immunity to opportunistic infections. Acquired immunodeficiency syndrome (AIDS) is the latest stage of HIV infection . At preshe world .General Situation of National Statutory Infectious Diseases in 2020 published by the Disease Control and Prevention Bureau of the National Health Commission, there were 62,167 new AIDS patients and 18,819 deaths from all causes of AIDS in 2020. AIDS has become an infectious disease with the sixth highest incidence rate and the highest mortality rate of Class A and B infectious diseases in China [Implementation Plan for Preventing the Spread of AIDS (2019–2022). It has promoted six major projects to curb the spread of AIDS, such as the AIDS prevention publicity and education project, the AIDS comprehensive intervention project, the AIDS expanded detection and treatment project, the AIDS prevention comprehensive social management project, the elimination of mother-to-child transmission of AIDS and the AIDS prevention education project for students.In China, AIDS is classified as a Class B infectious disease. According to the in China . As of Oin China . To effeUsing mathematical models to predict the epidemic trend of AIDS can provide a basis for better prevention and control of the spread of AIDS. To date, many scholars have used various mathematical models to predict the changing trend of AIDS incidence and mortality, such as the ARIMA model; this model was used to study the epidemic trend of AIDS and HIV in China, but the ARIMA model is an autocorrelation method. The ability to predict random frequent bursts is limited . StudiesTherefore, this study introduced the MGFM to explore the applicability of the model in AIDS disease prediction to enrich AIDS prediction methods and provide reference suggestions for AIDS prevention and control in China.China Health Statistics Yearbook compiled by China National Health Commission [Residents voluntarily undergo HIV antibody testing at the time of medical treatment and physical examination in medical institutions.Medical institutions report HIV antibody-positive residents to the local CDC, and the local CDC will conduct a diagnosis test on suspected patients.The local CDC reports confirmed positive tests to the medical institutions and urges the medical institutions to report the patient data for HIV/ARDS through the Chinese disease prevention and control information system.The Center for STD/AIDS Prevention and Control of China CDC uses the China Information System for Disease Control and Prevention to collect and publish HIV/AIDS data nationwide.In this research, we used AIDS incidence and mortality data from 2008 to 2019 collected in 31 provinces  and published in the mmission . The datAccording to the direction and location, Chinese geography can be divided into three regions , namely,The Eastern region includes 11 provinces: Beijing, Tianjin, Hebei, Liaoning, Shanghai, Jiangsu, Zhejiang, Fujian, Shandong, Guangdong, and Hainan.The Central region includes 8 provinces: Shanxi, Jilin, Heilongjiang, Anhui, Jiangxi, Henan, Hubei, and Hunan.The Western region includes 12 provinces: Inner Mongolia, Chongqing, Guangxi, Sichuan, Guizhou, Yunnan, Tibet, Shaanxi, Gansu, Qinghai, Ningxia, and Xinjiang.A descriptive statistics method was used to analyse the dynamic change trend of AIDS incidence and mortality, and the time series MGFM of AIDS incidence and mortality was constructed according to the collected data. Our research used DPS 17.10 software to help us build these models .Time series with equal interval sample size of N:The mean-generating function (MGF) of each order is constructed according to the following formula:N is the sample length of the sequence, t is the sampling time, l is the time interval length of the MGFM, M is the maximum period length, and INT is rounding. Cyclic extrapolation is carried out on MGF q is the prediction step, and M periodic function sequences of length N + q can be obtained. Taking these periodic continuation series as factors and performing stepwise regression analysis with the original series, we can establish a regression equation for simulation and prediction.Among them,Fitting the high-frequency components of the original data The corresponding sequence is recorded as To fit the upward increasing or downward decreasing trend in the original data sequence, the cumulative continuation sequence is further established:Among them,After the above process, finally, a total of 4 M continuation sequences of MGFMs are obtained:The purpose is to obtain a high-precision prediction model through screening, that is, there are more independent variables to be screened.The research results are as follows N is the sample length; k is the number of variables in the statistical model; and nij is the number in the contingency table of class i events and class j events.Qk is the sum of residual squares of the model, and the calculation formula is as follows:Qy is the sum of the squares of the total deviation of the model, and the calculation formula is as follows:Type, k is fixed as a small quantity, When the model is linear and R is a complex correlation coefficient,Therefore, the CSC value of the original sequence x(t) can be calculated for each MGFM as a fitting sequence when According to the above process, the CSC values of each sequence are calculated separately. The optimal subset method is used for screening based on the CSC value; that is, all possible combinations are listed, and the regression equation with the largest CSC value is selected as the optimal equation. When the number of variables is different and the CSC value is the same, the equation with fewer variables is taken as the optimal regression equation for simple calculation.From 2008 to 2019, the incidence and mortality of AIDS in China showed an increasing trend year by year, as shown in Fig. According to the public data of official Chinese departments, Table We used the optimal subset regression method, set M = 4 (the formula of M ≤ INT(N/3), where N = 12, then M ≤ 4), to build regression equations, calculate the estimated value of the original sequence, and then calculate the CSC value of each model.2 of each AIDS incidence model is relatively close: MAE, MAPE, MSE and RME4.Table To show the difference between the fitted value and the actual value of each AIDS incidence model more intuitively, we calculated the difference between the fitted value and the actual value of each AIDS incidence model and took the absolute value of the difference of each AIDS incidence model to draw Fig. 2 of each AIDS mortality model was relatively close: MAE, MAPE, MSE and RMS.Table Similar to the AIDS incidence model, we calculated the difference between the fitted value and the actual value of each AIDS mortality model and drew Fig. Using the optimal equation selected above, the short-term prediction of the incidence (K = 1) and mortality (K = 2) of AIDS in China from 2020 to 2023 was made. The results show that the predicted values of AIDS incidence in China were 5.2816/100,000, 5.4581/100,000, 5.9670/100,000 and 6.3201/100,000, and the predicted values of AIDS mortality in China were 1.4294/100,000, 1.4502/100,000, 1.6111/100,000 and 1.7092/100,000, respectively. The results show that the incidence and mortality of AIDS in China will continue to rise.At present, HIV is still a major global public health problem, which has claimed 36.3 million lives thus far . The sprAt present, the MGFM is widely used in disaster prediction, weather forecasting, preproduction of agricultural products and other disciplines , 21. HowThe results of the models constructed by our research showed that the determination coefficients of all models of AIDS incidence rate are above 0.95, and the determination coefficients of all models of AIDS mortality rate are above 0.94, and MAE, MAPE, MSE and RMSE of the two types of models are relatively close, indicating that the MGFM of AIDS incidence and mortality constructed by this study has good fitting effect and good accuracy. Therefore, MGFM can be used to predict the incidence and mortality of AIDS in China and can be used as a reliable method for disease prediction in the field of public health.As shown in Table The top five areas of AIDS incidence in 2019 were Sichuan , Guangxi , Guizhou , Chongqing  and Yunnan . The top five areas of AIDS mortality are Guangxi , Sichuan , Yunnan , Guizhou  and Xinjiang . As shown in Figs. The prediction results of our study showed that the incidence and mortality of AIDS in China will continue to rise from 2020 to 2023, which was similar to Hu et al. , Guo 2727, and XTo cope with the rising incidence and mortality of AIDS, Chinese public health departments should constantly promote AIDS prevention and treatment. First, Chinese health departments should increase publicity and education for Chinese citizens to popularize the main transmission routes of AIDS. Second, investment in research and development of AIDS vaccines should be increased, clinical treatment methods should be improved and early screening efforts should be strengthened as early as possible to identify patients in the incubation period. Finally, the government should constantly improve measures such as humanistic care for AIDS patients, psychological counselling and educational guidance –32.In summary, our research proved that the MGFM had a high fitting accuracy in the prediction of AIDS morbidity and mortality in China and can be applied to the prediction of AIDS mortality and morbidity in China. The research also found that the western region of China was the main area of the AIDS epidemic in the country and needs to be highly attended to by the Chinese health department, which can formulate intervention policies according to local conditions.There were some limitations in this research. First, this research only predicted the epidemic trend of AIDS from the perspective of macroscopic data and failed to analyse the influencing factors of the occurrence and spread of the disease. Tt is impossible to analyse the cultural, educational, and economic factors related to the AIDS epidemic in China. Second, the incidence of AIDS is related to region and season. In this research, we did not establish the epidemic model of monthly and quarterly data, which cannot reflect the changes in monthly or quarterly AIDS incidence and mortality in China. This will be the key research content of our team in the future. Finally, this study only discussed the prediction of AIDS incidence and mortality in China from a theoretical perspective. In fact, the AIDS epidemic may be influenced by various factors, and there may still be a gap between our research and the actual situation.Additional file 1.Table S1 Parameters of four MGFMs of AIDS incidence. Table S2 Parameters of four MGFMs of AIDS mortality.