Pyrrhoderma noxium (Corner) L.W. Zhou and Y.C. Dai is known to infect a large number of culturally and economically important plant species across the world. Although chemical control measures have been effective in managing this pathogen, their adverse effects on the ecosystem have limited their use. The use of biological control agents (BCAs) thus is generally accepted as an environmentally friendly way of managing various pathogens. Testing various consortia of the BCAs with different antagonistic mechanisms may even provide better disease protection than the use of a single BCA against aggressive plant pathogens such as the P. noxium. In the presented study, the wood decay experiment and the pot trial confirmed that the consortium of Trichoderma strains (#5029 and 5001) and streptomycetes (#USC−6914 and #USC−595-B) used was effective in protecting wood decay and plant disease caused by P. noxium. Among the treatments, complete elimination of the pathogen was observed when the BCAs were applied as a consortium. In addition, the BCAs used in this study promoted the plant growth. Therefore, Trichoderma and streptomycetes consortium could be used as a potential biocontrol measure to manage P. noxium infections in the field over the application of hazardous chemical control measures.The Brown root rot pathogen  Pyrrhoderma noxium (Corner) L.W. Zhou and Y.C. Dai can produce cellulolytic and ligninolytic enzymes, which can play a key role in root decay leading to tree failures and agricultural losses across the tropical and subtropical regions of the globe [P. noxium.The white root fungus, he globe ,2,3,4. She globe ,4,5,6. ATrichoderma and streptomycetes have been widely used in biological control applications and reported as being effective against many phytopathogenic fungi including Phytophthora capsici, Puccinia triticina, Magnaporthe oryzae, Pythium aphanidermatum, Phytophthora fragariae, Fusarium verticillioides, Rhizoctonia solani, Botrytis cinerea, P. noxium, Pythium ultimum, Fusarium oxysporum, Fusarium solani, Phytophthora nicotianae and Sclerotium rolfsii [In recent years, biological control of phytopathogens by the application of antagonistic microorganisms has gained more attention over the chemical-based control measures ,10,11,12 rolfsii ,23,24,25 rolfsii ,19,26,27Trichoderma and streptomycetes were tested as a single biocontrol agent against P. noxium in plate assays as well as in in vitro wood decay experiments and green house studies [Trichoderma and streptomycetes for the management of this pathogen.Previously, several  studies ,4,28,29.Trichoderma strains or the streptomycetes inoculum) and as a combined inoculum against P. noxium in soil. Therefore, the aims of this study were (1) to assess the biocontrol efficacy of the BCAs in preventing in vitro wood decay caused by P. noxium; (2) to test the BCAs for disease protection of plants growing in P. noxium infested soil; and (3) to evaluate the effect of P. noxium and the BCAs on plant growth.In biological control studies, in vitro evaluation of antagonist against the pathogen on an agar medium provides an initial screening for its antagonistic ability but does not mean that the same effect is reproduced in container media or in soil . TherefoStreptomyces cinereus and #USC−595-B: OP592220, closely related to Streptomyces platensis) [Trichoderma strains (#5001: SAMN30380231 and #5029: SAMN30380232) closely related to Trichoderma reesei [P. noxium consortium .Two streptomycetes   and two Ficus macrophylla  on 8 April 2020 were dried in an oven (10 April 2020 to 12 April 2020) at 105 °C until a constant weight was obtained. The dry weight of each wood block was recorded before autoclaving twice at 15 lbs of pressure at 121 °C for 15 min using the dry cycle program to prevent the accumulation of moisture. A final autoclave cycle was performed 24 h before the start of the experiment (14 April 2020). All blocks were re-wetted using sterile distilled water for 3 min under reduced pressure in a vacuum desiccator to increase their moisture content to 50% (by dry weight) [The wood decay experiment was conducted using the method described by Schwarze et al.  with sli weight) .Trichoderma strains (#5001 and #5029) and streptomycetes (#USC−6914 and #USC−595-B) previously confirmed for their antagonistic activity against P. noxium in vitro were selected as the test organisms [P. noxium consortium was prepared by inoculating 6 plugs (0.5 cm radius) of P. noxium strains A, B, D, E and F onto sterile millet seeds on 15 March 2020). Trichoderma consortium was prepared by the co-inoculation of Trichoderma strains #5001 and #5029 (15 plugs/strain used for inoculation), while a streptomycetes consortium was prepared by the co-inoculation of streptomycete #USC−6914 and streptomycete #USC−595−B (15 plugs/species used for inoculation) on 15 March 2020. After visual confirmation of the inoculum establishment on the millet seeds (after 30 days), inoculum (2% w/w) was thoroughly mixed with the sterile soil packed in the sterile containers. The inocula combinations used in this experiment are listed in Gamma irradiated  topsoil  was packed in sterile polyproplene containers with air filter embedded lids, and the soil moisture level was maintained at 65% WHC by adding sterile distilled water. Two rganisms . P. noxiP. noxium and streptomycetes were inoculated seven days before wood blocks placed on the packed soil (8 April 2020). On the other hand, Trichoderma was inoculated on the start of the experiment date (15 April 2020) due to its fast growth rate. Finally, two sterile wood blocks were aseptically placed into each sterile container and incubated for a period of three months (15 April 2020 to 15 July 2020) at 28 ± 1 °C. The treatments  were cleaned and oven-dried at 70 °C until a constant weight was obtained. Final dry weights were recorded and used for calculating the percentage weight loss of each block .P. noxium was studied using a Longitudinal Compression Strength (LCS) test [The ability of the BCAs to protect the wood strength by controlling the growth of CS) test . Five raCS) test . All theCS) test . LCS valP. noxium on wood blocks was evaluated in terms of the frequency of pathogen re-isolation from inner and outer wood surfaces. Five wood blocks randomly selected from the treatments of P, SP, TP and TSP were cleaned (to remove mycelium and soil particles) inside the biosafety cabinet (on 15 July 2020). A total of 25 wood fragments per treatment comprising five fragments per block were removed from outer surface. For outer surface sampling, one wood fragment was taken from top and four sides of the blocks facing upwards. A specimen was not taken from the bottom surface since it was already in contact with the soil inoculated with pathogen/BCAs. After splitting the wood blocks at the center, 25 wood segments were taken from the center part of blocks for each treatment comprising five wood segments per block. This experiment was performed only once. The wood fragments were inoculated on modified P. noxium selective media [P. noxium growth was recorded. Finally, effectiveness of BCAs on controlling P. noxium was expressed as percentage of eradicated P. noxium [The inhibitory effect of the biological control agents on establishment of ve media . The ino. noxium .Trichoderma strains and streptomycetes singly or combined against P. noxium in the greenhouse settings. F. macrophylla was selected as the test plant due to its cultural significance in Queensland, Australia. The experiment was conducted in a shade house located at Seventeen Mile Rocks, Brisbane, Australia for a period of six months between 10 December 2018 and 10 June 2019.A pot experiment was carried out to evaluate the antagonistic abilities of w/w) was thoroughly mixed with 2.5 kg of gamma irradiated  potting mix as per treatment requirements at the time of planting. Approximately same sized (15 cm) and aged (six months) F. macrophylla plants produced from cuttings were purchased from Ibrox Park Nursery, Burbank, Australia on 10 December 2018 were used in this study. Roots were surface sterilized on 10 December 2018 in 0.5% sodium hypochlorite solution for 2 min to prevent any existing pathogenic fungi from flourishing during the experiment. After the surface sterilization of roots, plants were re-potted in 200 mm plastic pots filled with potting mix and inocula combinations  [Trichoderma strains, Trichoderma agar media, were used [Trichoderma and P. noxium and at 25 °C for streptomycetes for seven days. Triplicate plates were used for each treatment. The Trichoderma, streptomycetes and P. noxium resembling colonies were isolated from the plates and were identified based on the morphological appearance of the original cultures used in the study.After two weeks (on 24 December 2018) of inoculum application, re-isolation of sulfate) , for strsulfate)  and for ere used ,39. PlatP. noxium and the plants treated only with P. noxium.At the end of six months (on 10 June 2019), fig plants were carefully removed from pots, and soil particles attached on the roots were cleaned without damaging the roots. Shoot length was measured between plant collar (root initiation point) and terminal shoot point. Roots were straightened, and the length was measured between root initiation point to the terminal root. After separation, fresh root and shoot weight of each plant were recorded. Shoots and roots were dried at 70 °C for 7 days before obtaining the dry shoot and root weight. Shoot length, root length, fresh shoot weight, dry shoot weight, fresh root weight and dry root weight of inoculated plants were compared with the un-inoculated control plants to study the effect of inocula on the plant growth parameters. Similarly, disease control ability of the BCAs was evaluated by comparing the growth parameters of plants treated with the BCAs and P. noxium and prevent the disease was evaluated in terms of frequency of P. noxium re-isolation from the root samples. At the end of the experiment (on 10 June 2019), 15 root cuttings were collected from two main and five fine roots [P. noxium selective medium [Trichoderma strain, streptomycetes and combined BCAs consortia were expressed as a percentage of eradicated P. noxium from the roots [The ability of the BCAs to control the growth of ne roots  of each e medium . A totalhe roots .For the wood block study, the effect of treatments on the wood weight and strength was evaluated via one-way ANOVA followed by Tukey’s test using SPSS statistics 26 . SimilarP. noxium showed decayed signs with a conspicuous network of brown lines while the blocks treated with BCAs did not show any sign of P. noxium degradation  = 1392.9, p = 0.001) and wood strength loss  = 49.13, p < 0.001). The wood blocks from all the treatment showed some amount of weight and strength loss compared to control (P. noxium was significantly (p = 0.001) higher than the blocks treated with BCA (T or S or TS). On the other hand, the weight and strength loss in the treatments with BCAs and P. noxium (SP or TP or TSP) was significantly lower (p = 0.001) than the weight loss measured in P. noxium (P) treatment (P. noxium was ~5 times lower than the weight loss percentage measured in treatment with P. noxium (P. noxium was approximately ~1.6 times higher than the wood strength measured in P. noxium treatment (P. noxium in preventing the wood decay.There was significant difference between the treatments in weight loss percentage ( control . Howeverreatment . Weight . noxium . Meanwhireatment . The weiP. noxium was low in the blocks treated with BCAs and P. noxium (TP or SP or TSP) compared to the wood blocks treated with P. noxium  compared to Trichoderma consortium with P. noxium (TP) or streptomycetes consortium with P. noxium (SP). Penetration of P. noxium into the inner wood surface also prevented in the treatments of TP, SP and TSP, while P. noxium was detected in the inner wood surface in the treatment with P. noxium (P) (The re-isolation frequency of . noxium . Completxium (P) . P. noxium in the potting mix  confirmed the establishment of the BCAs and ting mix . During F = 65.938, p < 0.001), shoot length  = 169.5, p < 0.001), fresh root weight  = 191.53, p < 0.001), dry root weight  = 93.89, p < 0.001), fresh shoot weight  = 364.326, p < 0.001) and dry shoot weight  = 157.93, p < 0.001) of plants. The root length, shoot length, fresh root weight, dry root weight, fresh shoot weight and dry shoot weight of the plants treated with P. noxium were significantly (p < 0.001) lower than the controls and the plants grown in the presence of BCAs (T or S or TS or TP or SP or TSP) (Trichoderma consortium (T), streptomycetes consortium (S), streptomycetes with Trichoderma consortium (TS), Trichoderma consortium with P. noxium (TP), streptomycetes consortium with P. noxium (SP) and P. noxium with all the BCAs (TSP) were significantly (p < 0.001) higher than control plants  was significantly (p < 0.001) higher than the plants inoculated with Trichoderma consortium or streptomycetes consortium. Similarly, in the presence of P. noxium, shoot length and root length of plants treated with the combined BCAs (TSP) were significantly (p < 0.001) higher than the plants treated with Trichoderma or streptomycetes consortium (There was a significant variation among the treatments in terms of root length ( or TSP) . On the l plants . In the nsortium .P. noxium from the plants grown in the P. noxium treated soil was higher than all the other treatments  (Trichoderma consortium with P. noxium (TP) and streptomycetes consortium with P. noxium (AP). Furthermore, P. noxium re-isolation frequency was slightly higher in treatment with streptomycetes consortium compared to the treatment with Trichoderma consortium.Isolation frequency of and TSP) . The comP. noxium on wood and plants using laboratory and green house assays. P. noxium treated wood blocks in the absence of BCAs showed ~23% of weight loss and possibly cell wall degrading enzymes produced by P. noxium played an important role in the wood degradation [P. noxium ranged from 0.94 to 34.78%, 3.5 to 11.5%, 12.7 and 10.8%, respectively. The variation in the weight loss percentage could be due to the P. noxium strains and the wood species used in the experiments. The wood degradation also correlates with the decline of properties such as tension or bending [P. noxium (P) compared to other treatments.The presented study evaluated the impact of radation . Similarradation  and Burcradation , and the bending ,44. ThisTrichoderma strains (#5001 and #5029) and streptomycetes (#USC−6914 and #USC−595−B) indicates that the BCAs used in this study were effective in preventing the establishment of pathogen on the blocks, thus protecting the wood blocks from weight and strength loss. Similarly, T. virens, T. asperellum. T. koningiopsis, T. harzianum, T. reesei and T. saturniporum used in previous studies were also reported as effective in controlling P. noxium growth and protecting wood blocks [Streptomyces atratus and S. tsukiyonensis were effective against brown rot fungus, Gloeophyllum trabeum, and protected the wood properties [Trichoderma strains (#5001 and #5029) and streptomycetes (#USC-6914 and #USC-595-B) were used together, possibly the combined biological control mechanisms [P. noxium. In addition, these control agents may be used as environmentally friendly wood preservatives over toxic chemicals used in the protection of construction materials.The lowest re-isolation percentage of the pathogen in the presence of d blocks ,4,42. Inoperties . When thchanisms ,50,51,52P. noxium (P) treated plants were significantly reduced compared to other treatments. Similar negative impact by Phythium ultimum, P. aphanidermatum, Phytopthora capsici, F. solani, R. solani, on plant growth parameters of different plant varieties were previously reported in a number of greenhouse experiments [Trichoderma strains (#5001 and #5029) used in this study were effective in protecting the plants from P. noxium infections. Similar results were also previously reported with antagonistic T. asperellum against P. noxium [T. longibrachiatum against Fusarium solani [T. viride against R. solani [Trichoderma inoculants, streptomycetes (#USC-6914 and #USC-595-B) used in this study were also found to be effective in protecting the plants from P. noxium infection. Similarly, antagonistic ability of S. tsusimaensis, S. caviscabies, S. setonii, S. goshikiensis, S. africanus against Fusarium oxysporum [S. philanthi against Rhizoctonia solani, S. sindeneusis isolate #263 and S. globisporus JK-1 against Magnaporthe oryzae [S. cinereus against F. oxysporum, R. solani, Aspergillus niger, Alternaria brassicicola and Phytophthora dresclea [In this study, the growth parameters of the eriments ,22,25,53. noxium , with T.m solani  and with. solani  in greenxysporum , S. phile oryzae  and S. cdresclea  have alsTrichoderma and streptomycetes have different modes of antagonistic mechanisms against plant pathogens and various benefits for the plant growth and development, a number of studies have focused on developing a consortium of Trichoderma and streptomycetes for biological control applications [Trichoderma strains (#5001 and #5029) and streptomycetes (#USC-6914 and #USC-595-B) were inoculated together, and enhanced antagonistic activity was displayed against P. noxium, which resulted in complete inhibition of P. noxium growth. Similarly, a consortium of S. viridosporus and T. harzianum and consortium of T. harzianum and S. rochei used in previous studies showed an enhanced inhibitory effect against Puccinia triticina and Phytophthora capsici, respectively, compared to a single strain application [P. noxium. The presented study clearly showed that consortium of Trichoderma strains (#5001 and #5029) with the streptomycetes (#USC-6914 and #USC-595-B) may be considered as a potential biological control measure to manage the pathogen and protect the plants growing in the P. noxium infested soils. In addition, application of consortia inoculum can also provide a greater protection against genetically diverse P. noxium strains in the field. Further studies are underway to evaluate their biological control efficacy in the field.Since the ications ,26. Micrications ,57,58. Tlication ,19. HoweTrichoderma and streptomycetes is due to the production of plant regulators or production of secondary metabolites which might be associated with solubilisation and mobilisation of micronutrients and mineral ions [The BCAs used in this study were also found to promote the plant growth. The plant growth promotion by ral ions ,61,62,63Trichoderma strains and streptomycetes can be used as an effective way of controlling P. noxium. This can be more effective than the use of individual biocontrol agents in the Brisbane Hinterland and other geographical settings to control similar infections. In addition, the plant growth promoting ability of the BCAs observed in this study also suggests that these BCAs can facilitate the recovery of P. noxium infected plants in soil.The wood decay study and pot experiment results indicate that the application of a consortium of


Schizosaccharomyces pombe, where they regulate intracellular Ca2+ and cell volume under hypo-osmotic conditions. Here we characterize two MscS-like putative channels, named MscA and MscB, from the model filamentous fungus Aspergillus nidulans. Orthologues of MscA and MscB are present in most fungi, including relative plant and animal pathogens. MscA/MscB and other fungal MscS-like proteins share the three transmembrane helices and the extended C-terminal cytosolic domain that form the structural fingerprint of MscS-like channels with at least three additional transmembrane segments than Escherichia coli MscS. We show that MscA and MscB localize in Endoplasmic Reticulum and the Plasma Membrane, respectively, whereas their overexpression leads to increased CaCl2 toxicity or/and reduction of asexual spore formation. Our findings contribute to understanding the role of MscS-like channels in filamentous fungi and relative pathogens.Mechanosensitive ion channels are integral membrane proteins ubiquitously present in bacteria, archaea, and eukarya. They act as molecular sensors of mechanical stress to serve vital functions such as touch, hearing, osmotic pressure, proprioception and balance, while their malfunction is often associated with pathologies. Amongst them, the structurally distinct MscL and MscS channels from bacteria are the most extensively studied. MscS-like channels have been found in plants and  Escherichia coli and form two structurally distinctive families, MscL  for 1–2 h. FM4-64  staining was according to  and then shifted to various conditions [dHrding to . Images http://www.phylogeny.fr/ [http://multalin.toulouse.inra.fr/multalin/ [Phylogenetic analysis of Msc-like proteins was performed by geny.fr/ . Multiplultalin/ .https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb) [https://fungidb.org/). Structure prediction was attempted for both sequences with 1–780 amino acids, as longer amino acids length attempts failed during server processing. S. pompe Msy1 and Msy2, A. thaliana MSL9 and MSL10 and E. coli MscM monomers were previously predicted by AlphaFold, and the PBD files were downloaded from the AlphaFold Protein Structure Database . Previously determined cryo-EM structures of MSL1 (PDB: 6VXM) [E. coli MscS (PDB: 5AJI) [https://www.rcsb.org/). For all the predicted structures, residues with low confidence scores (<70 pLDDT) were not included in our analysis and figure illustrations. The monomer of the E.coli MscS was used as model for structural alignment for the nine proteins included in AlphaFold modeling was performed using Alphafold Colab prediction tools (d.ipynb) , 43. ProB: 6VXM) , FLYC1 (B: 6VXM) , and an Click here for additional data file.


Listeria monocytogenes can cause listeriosis, and people with hypoimmunity such as pregnant women, infants and fetuses are at high risk of invasive infection. Although the incidence of listeriosis is low, the fatality rate is high. Therefore, continual surveillance and rapid epidemiological investigation are crucial for addressing L. monocytogenes. Because of the popularity of next-generation sequencing, obtaining the whole-genome sequence of a bacterium is easy. Several genome-based typing methods are available, and core-genome multilocus sequence typing (cgMLST) is the most recognized methods. Using cgMLST typing to compare L. monocytogenes whole-genome sequences (WGS) with those obtained across distinct regions is beneficial. However, the concern is how to incorporate the powerful cgMLST method into investigations, such as by using source tracing. Herein, we present an easy-to-use web service called–LmTraceMap  that can help public-health professionals rapidly trace closely related isolates worldwide and visually inspect them in search results on a world map with labeled epidemiological data. We expect the proposed service to improve the convenience of public health investigations. Listeria monocytogenes [L. monocytogenes infection are diarrhea, nausea and gastrointestinal disorders [Listeriosis is an infectious disease caused by ytogenes . The cliytogenes . The sevytogenes . The comisorders . Howeverisorders –7. Invasisorders . Althougisorders . Listeriisorders . Historiisorders , 12 causL. monocytogenes, which is a facultative anaerobic gram-positive bacterium that can be cultured from the environment [L. monocytogenes can grow at low temperatures (e.g. -1.5°C) and is tolerant to low humidity and high-temperature (e.g. 72°C), acid, salt, and alcohol conditions [L. monocytogenes can be classified into 14 serotypes according to O and H antigens [The infectious agent of listeriosis is ironment . L. mononditions –17. Thernditions . L. monoantigens , and 1/2antigens .L. monocytogenes WGS with sequencing systems from other countries is beneficial.With the decreasing price of next-generation sequencing systems, public-health researchers are increasingly applying genomic epidemiology methods such as wgSNP , MLST 22, wgMLSTLmTraceMap (http://lmtracemap.cgu.edu.tw/hua_map/test/upload.php) that can provide public-health researchers fast access to compare their L. monocytogenes WGS results with isolates from other countries to track potential sources. A large database containing > 20,000 L. monocytogenes WGS results was created (Lm trace database). The gene-by-gene comparison method [Herein, we propose a platform–n method  was applL. monocytogenes WGS from the NCBI SRA database . The allelic profiling , an open source JavaScript library for constructing web-mapping applications, is used to visualize the tracking results. For email management, the Mailutils general-purpose mail package (https://mailutils.org) is used to automatically send the results through email to inform the user. For browsing the LmTraceMap, we recommend using Chrome, Safari and Firefox browsers to avoid the problem.The web server was constructed using PHP, Python, and Perl scripts and the MySQL database. The Bootstrap framework is used to render the result form downloadable. Leaflet  is required as the input data for initializing the service. Only one genome at a time is available.Users can acquire a test file from our “Input/Output Sample” icon in the upper right corner of our web server to sample how LmTraceMap works. The genome assembly of select file option. Country data must be selected from the pull-down list. A user’s email address is required to receive emailed results. The result page contains 2 tags: MAP . This isolate was collected from regular surveillance, and the whole-genome raw reads were submitted by Statens Serum Institute, Denmark in 2016. We assembled the downloaded raw reads by using SPAdes software [MAP result page is presented in FORM result page is presented in Mom-a. This confirmation is an example of how to use LmTraceMap to trace all of the possible epidemiological related isolates across distinct regions wordwide.For demonstration of our web server, we downloaded an software  and submL. monocytogenes epidemiological data items and cgMLST profiles, and backend data are continually updated. Although only an L. monocytogenes database was created in this study, additional crucial foodborne pathogens could be added in the future.The investigation of foodborne illness clusters is a labor- and time-consuming process. Large-scale comparison among isolates collected from various regions are generally required; therefore, an easy-to-use platform that assists public-health professionals in epidemiological investigations is imperative. Currently, the LmTraceMap system maintains approximately 20 000 S1 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S1 File(CSV)Click here for additional data file.S2 File(ZIP)Click here for additional data file.


Pseudomonas aeruginosa is a bacterial pathogen of global concern and is responsible for 10–15% of nosocomial infections worldwide. This opportunistic bacterial pathogen is known to cause serious complications in immunocompromised patients and is notably the leading cause of morbidity and mortality in patients suffering from cystic fibrosis. Currently, the only line of defense against P. aeruginosa infections is antibiotic treatment. Due to the acquired and adaptive resistance mechanisms of this pathogen, the prevalence of multidrug resistant P. aeruginosa strains has increased, presenting a major problem in healthcare settings. To date, there are no approved licensed vaccines to protect against P. aeruginosa infections, prompting the urgent need alternative treatment options. An alternative to traditional vaccines is vectored immunoprophylaxis (VIP), which utilizes a safe and effective adeno-associated virus (AAV) gene therapy vector to produce sustained levels of therapeutic monoclonal antibodies (mAbs) in vivo from a single intramuscular injection. In this review, we will provide an overview of P. aeruginosa biology and key mechanisms of pathogenesis, discuss current and emerging treatment strategies for P. aeruginosa infections and highlight AAV-VIP as a promising novel therapeutic platform. Pseudomonas aeruginosa (P. aeruginosa) is recognized as a pathogen of global importance and is among the list of ESKAPE pathogens  that exhibit both multidrug resistance and virulence [P. aeruginosa is a nosocomial bacterial pathogen that is particularly prevalent in patients suffering from cystic fibrosis (CF). Roughly 40% of patients with CF have chronic P. aeruginosa infections by the time they reach adulthood [P. aeruginosa is first detected, although successful eradication is variable and not sustained [P. aeruginosa has acquired intrinsic resistance to multiple classes of antibiotics, and there are currently no licensed vaccines for the prevention and treatment of this pathogen, alternative treatment approaches, such as antibody-based strategies, to prevent P. aeruginosa infection and persistence are needed. In this review, we will discuss the history, pathogenesis, and current treatment strategies of P. aeruginosa, emphasize the connection between nosocomial infections and chronic infections in patients with CF, examine the application of antibody therapy for P. aeruginosa infections, and delve into the potential use of adeno-associated virus vector-mediated expression of monoclonal antibodies (mAbs) as an alternative prophylactic platform for long-term passive immunity against P. aeruginosa infections.irulence . Taking irulence . P. aerudulthood , which idulthood . Antibioustained . Since PP. aeruginosa was first observed in 1850 by Sédillot [P. aeruginosa, were described [P. aeruginosa is a ubiquitous microbe that can survive in diverse environments, such as living plants, animals, and humans, due to its robust ability to thrive off minimal nutrients and tolerate harsh conditions. This vigorous survival has led this pathogen to persist in both community and hospital settings. P. aeruginosa has been isolated in communities from swimming pools, hot tubs, home-use humidifiers, soil, and vegetation, and has been found in respiratory therapy equipment, antiseptics, sinks, cleaning equipment, medicine and hydrotherapy pools [P. aeruginosa in nature and in communities, most serious infections with P. aeruginosa are hospital acquired.Sédillot ; howeverpy pools ,9,10. EvP. aeruginosa, are of particular concern due to the low permeability of the bacterial cell wall [A hospital acquired infection (HAI) is defined as an infection a patient develops while receiving medical care that is not associated with the patient’s original diagnosis. In recent years, the financial burden attributed to HAIs in healthcare settings has increased substantially. It is estimated that the overall direct cost of HAIs in the United States ranges from $28 billion to 45 billion USD, annually . While tell wall , and capell wall .P. aeruginosa is an opportunistic pathogen frequently associated with persistence in healthcare settings, given its presence in hospital water, healthcare equipment, and staying power on dry inanimate objects from 6 h to 6 months [P. aeruginosa as the fifth most isolated nosocomial pathogen and the cause for 9% of all nosocomial-acquired infection in the United States [P. aeruginosa [P. aeruginosa on the healthcare system were heavily monitored up until the early 2000s, the majority of the P. aeruginosa reporting since has heavily described the impact of antimicrobial resistance (AMR), thus limiting our knowledge of the true impact of P. aeruginosa infections.6 months . Surveild States . Furtherruginosa . ThroughGammaproteobacteria class, and Pseudomonadaceae family, the Pseudomonas genus is among a class of Gram-negative bacteria containing 302 validly named species . Pseudomonas is one of the largest and most complex Gram-negative bacteria with the first complete genome sequence of strain PA01 being reported in 2000 [Pseudomonas species were reported, with continuous reporting of new species [Belonging to the  in 2000 . From Ma species . Though  species .P. aeruginosa is a Gram-negative, facultative aerobic, non-spore forming and bacillus-shaped bacterium, considered to be saprophytic in nature [P. aeruginosa has a circular genome  containing over 5000 core genes conserved amongst all isolates [P. aeruginosa to acquire genetic elements through horizontal gene transfer  [P. aeruginosa are P. aeruginosa pathogenicity island 1 (PAPI-1) and PAPI-2, which both encode numerous virulence genes and are well characterized in the highly virulent clinical strain PA14 [Pseudomonas to persist in various environments, especially in clinical settings which undergo continuous sanitation [n nature ,20. P. aisolates . These cisolates . Accessoduction) . For exaain PA14 . Furthernitation ,23. Intrnitation . The acqP. aeruginosa pathogenesis. LPS consists of a lipid A domain, a core oligosaccharide and O-antigen, all of which play crucial roles in bacterial physiology and protection against the host defense system [P. aeruginosa consists of a very similar structure to other Gram-negative bacteria, with a cell wall consisting of an inner membrane, peptidoglycan layer and outer membrane [Lipopolysaccharide (LPS) is a structural component of the cell envelope seen in Gram-negative bacteria and is considered a major virulence factor in e system . The maimembrane .P. aeruginosa enables it to cause disease in a variety of organisms including plants, insects, and mammals. P. aeruginosa is of particular concern in hospital settings due to its capacity to colonize and persist on artificial surfaces, posing an extreme risk to elderly and immunocompromised individuals [P. aeruginosa enters the host, it deploys a diverse arsenal of mechanisms that can be exploited for successful infection and evasion of the host immune system. Many gene functions have been shown to contribute to P. aeruginosa pathogenesis, including those involved in biofilm formation, quorum sensing, protein secretion, LPS, outer membrane vesicle (OMV) formation, and bacterial motility [The opportunistic nature of ividuals . This opividuals . Once P.motility .P. aeruginosa facilitates the rapid release of effector toxins resulting in an acute infection, while the sessile lifestyle is attributed to chronic infections through biofilm formation, capable of attaching to both biotic and abiotic surfaces. Both lifestyles are influenced by bacterial motility and are understood to play an important role in the sustained virulence of P. aeruginosa [During infection, LPS induces toll-like receptor 4 (TLR-4) dependent and independent inflammatory responses, and also stimulates host immune responses by activating the complement system and neutrophil extracellular trap formation (NETosis), resulting in programmed cell death and inhibition of bacterial dissemination ,28. The ruginosa .P. aeruginosa OMVs have been linked to the priming of host tissue surfaces for bacterial adhesion, acquisition of drug resistance, immune evasion, as well as the removal of competing bacteria from the environment during an infection [OMVs are bacterial components approximately 10 to 300 nm in diameter that function to deliver a variety of bacterial proteins, lipids, nucleic acids, ion metabolites, and signaling molecules in order to increase virulence capacity . P. aerunfection ,28. InteP. aeruginosa biofilm matrix is comprised of polysaccharides, extracellular DNA, lipids and proteins. Three exopolysaccharides  are produced by P. aeruginosa and play crucial roles in biofilm formation, surface attachment, biofilm stability, and bacterial clearance [P. aeruginosa biofilm development is typically slow growing, as unattached cell aggregates thrive under hypoxic and anoxic conditions. These conditions are commonly seen in the airways of people with CF (pwCF), which may play a role in their heightened susceptibility to P. aeruginosa infections [Biofilm formation is advantageous to the survival and protection of bacteria in the face of host immune responses, antibiotics, as well as other adverse environmental conditions including fluctuations in pH, temperature and nutrient availability . It is ilearance ,27. P. afections . Studiesfections ,29.P. aeruginosa autoinducer signal molecules N-homoserine lactone and N-butyryl homoserine lactone accumulate as the bacterial population increases. Once the intracellular threshold is surpassed, these molecules bind and activate their transcription factors LasR and RhlR, which regulate hundreds of genes [Quorum sensing is the regulation of gene expression in response to fluctuations of cell density within a population . These cof genes . LasR anof genes ,32.P. aeruginosa utilizes a complex set of secretion systems to transport virulence factors from the bacterial cell envelope across the host cell plasma membrane. These processes are critical for bacterial invasion of host tissue sites and manipulation of the host immune responses. Secretion systems function to secrete proteins across the inner and outer membrane of the cell envelope of the phospholipid membrane, contributing to the growth and survival of Gram-negative bacteria [P. aeruginosa pathogens possess six known secretion systems found in Gram-negative bacteria, sometimes with multiple copies per bacterium [P. aeruginosa possesses Type II secretion system (T2SS) and T5SS, which are classified as one-step secretion systems while the T1SS, T3SS, T4SS, and T6SS are classified as two-step secretion systems [To successfully invade the mammalian host cell, bacteria . Exoprotacterium ,33. Secracterium . These sacterium . P. aeru systems ,34. The P. aeruginosa, namely T2SS and T3SS. The T2SS is responsible for the secretion of the most toxic virulent factor, exotoxin A (ETA). This toxin is an ADP-ribosyl transferase, capable of decreasing amino acid uptake and inactivating the eukaryotic elongation factor 2 (eEF-2), thereby inhibiting host protein synthesis [Two protein secretion systems are highlighted as major contributors to virulence in ynthesis ,36. ETA ynthesis .P. aeruginosa infections [P. aeruginosa V-antigen (PcrV), has therapeutic applications as it has been shown to be successful in blocking the release of these effector proteins [The T3SS is highlighted as a contact-dependent protein secretion pathway and is heavily implicated in the pathogenesis of acute fections . A needlfections ,39. ExoSP. aeruginosa has led to this pathogen as one of the most common Gram-negative bacteria causing nosocomial infections and is reported to be responsible for 10–15% of nosocomial infections worldwide [P. aeruginosa can result in a variety of clinical manifestations, such as urinary tract infections, respiratory infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, and gastrointestinal infections [P. aeruginosa [The diverse repertoire of virulence factors utilized by orldwide . Infectifections . These cruginosa .P. aeruginosa. A study conducted by Douglas and colleagues reported that 28% of children with CF acquired pulmonary infections with P. aeruginosa within the first 6 years of life [P. aeruginosa present and increased by eightfold when P. aeruginosa acquired antibiotic resistance [P. aeruginosa is frequently the most dominant organism in CF airways during the end-stage of the disease [P. aeruginosa infections are diagnosed and eradicated prior to the overproduction of polysaccharide alginate, which results in the transition from non-mucoid to mucoid phenotype. The mucoid phenotype is suggested to enhance biofilm formation and is a hallmark of chronic and persistent infections [CF is the most common monogenic disorder in North America, caused by recessive mutations in the CFTR gene . This ge of life . Anothersistance . As P. a disease , it is cfections .P. aeruginosa is an extremely successful pathogen due in part to its ability to adapt and proliferate in niche environments, which can be attributed to its intrinsic and acquired resistance capabilities [P. aeruginosa to support the survival of persister cells, an antibiotic tolerant phenotype of bacteria which serves to repopulate the bacterial population after the course of antibiotic treatment has been completed [P. aeruginosa strains from burn patients, 90% were resistant to at least one antibiotic, while 94% were considered multidrug resistant (MDR) [bilities . Variousompleted . Classicompleted . Adaptivnt (MDR) .P. aeruginosa from the lungs, a combined effort of both host innate and adaptive immune recognition is required to clear infection. Invasion of P. aeruginosa triggers a robust immune response during the acute phase of the infection [In order to effectively eradicate nfection . Althougnfection .P. aeruginosa infections. Upon host recognition, the LPS can activate TLR-4, followed by TLR-5 activation through P. aeruginosa flagella. TLR-9 is activated by unmethylated bacterial CpG DNA, which is suggested to contribute to the sensing of P. aeruginosa bacteria and extracellular-DNA-containing biofilms [P. aeruginosa can also facilitate the release of OMVs. These OMVs can deliver peptidoglycan to nucleotide–binding oligomerization main containing protein-1 (NOD-1) receptors, which can then interact with the receptor interaction protein-2 (RIP-2) adaptor protein to activate NF-κB and mitogen-activated protein kinase pathways in epithelial cells [P. aeruginosa has been found to cause cytotoxicity in macrophages [TLRs are understood to play an important role in the activation of innate immunity in biofilms . P. aerual cells . The bacrophages .P. aeruginosa virulence due to its ability to directly inject bacterial effectors into host cells and mediate bacterial recognition through PRRs, leading to the activation of inflammasomes. In addition to the T3SS effector proteins stimulating an innate immune response, the T3SS needle can activate the NLR Family Card Domain 4 (NLRC4) inflammasome through neuronal apoptosis inhibitory protein (NAIP) recognition, leading to pyroptotic cell death and the secretion of IL-1ß and IL-18 [P. aeruginosa infections, antibodies against T3SS-dependent effector proteins are present, although strains isolated from chronic infections are typically T3SS-negative. This suggests that CF-adapted P. aeruginosa can downregulate T3SS expression in order to enhance immune evasion and promote bacterial persistence, and further suggesting that the T3SS may not play as important a role in virulence during chronic infections as during acute infections [The T3SS plays a major contributor to nd IL-18 . Furtherfections .Pseudomonas lung infections. One caveat to this effective host defense mechanism is that chronic stimulation of neutrophils by bacterial pathogen-associated molecular patterns (PAMPs) and/or cytokines can greatly increase local damage through granulation and inflammation [P. aeruginosa employs numerous mechanisms to evade and eliminate neutrophils, including bacterial adsorption of host sialo-glycoproteins to inhibit degranulation, reactive oxygen species (ROS) production and NETosis. Interestingly, studies have shown that in cases of chronic CF, P. aeruginosa is capable of not only developing resistance to neutrophil effector mechanisms, but also using neutrophils to promote biofilm growth in CF airways. Extracellular DNA (eDNA) obtained from necrotic neutrophils has been shown to support the growth of P. aeruginosa aggregates, which is required for biofilm formation [Neutrophil recruitment is considered a vital initial innate immune response that is commonly seen in chronic ammation . Overstiormation .P. aeruginosa pathogenesis [h)1 and Th2 cells are typically skewed, thereby impairing or completely inhibiting bacterial clearance [h2 marker IL-4 were significantly higher in chronically infected patients, as opposed to non-chronically infected patients who showed elevated levels of IFN-γ, produced by macrophages and Th1 helper cells [P. aeruginosa infections can skew the immune response in favor of the pathogen.In addition to host innate immunity, adaptive immunity is commonly seen in the presence of biofilm formation, and plays critical roles in ogenesis . In bactlearance . A studyer cells . This suP. aeruginosa infections, this opportunistic pathogen is capable of quickly adapting to its environment, which presents a major problem in nosocomial settings where previously effective treatment strategies are becoming less effective with the rise of antimicrobial resistance.Despite a wide array of host immune defense mechanisms that are understood to play a role in the clearance of P. aeruginosa infections is antibiotic therapy. Known risk factors and the progression of infection both play vital roles in treatment plan development and should be evaluated on a case-by-case basis. Typically, if infection is severe, empirical antibiotic treatment will be started, which should include two different classes of antibiotics to increase the likelihood of effective treatment, especially with the rise in antimicrobial resistance. Typically, treatment relies on in vitro susceptibility testing, and should be tailored accordingly whenever possible [To date, the only approved treatment strategy for possible . CombinaP. aeruginosa infections [P. aeruginosa infections  type efflux pumps that play crucial roles in the reduction of antibiotic susceptibility. These MDR efflux pumps have also been shown to play a role in the acquisition of MDR [Six bacterial pathogens possessing multidrug resistance and extreme virulence have been termed ‘ESKAPE’ pathogens—an acronym for ter spp. . These pneumonia . Recentleumoniae ,61. Varipatients . P. aerun of MDR . Studiesn of MDR .P. aeruginosa are quickly becoming a major concern in clinical settings, especially due to the increasing development of multidrug resistant (MDR) strains. Intrinsic resistance to antimicrobials including B-lactam and –penem group of antibiotics is attributed to the low permeability of its outer membrane. Upregulation of efflux pump systems and intrinsic production of antibiotic inactivating enzymes are also mechanisms utilized by this opportunistic pathogen to confer intrinsic drug resistance [P. aeruginosa strains from burn patients, 90% were resistant to at least one antibiotic, while 94% were multidrug resistant [Antimicrobial resistant strains of sistance . Classicsistance . Adaptivesistant .P. aeruginosa has been proven difficult to treat. A total of three vaccines against P. aeruginosa had been evaluated in Phase III clinical trials, none of which were deemed successful [P. aeruginosa pathogenesis have been explored, the high mutation and variability rate in targets such as LPS and flagellum present a major obstacle in vaccine design. The development of novel antibiotics and alternative therapeutics is extremely slow, in part due to the complexity of drug resistance, as well as certain roles in P. aeruginosa pathogenesis, which remain unclear [Despite the urgent need for effective therapeutics, ccessful . While n unclear . This baP. aeruginosa infections involved the infusion of anti-pseudomonas immune plasma to granulocytopenia dogs in 1976 [P. aeruginosa that can be adapted for human use. An extremely potent and clear candidate for pathogenic targeting is the PcrV protein, which is located at the tip of the T3SS and responsible for the transportation of cytotoxic effector proteins into the host cytoplasm [P. aeruginosa [The first attempt at passive immunization for treatment against  in 1976 . Currentytoplasm . Generatytoplasm . In an aruginosa . Adminisruginosa .P. aeruginosa mAbs have shown promise in animal models, they are limited therapeutically due to their potential immunogenicity in humans [P. aeruginosa have been identified through high-throughput screening of B cells from convalescent patients. Lymphocytes from healthy volunteers and CF patients were transformed and cultured to generate lymphoblastoid cell lines secreting human monoclonal antibodies MAB-RM5, MAB-FDD7 and MAB-11F9 against LPS from P. aeruginosa [P. aeruginosa immunotypes 2, 4 and 5, whereas MAB-9H10 provided protection against immunotypes 3 and 6 [P. aeruginosa serotype 5 was generated by cell fusion between human tonsillar lymphocytes and a mouse plasmacytoma cell P3-X63-Ag8-U1. This mAb, termed mAb P3D9, was shown to be highly protective in a mouse model experiment with a 50% protective dose (PD50) of 2.4 µg/mouse [P. aeruginosa and identified mAb F429γ1 that was able to induce phagocyte dependent- killing and provide protection against lethal P. aeruginosa pneumonia challenge with both mucoid and nonmucoid strains in a murine model [P. aeruginosa infections for OPK and cell attachment interference and identified Cam-003 [P. aeruginosa infection, including pneumonia, thermal injury, and ocular keratitis. The anti-PcrV human mAb, V2L2MD, demonstrated good prophylactic protection in several models of P. aeruginosa infection as well as in a post infection therapeutic model [p < 0.0001) better in vivo protection than that provided by another potent anti-PcrV murine mAb, mAb166 [While murine anti-n humans . Alternaruginosa . These m 3 and 6 . The spe 3 and 6 . Anotherµg/mouse . A studyne model . A study Cam-003 . This huic model . Interes, mAb166 .P. aeruginosa, combining the single-chain variable fragments (scFvs) of both anti-Psl and anti-PcrV mAbs. MEDI3902 has been shown to be efficacious in the prevention and treatment of P. aeruginosa infections in vivo. Treatment with MEDI3902 has shown decreased bacterial burden and decreased expression of pro-inflammatory mediators in lung tissue of multiple animal models [The concept of a bispecific antibody (bsAb) was first described by Nisonoff and colleagues in the 1960s, although it was not until 1996 that platforms for the generation of bispecific antibodies were developed . Bispecil models .P. aeruginosa mAbs have been evaluated in clinical trials, with none currently approved for the market  [To date, a total of six e market . Past anbo group ,84,85. Aia (HAP) . An IgY ia (HAP) . Phase Iia (HAP) .P. aeruginosa infections [P. aeruginosa clinical isolates demonstrated that between 97.3–100% of isolates expressed either one or both targets [P. aeruginosa infections in a nosocomial setting. MEDI3902 was found to be safe in a Phase 1 dose escalation study in healthy subjects [P. aeruginosa pneumonia [P. aeruginosa infections.One bispecific hIgG antibody MEDI3902) holds great promise due to its antigenic binding affinity to the potent PcrV protein, and the abundantly expressed Psl exopolysaccharide that is crucial for biofilm formation in fections . MEDI390fections . A geogr targets , therefosubjects , and was2 holds gneumonia . Furthertrans, allowing for production of replication defective AAV particles.AAV gene therapy vectors are currently the most promising in vivo gene delivery vehicles. AAV has gained significant traction as a gene therapy vector due to its lack of pathogenicity, low-level genomic integration, and favorable delivery and sustained transgene expressing properties . To dateAAV-vectored expression of highly potent, pathogen-specific mAbs is a promising method to enable long-term expression of therapeutic antibodies in vivo following intramuscular (IM) injection. The AAV genome is engineered to express the heavy and light chain domains of the mAb as one polyprotein separated by a 2A self-processing peptide sequence  that conC. difficile [Although the initial application of VIP was directed towards prevention of HIV, this strategy has now been tested against an ever-expanding list of infectious diseases, including more recently, bacterial infections like ifficile . Based o12 vg to 1.2 × 1014 vg of rAAV1-PG9DP intramuscularly. No serious adverse events were noted, with only a singular mild adverse event attributed to the scheduled treatment regimens. PG9 mRNA transcripts were detectable in muscle biopsies as was IgG expression within muscle cells and extracellular tissues via immunohistochemistry. However, serum concentrations of PG9 were below the limit of detection of the ELISA, which was 2.5 µg/mL. Anti-drug antibody (ADA) responses were detected in many of the participants, particularly in the high dose group, which likely contributed to the lack of PG9 in the serum [Numerous advanced-stage clinical trials are currently ongoing to evaluate the efficacy of AAV-therapies for a variety of genetic disorders, including alpha-1 antitrypsin deficiency, choroideremia, and hemophilia. Along with these clinical trials are two AAV-mAb platforms that are being evaluated for safety and efficacy with respect to prevention or treatment of HIV infections. The first clinical trial (NCT01937455) evaluating AAV-mediated antibody gene delivery for the prevention of HIV infection utilized an AAV1 vector expressing the broadly neutralizing anti-HIV antibody (bnAb) PG9 , that hahe serum . Further10 to 2.5 × 1012 vg/kg of AAV8-VRC07 via intramuscular injection. For this study, 50 μg/mL of VRC07 was chosen as the target serum concentration based on previous data in mice and NHPs [A second Phase I AAV IP clinical trial (NCT03374202) evaluating AAV-mAb expression, conducted by David Baltimore and colleagues at the Ragon Institute of MIT and Harvard and Massachusetts General Hospital, evaluated the safety and efficacy of AAV8-mediated VRC07 expression in HIV-infected adults (ages 18–60) . In thisand NHPs , and theand NHPs . No adveand NHPs . This isAAV-mediated expression of mAbs either as a prophylactic or therapeutic provides an alternative to traditional vaccines or passive mAb therapies, respectively. Development of such AAV-mAb therapies can begin as soon as sequences for pathogen-specific neutralizing mAbs become available and may be an ideal stop gap measure for intervention in outbreaks where a vaccine is unavailable and could potentially be used in combination with a traditional vaccine to combine the benefits of both vaccination strategies. Through a singular administration, AAV-mAb potentially provides a longer-lasting therapeutic level of mAbs compared to passive administration. Since AAV-mAb expression leads to production of protective mAbs independent of the immune system, it could be utilized in people who do not respond to traditional vaccines or whose immune system is functioning poorly, including immunocompromised or elderly individuals The AAV capsid can be swapped or modified to target specific cell types and tissues, or to evade pre-existing immunity to the capsid . Lastly,P. aeruginosa infections, hurdles to this approach still exist. Prior exposure to wild-type AAV can lead to the development of both humoral and T cell immunity that may result in neutralization of the vector or immune-mediated clearance of vector transduced cells, which can affect the duration of transgene expression. De novo immune responses to the administered AAV vector represent another impediment to the success of AAV VIP [While AAV VIP therapy holds great promise as an alternative or adjunctive therapeutic for  AAV VIP . TLR2 an AAV VIP ,123. How AAV VIP ,125. For AAV VIP ,127 and  AAV VIP . With AAAs previously mentioned, ADA responses have been documented in NHP studies and human clinical trials, which can be attributed to vector design and the immunogenicity of the transgene product itself . The cavFinally, it may be necessary to implement genomic safety measures that can excise or invert sections of the AAV genome upon transient drug administration to turn off AAV-mAb expression should long-term mAb expression be undesirable ,132,133.P. aeruginosa is a clinically relevant bacterial pathogen associated with nosocomial infections, high infection rates in immunocompromised individuals, and chronic infections in CF patients. The stealth virulence factors utilized by P. aeruginosa, in addition to increasing antimicrobial resistance, have resulted in a pathogen of public health concern lacking an effective vaccine.Since the introduction AAV-VIP in the early 2000s , there hP. aeruginosa at multiple pathogenic epitopes from a single product.The field of antibody engineering has resulted in an ever-increasing number of designs for improved therapeutic antibody expression. Multidomain antibodies, such as diabodies or bispecific mAbs, allow for variable domains of multiple potent antibodies to be structurally bound together, for binding and neutralizing of multiple epitopes from the same protein ,137. TheC. difficile toxin-specific antibodies protects mice from toxin challenge demonstrating the potential of AAV VIP as an alternative or adjunctive treatment for recurring bacterial infections [P. aeruginosa-specific mAbs with AAV provides an additional tool in the currently limited arsenal of P. aeruginosa treatments. Here, we delve into the use of VIP for prevention and treatment of P. aeruginosa; however, this platform could be applied to other bacterial infections where effective treatments remain elusive. With the recent FDA approvals of Luxturna and Zolgensma, AAV gene therapies are rapidly becoming tools for mainstream medicine, which holds great promise for the field of VIP.We previously demonstrated AAV-mediated expression of fections . The cou


Escherichia coli and necrotic enteritis due to Clostridium perfringens remain major problems for poultry producers, despite substantial efforts in prevention and control. Antibiotics have been used not only for the treatment and prevention of such diseases, but also for growth promotion. Consequently, these practices have been linked to the selection and spread of antimicrobial resistant bacteria which constitute a significant global threat to humans, animals, and the environment. To break down the antimicrobial resistance (AMR), poultry producers are restricting the antimicrobial use (AMU) while adopting the antibiotic-free (ABF) and organic production practices to satisfy consumers’ demands. However, it is not well understood how ABF and organic poultry production practices influence AMR profiles in the poultry gut microbiome. Various Gram-negative  and Gram-positive  bacteria harboring multiple AMR determinants have been reported in poultry including organically- and ABF-raised chickens. In this review, we discussed major poultry production systems  and their impacts on AMR in some potential pathogenic Gram-negative and Gram-positive bacteria which could allow identifying issues and opportunities to develop efficient and safe production practices in controlling pathogens.Economic losses and market constraints caused by bacterial diseases such as colibacillosis due to avian pathogenic  Poultry meat is an important animal protein and one of the most popular meat consumed by humans worldwide. Its consumption is projected to increase 17.8% by 2030 according to the OECD-FAO; the highest increase among all types of animal meats .Lactobacillus, Clostridium, Ruminococcus, Salmonella enterica serovars, Enterococcus spp., and E. coli inhabit the GIT to constitute the gut microbiota. Through horizontal gene transfer of mobile genetic elements such as transposons and plasmids, the gut microbiota can be a reservoir for antimicrobial resistance genes (ARGs). The addition of antibiotics to poultry diets can modulate the gut microbiota by decreasing the pathogenic bacteria load, increased the intestinal nutrient absorption, and ultimately improved growth parameters [The gastrointestinal tract (GIT) plays a crucial role in poultry health as it provides the first-line of defense against foreign pathogens while also allowing nutrient absorption . In addirameters . Thus, irameters , 8.C. perfringens is one of the major poultry diseases costing $6 billion per year to the global poultry industry [C. perfringens give bacteria access to the bile duct and blood stream, consequently damaging additional organs in birds [Eimeria spp. parasites which, invade and replicate in the intestine [Eimeria infections have also been associated with the promotion of NE [Necrotic enteritis (NE) caused by a Gram-positive anaerobic spore-forming bacterium industry . This baindustry . Sub-cliindustry . Intestiin birds . Typicalin birds . Coccidintestine . This pantestine , 14. Eimon of NE , 16.In poultry production, the type of feeding program is extremely important to ensure nutrient and health requirements are met. Feeding programs are selected based on cost efficiency, effectiveness to improve health and growth. Conventional feeding programs relied on AMU to improve growth performance while simultaneously preventing infectious diseases. However, increasing concerns over AMR resulted in the development and adoption of alternative productions known as antibiotic-free (ABF) productions, also called no antibiotic ever (NAE) or raised without antibiotic (RWA) and organic productions. Harmonization of the definitions used for the terms ABF, NAE and RWA is necessary, so “ABF” will be used for consistency in this review. Research on the effectiveness of alternative feeding programs to reduce AMR is needed to identify best production practices in preserving gut heath –19. SubsIn this review, we will discuss the significance of conventional, ABF and organic poultry productions by outlining the AMU concerns and the use of alternatives to antibiotics. Moreover, a summarization of some important antimicrobial agents and alternative products in poultry production is presented to highlight pathogenic bacteria of concern and opportunity for improvements for their efficient control while highlighting that AMR issues should be addressed by a “One Health” approach.Feed is a significant component in poultry production. Feed quality, nutrient composition, and consumption rate by birds are critical parameters to monitor for their health and productivity. Nutrients of poultry feed can be categorized into five different groups: carbohydrates, lipids, proteins, minerals and vitamins. High quantity and quality water is also essential. Each feed ingredient plays a vital role in either, energy acquisition and utilization, metabolism or health of poultry. For several years, sub-therapeutic levels of antibiotics have been used in broiler feed to maximize their productivity . This prConventional production practices were widely adopted to shape the livestock industry to what it is today. One key difference between conventional production and ABF or organic production is the use of antibiotics in healthy animal during conventional production. Justifications of antibiotics used in poultry production include growth promotion and prevention of important diseases. The World Health Organization (WHO) created a global critically important antibiotics (CIA) list that categorize antibiotics into three different classes based on their importance in human medicine; important, highly important, and critically important . In addiSalmonella, Campylobacter and C. perfringens [Campylobacter spp. than conventional broiler carcasses, possibly due to the organic bird’s free access to pasture where they could be more exposured to environmental of bacterial pathogens [Campylobacter illnesses following consumption of contaminated organic broiler meat, compared to conventional broiler meat in Denmark. These reports on organic poultry production indicate that investigation are needed to develop cost-efficient methods to improve the gut health, reduce risks to consumers, and minimize negative impacts of production on the environment.Organic production typically raise animals naturally while maintaining optimal health, welfare and living conditions. More and more poultry producers are opting for organic production due to the sustainability and harmony with the environment. Each country has their own standards and regulations of organic production systems, such as the United States Department of Agriculture’s National Organic Program and the Standards Council of Canada’s CAN/CGSB-32-Organic Production Systems , 24. Desfringens , which afringens , 27. Howfringens . Furtherfringens . Colonizathogens . These aAntibiotic-free production is similar to conventional production, with the exception of AMU as a prophylactic and for growth promotion. Thus, the potential health and production issues in ABF production requires alternative solutions Section . ConsumeMany different alternatives to antibiotics have been investigated in poultry production at an attempt to replicate their multifunctions. A list of alternatives investigated and/or used in poultry production include probiotics, prebiotics, organic acids, phytochemicals, vaccines, in-feed enzymes, and essential oils Table . A descrLactobacillus spp. , Enterococcus faecium and E. faecalis, Streptococcus thermophilus, and Bacillus subtilis in poultry. They function by interfering with the colonization of the gut by pathogenic bacteria through competitive exclusion [+, B cells, and anti-inflammatory cytokine production [E. coli and Salmonella spp. [B. subtilis was found to improve hen’s performance and egg quality at a lower dose while improving the protein quality in the eggs at a high dose [They are live microbial feed additives to help maintain intestinal microbial balance and benefit the host’s health –37. Mostxclusion –37. Moreoduction , 56. Prooduction . Howeveroduction . Probiotigh dose . In contigh dose  did not Bifidobacterium and other lactic acid bacteria. The prebiotic FOS indirectly alters the gut microbiota community through increased production of short-chain fatty acids (SCFA), some of which favour fermentation. The SCFAs are important in the GIT and immune function but, they also elicit acid stress to pathogenic bacteria [E. coli and Salmonella to intestinal cells by binding to their flagella, which are important in their motility and attachment to intestinal epithelial cells [They are carbohydrate-based polymers such as fructooligosaccharides (FOS), galactooligosaccharides, and mannanoligosaccharides (MOS) that function to promote beneficial bacteria in the gut, aid in digestion, and inhibit colonization by pathogenic bacteria . Prebiotbacteria , 38. Howal cells , indicatal cells .Salmonella pathogenicity islands which are important virulence-encoding genes in Salmonella. However, lactate has been identified to fuel Salmonella growth due to the utilization of the lactate-degrading respiratory lactate dehydrogenases LldD (converts L-lactate into pyruvate) and Dld (oxidates D-lactate to quinones) [barA gene function in mutant S. typhimurium barA−; this gene encodes for the BarA sensor kinase important in the interaction with SirA to shift Salmonella from mobility to virulence [These compounds have acquired a favourable reputation in the poultry industry due to their strong nutritional and antimicrobial properties. Moreover, organic acids have already been applied for feed preservation and performance enhancement in livestock production. These compounds have a carboxylic acid (R-COOH) group in common, in the simple monocarboxylic , hydroxyl , or short-chain  form . They acuinones) . Moreoveirulence .Lactobacillus population in LBBP-fed birds were more abundant than those fed a control diet from 21 to 42 days old birds. Moreover, Das et al. [The immune-stimulatory potential of fruit products in poultry have been recently reviewed . Berry fs et al.  reporteds et al. .E. coli while improving the intestinal digestibility, the overall performance and the meat quality in broilers [They are known to have antioxidant and antimicrobial properties . With thbroilers , 62. Brobroilers .These biological catalysts are typically administered to assist in digestion of certain feedstuffs . EnzymesSeveral health and economic challenges arise when implementing ABF productions using the alternative products. Despite promised beneficial activities, the efficacy of antibiotic alternatives is quite variable by study. It is hypothesized that combination of these individual alternatives may provide optimal activities. This probably could explain in the decrease of overall performance  with a single alternative product when compared to traditional antibiotics , 19. ReqAntimicrobials significantly contribute to the treatment and prevention of infectious diseases, the improvement of poultry performance, and overall yield –69. HoweblaCMY-2 genes harboring Salmonella enterica and E. coli from infected humans were positively correlated to ceftiofur-resistant Salmonella and E. coli from chicken meat [Several studies reported links between AMU in food animal production and the prevalence of AMR in bacteria , 78. Theken meat . Action ken meat . Due to ken meat , 82.Typical conventional broilers are raised in barns from hatching to 36–42 d during which each bird can consume 3.2 to 4.0 kg of feed to reach a body weight of about 1.8 to 2.2 kg . ConventStreptomyces viridochromogenes, targeting Gram-positive bacteria such as C. perfringens to prevent NE in broiler chickens [Enterococcus faecium have been reported in broiler fecal from several farms in Denmark and France [Is an orthosomycin antibiotic from chickens . It has chickens , 68. Avichickens . Avilamychickens , 86. Reschickens , 88. Vard France , 90.Streptomyces including S. bambergiensis and S. ghanaensis is not categorized in the WHO/CIA list as important in human medicine, but used in poultry production [Is also known as flavomycin, flavophospholipol or moenomycin. This phosphoglycolipid antibiotic originating from various strains of oduction . Targetioduction , 92. ResBacillus licheniformis and B. subtilis strains is categorized as important in the WHO/CIA list [55-isoprenyl pyrophosphate which is a lipid carrier involved in bacterial peptidoglycan synthesis [55-isoprenyl pyrophosphate results in the prevention of transport of N-acetylglucosamine (NAG) and β-(1–4)-N-acetylmuramic acid (NAM) to build the peptidoglycan wall [bcrR gene encoding a unique membrane-bound one-component system through a putative ATP-binding cassette (bcrAB) transporter [This cyclic polypeptide antibiotic produced by CIA list . Primariynthesis , 94. Depcan wall . Bacitracan wall , 96. Bacnsporter , 98.Streptomyces albus, S. aureofaciens, and S. cinnamonensis, respectively. Salinomycin use dates back to its discovery in 1974, demonstrating effectiveness against Gram-positive bacteria and coccidiosis [Eimeria spp. by disrupting their osmoregulation [Salinomycin, narasin, and monensin belonged to this group and are unclassified in the WHO/CIA list . They arcidiosis . Salinomcidiosis . Ionophogulation . The mecgulation .Staphylococcus aureus cells and are used to treat humans [S. enterica serovar Heidelberg showed resistance to the third-generation cephalosporin ceftiofur  and ceftriaxone  [Antibiotics and their alternatives on livestock or poultry may have complex direct or indirect mechanism of actions . Antibiot humans , 105. Isn human) . This ran human) , 107.CATH2, MPO, and IL-5 genes respectively, in immune organs of bacitracin-fed birds compared to a control diet . A positive association between consumption of antibiotics in poultry and corresponding antibiotic resistance in some bacteria has been reported in Europe [E. coli, Salmonella spp., and Campylobacter spp. reported worldwide in poultry is presented in Fig. Various studies have been conducted on the AMU in food animals and their consequences on AMR in foodborne bacteria , 109–111ed Table . Antibion Europe . It is cE. coli, Salmonella Pullorum/Gallinarum, Gallibacterium anatis, Pasteurella multocida, Klebsiella spp.) and foodborne illness in human (S. enterica serovars and Campylobacter jejuni/coli). The complexity of the Gram-negative bacterial cell surface provides intrinsic resistance against various antibiotics [A wide variety of Gram-negative bacteria can cause diseases in poultry  strains are epidemiologically and phylogenetically distinct from both intestinal pathogenic and commensal strains [E. coli (APEC) is an ExPEC responsible for significant economic losses in the poultry industry [ factors . The ext strains . Avian pindustry  and was industry .E. coli [E. coli infections. Multiple resistance to amoxicillin–clavulanic acid, ceftiofur, ceftriaxone, cefoxitin, gentamicin, sulfonamide and tetracycline in commensal and APEC isolates have been documented in poultry [blaTEM, blaSHV, blaCMY-2, aac (3)-Via, aadA1, aph (3)-Ib, aph (6)-Id, sul1, sul2, tet(A) and tet(B), were observed in corresponding resistance phenotypes. Mobile genetic elements including pAPEC-O2-R, IncA/C2 and IncI1 plasmids as well as Class I integrons carrying antibiotic resistance genes tet(A), sul1, and blaTEM able to be transferred to a recipient bacterium have been also observed in E. coli from broilers [E. coli isolates than those from the non-medicated feeds [sul1, aadA, and Class 1 integrons in salinomycin-fed chickens than in control or other treatment groups [E. coli isolates harboring corresponding genes such as blaCMY-2, blaTEM, tetB, sul1, and aadA [E. coli harboring tet(A) or tet(B) increased [E. coli and Klebsiella pneumoniae were reported in local and imported chicken meat [E. fergusonii harboring resistance to ampicillin, streptomycin and tetracycline were isolated, but the antibiotic usage from the studied farm was unknown [E. fergusonii isolates tested contained the β-lactam (blaCMY-2), aminoglycoside , trimethoprim , tetracycline (tet(A), tet(B), tet(C), tet(E)), and sulfonamide  resistance genes [In conventional poultry production, AMU has been correlated to the increased prevalence of AMR in E. coli , 154. An poultry , 154. Anbroilers . Dietaryed feeds . Interest groups . Regardland aadA . Oral adncreased . Extendeken meat . From co unknown . These ace genes .E. coli was found to be lower in organic and ABF broilers compared to conventionally produced ones [E. coli isolated from organic, ABF and conventional production systems demonstrated high frequencies of resistance (> 50%) to ampicillin, cefazolin, sulfonamides, streptomycin and tetracycline [E. coli isolates from conventional poultry meat were more MDR than those from organic poultry meat [E. coli and Lactobacilli can survive in acidic environments due to their innate acid resistance properties [E. coli in chicken and turkeys, although differences were noted in specific antibiotic resistant phenotypes. Moreover, a higher prevalence of resistant E. coli to ampicillin, ampicillin-sulbactam, cefazolin, cefoxitin, ceftriaxone, and trimethoprim-sulfamethoxazole was found in conventionally raised turkeys compared to organic and RWA produced ones [E. coli when fed conventional and ABF, respectively [E. coli harboring blaCTX-M-1 and blaCMY-2 genes have been isolated from commercial organic broilers [As organic and ABF poultry production systems are becoming popular, their efficacy to reduce AMR deserves investigations. Recently, the prevalence of antimicrobial resistant commensal ced ones . Howeveracycline . It has try meat . Even thoperties . Davis eoperties  reportedced ones . Howeverced ones  reportedectively . In the broilers .aadA1, aadA2, strA), β-lactams , tetracycline (tet(A), tet(B)) and sulfonamides (sul1) were detected in these Salmonella isolates [fosA7, was identified in S. enterica serovar Heidelberg isolated from conventional broilers [Salmonella isolated from retail raw poultry meats were MDR to at least three different classes of antimicrobials, which included nalidixic acid, ampicillin and streptomycin [S. enterica serovar Indiana [S. enteritidis known as SE86, a frequently identified poultry Salmonella isolate in Brazil associated with foodborne outbreaks, has been reported to be resistant to ciprofloxacin (41.9%) and sulfafurazole (75%) [S. enteritidis (SE_TAU19) resistant to nalidixic acid and sulfadimethaxine was reported [mcr-1 gene (colistin resistance) in E. coli and S. enterica from poultry and swine. From commercial poultry farms, Liljebjelke et al. [Salmonella resistant to streptomycin, gentamycin, sulfadimethaxine, trimethoprim, and tetracycline. A recent report from Europe indicated no associations between consumption of cephalosporins and quinolone in poultry and resistance to these antibiotics in Salmonella isolates from humans [In food production animals, NTS induces diarrhea with fever, anorexia, and dehydration. However, poultry can be asymptomatic carriers of these pathogens and exposure to antimicrobials could promote AMR isolates that may be transmitted to humans , 159. Itisolates . Fosfomybroilers . In Chinptomycin . Co-resi Indiana . A clonale (75%) . A persireported . Quesadareported  found the et al.  reportedm humans .Salmonella have been conducted in organic and ABF poultry production systems [Salmonella isolates from large-scale organic poultry production farms was significantly lower than isolates from conventional broiler production [Salmonella isolates were found in ABF broiler than in those from conventional production [Salmonella isolated from US conventional retail poultry meat showed 2.6 times higher resistance prevalence compared to those from organic retail meats [A few studies on AMR  systems , 164. Thoduction , 163. Inoduction . SalmoneCampylobacter spp. (C. jejuni and C. coli) are important foodborne pathogenic bacteria associated with poultry. These bacteria are microaerophilic and certain environmental stresses such as exposure to air, drying, low pH, and prolonged storage can be detrimental to their survival. In humans, these pathogens cause a self-limiting diarrheal disease from improperly prepared or contaminated food, including poultry products. In the United States, Campylobacter are responsible for an estimated 2 million cases of gastroenteritis annually. Antibiotics belonging to the macrolides (erythromycin), fluoroquinolone, tetracyclines and aminoglycosides (gentamicin) classes are used against Campylobacteriosis [Campylobacter isolates from chicken could be originated from upstream [Campylobacter isolated in British Columbia, while those isolated in Quebec and Ontario provinces were predominantly resistant to tetracyclines, macrolides, ketolides, and lincosamides [Campylobacter from poultry led to the restriction or ban of sarafloxacin and enrofloxacin used in poultry [Campylobacter spp. isolated from conventionally-raised broiler chickens [Campylobacter spp. isolated from turkeys were resistant to over seven antimicrobials [Campylobacter and the use of macrolides along with a trend of increasing prevalence of erm(B) gene in isolates were observed in poultry [teriosis . Despiteupstream . These aosamides . The eme poultry . An extrchickens . Moreovecrobials . Correla poultry , 171.Campylobacter in ABF and organic poultry productions. However, a study reported a significantly lower fluoroquinolone resistant Campylobacter prevalence (< 2%) in organic than in conventional (46%) poultry farms [Campylobacter isolates from organic (n = 77) and conventional (n = 80) chickens showed that all organic isolates were sensitive to all antibiotics, except two that were resistant to tetracycline, while resistance to quinolones and tetracycline were observed among the 80 isolates from conventional chickens [Campylobacter in poultry products [Salmonella, Escherichia coli, and Campylobacter) showed the potential for progressive transitions from conventional to antibiotic-free broiler production [Limited studies investigated the presence of antibiotic resistant ry farms . Susceptchickens . Despiteproducts . Howeveroduction . The aboEnterococcus spp., Staphylococcus spp., and C. perfringens are common in poultry and can be commensal or pathogenic. According to a meta-analysis performed by Cardinal et al. [S. aureus (MRSA), vancomycin-resistant S. aureus (VRSA), MDR Streptococcus pneumoniae, and vancomycin-resistant E. faecium (VRE). Several MDR Gram-positive bacteria have been isolated from conventional, organic and ABF poultry productions [Gram-positive bacteria including l et al. , the mosl et al. . Exampleductions , 177.Micrococcus and fecal streptococci more than 113 years ago [Enterococcus genus (more than 40 species). Enterococcus spp. particularly E. faecalis, E. faecium and E. cecorum have been associated with diseases in both human and poultry [E. faecium only). Newer antibiotics  or older antibiotics  have been also considered to fight against Enterococcus. However, these bacteria are characterised by intrinsic resistance to important antibiotic classes and to tolerate low concentrations of β-lactams, quinolones, aminoglycosides, and lincosamides, as well as being able to metabolise preformed folic acid (trimethoprim and sulphonamides). Furthermore, Enterococci have developed a high ability to acquire exogenous resistance genes via conjugative transposons and plasmids [Enterococci isolates and a potential zoonotic transmission of AMR isolates has been suggested [E. faecium was reported in a surveillance study conducted by Aarestrup et al. [E. faecium in humans and the use of virginiamycin [E. faecium and avilamycin use during broiler production [E. faecium has also been reported to be cross-resistance to evernimicin [E. faecium isolates contained the emtA gene encoding a methyltransferase which inhibits avilamycin and evernimicin function [emtA positive E. faecium has also been found to harbor vancomycin, gentamicin, tetracyclines, and erythromycin and streptogramin resistance genes in chickens [Enterococcus faecium isolated from broiler chickens treated with virginiamycin demonstrated resistance to quinupristin-dalfopristin, supporting previous observations on the induction of quinupristin-dalfopristin resistance from the use of virginiamycin [E. faecium strains were isolated from broilers fed bambermycin, penicillin, salinomycin, bacitracin, or a salinomycin/bacitracin combination [E. faecium and E. faecalis isolates showing resistance phenotypes and genotypes against bacitracin, erythromycin, tylosin, lincomycin, streptomycin, gentamycin, tetracycline and ciprofloxacin were reported in commercial broiler [They were initially described as ears ago  belongin poultry , 180. Thplasmids . In convuggested , 182. Asp et al. . Subsequniamycin . A case-oduction . Avilamyrnimicin , 105 andrnimicin . All avifunction , 184. Suchickens . Enterocniamycin , 186. Ci broiler .Enterococcus spp. from organic chickens compared to conventional chickens. Moreover, prevalence of MDR Enterococcus spp. was higher in conventional chickens compared to organic chickens [Enterococcus spp. but less AMR Enterococcus spp. in organic chicken compared to conventional chickens, predominantly showing resistance to streptomycin and erythromycin.As there are a limited number of studies that investigated resistance profiles of important Gram-positive bacteria in organic and ABF poultry production, it is imperative to broaden this topic of research. Miranda et al.  reportedchickens . In Soutchickens . Intereschickens  reportedStaphylococci are widespread in nature and comprise of coagulase-positive and coagulase-negative species able to induce minor and major infections in poultry and human [Staphylococcus aureus can cause infections such as omphalitis, pneumonia and arthritis [S. aureus infections becomes difficult due to the emergency of multiple antibiotic resistant isolates including MRSA resulting from AMU in both animal and human. In Belgium, MRSA also resistant to antimicrobials including tylosin, amoxicillin, trimethoprim-sulfamethoxazole, lincomycin, tetracycline, and colistin were isolated from broiler [S. aureus strains have been reported in different conventional broiler production operations in Korea along with four MRSA isolates from three different operations [S. aureus from poultry and their products using the “Farm to Fork” approach showed a prevalence of 31.25% (n = 120/384) of S. aureus in analyzed samples: farm (40), transport (15), abattoir (30), and retail point (35) [S. aureus isolated from poultry demonstrated increased resistance against antibiotics compared to S. aureus isolates from 1970s [S. aureus strains in farm could contaminate chicken meat during processing. Accordingly, MDR S. aureus isolated from raw poultry meats were reported, with a highest resistance prevalence being observed towards β-lactams, macrolides, quinolones, and fluoroquinolones [S. aureus from conventional poultry meat than from organic poultry meat [S. xylosus isolates resistant to nalidixic acid, novobiocin, penicillin, oxacillin, ampicillin, lincomycin, tetracycline, erythromycin, bacitracin, and streptomycin were observed [tetK (tetracycline), linA (lincomycin), ermB (erythromycin) and blaZ (β-lactam) genes.nd human . The coarthritis . The tre broiler . Penicilerations . South Aerations  in S. auint (35) . The autint (35) . In 2006om 1970s . Multidrinolones . There wtry meat . From coobserved . These rC. perfringens resistant to bacitracin, penicillin, streptomycin, tetracyclines and gentamicin have been reported in poultry [C. perfringens isolated from conventionally produced broilers demonstrated resistance to tetracycline and bacitracin, and intermediate resistance to lincomycin [C. perfringens from poultry, pig, and cattle in Belgium farms [Clostridium including C. perfringens and other anaerobes has been reviewed recently [C. perfringens isolated from broiler chickens in Egypt, namely neomycin, colistin, pefloxacin, trimethoprim-sulfamethoxazole, gentamicin, streptomycin, lincomycin, oxalinic acid, erythromycin and spiramycin [C. perfringens on poultry health, there are a lack of studies that investigated the prevalence of AMR C. perfringens in organic and ABF poultry production. Thus, more research is warranted to better understand the impact of organic and ABF poultry production on AMR in C. perfringens.Isolates of  poultry . Moreovencomycin . Bambermum farms . Antimicrecently . High priramycin . DespiteThe poultry industry is rapidly growing due to market and consumer’s demand. However, adopting alternative poultry production practices to improve bird health and performance while decreasing AMU is imperative due to AMR concerns. In poultry production systems, complex environmental and genetic factors could contribute to the prevalence and spread of AMR and their related ARGs despite existence of correlations between AMU and AMR. In ABF and organic poultry productions, several antibiotic alternatives and vaccines are currently being applied. However, cost-effective benefits for most of the alternatives to antibiotics in poultry remain to be established. These alternative products appear to have pleotropic activities including antimicrobial, antioxidant, immune stimulatory and anti-inflammatory actions indicating that more investigations are required to determine their mechanism of action both against bacteria including their AMR profiles and birds. Overall this review indicates that AMR was present in poultry production systems that did not use any antimicrobials but a significant lower prevalence than in conventional poultry. However, more studies to investigate AMR in organic/ABF poultry production need to be done. Furthermore, understanding how feeding programs impact the commensal gut microbiota, pathogenic bacteria, and AMR will help guide dietary and bird health management practices. Accordingly, extensive efforts using integrative One Health approaches are imperative to breakdown the emergence and spread of AMR in poultry.


Anaplasma spp. and Coxiella burnetii and the tick-borne encephalitis virus (TBEV) are tick-transmitted pathogens circulating in the southern German sheep population. Knowledge of interaction among Anaplasma spp., C. burnetii and TBEV in sheep is lacking, but together they might promote and reinforce disease progression. The current study aimed to identify co-exposure of sheep to Anaplasma spp., C. burnetii and TBEV. For this purpose, 1,406 serum samples from 36 sheep flocks located in both southern German federal states, Baden-Wuerttemberg and Bavaria, were analysed by ELISAs to determine the antibody levels of the three pathogens. Inconclusive and positive results from the TBEV ELISA were additionally confirmed by a serum neutralisation assay. The proportion of sheep with antibodies against Anaplasma spp. (47.2%), C. burnetii (3.7%) and TBEV (4.7%) differed significantly. Significantly more flocks with Anaplasma spp. seropositive sheep (91.7%) were detected than flocks with antibodies against TBEV (58.3%) and C. burnetii (41.7%), but there was no significant difference between the number of flocks which contained TBEV and C. burnetii seropositive sheep. Seropositivity against at least two pathogens was detected in 4.7% of sheep from 20 flocks. Most co-exposed sheep had antibodies against Anaplasma spp./TBEV (n = 36), followed by Anaplasma spp./C. burnetii (n = 27) and Anaplasma spp./C. burnetii/TBEV (n = 2). Only one sheep showed an immune response against C. burnetii and TBEV. Flocks with sheep being positive against more than one pathogen were widely distributed throughout southern Germany. The descriptive analysis revealed no association between the antibody response of the three pathogens at animal level. Taking the flocks as a cluster variable into account, the exposure to TBEV reduced the probability of identifying C. burnetii antibodies in sheep significantly , but the reason for this is unknown. The presence of Anaplasma spp. antibodies did not influence the detection of antibodies against C. burnetii and TBEV. Studies under controlled conditions are necessary to evaluate any possible adverse impact of co-exposure to tick-borne pathogens on sheep health. This can help to clarify rare disease patterns. Research in this field may also support the One Health approach due to the zoonotic potential of Anaplasma spp., C. burnetii and TBEV.The intracellular bacteria  Coxiella burnetii, Anaplasma phagocytophilum and Anaplasma ovis, and the tick-borne encephalitis virus  are tick-transmitted pathogens and circulate in sheep flocks in the southern German federal states, Baden-Wuerttemberg (BW) and Bavaria (BAV) [A. phagocytophilum and TBEV is Ixodes ricinus and this tick species is widely distributed throughout Germany [C. burnetii has also been found in I. ricinus [Dermacentor marginatus is considered to transmit this pathogen to sheep [D. marginatus is limited to certain areas in southern Germany [A. ovis was identified in engorged D. marginatus from Bavarian sheep, but this does not prove its vector competence, and solid data about A. ovis vectors are still lacking [C. burnetii can result in reproductive disorders [A. ovis, whereas an A. phagocytophilum infection results in tick-borne fever [A. phagocytophilum is an immunosuppressive agent and negatively affects the function of neutrophils, resulting in a higher susceptibility to secondary infections [Flaviviridae) with A. phagocytophilum promote the onset of severe LIV-associated neurological disorders [A. phagocytophilum and TBEV resulted in a significantly higher TBEV antibody response compared to a consecutive infection [Anaplasma spp., C. burnetii and TBEV in sheep is lacking, but together they might promote and reinforce disease progression.The intracellular bacteria ia (BAV) –3. Theseia (BAV) –7. The m Germany , 8, 9. C ricinus , but Derto sheep , 12. Theisorders . Haemolyne fever . Moreovefections . TBEV infections . Concurrisorders . Furthernfection . HoweverAnaplasma spp., C. burnetii and TBEV. For this purpose, 1,406 serum samples from 36 sheep flocks located in BW and BAV were analysed to detect antibodies against the three tick-borne pathogens. Initially, the blood samples were collected for a Q fever study, and the number of specimens required from each flock to estimate the positivity rate was calculated on the assumption of 3% expected prevalence, 95% confidence interval, 80% power and 5% precision [The current study aimed to identify to which extent grazing sheep had antibodies against Anaplasma spp., C. burnetii and TBEV were determined by three different commercial ELISAs in accordance with the manufacturers’ instructions and described in detail elsewhere [Anaplasma spp. assay , but this ELISA does not differentiate between antibodies against A. phagocytophilum and A. ovis. A sensitivity of 91.9% and a specificity of 86.9% were assumed according to Shabana et al. [C. burnetii ELISA was considered positive , in accordance with the sensitivity and specificity of 100% each stated by the manufacturer. Regarding the TBEV ELISA , the manufacturer specified samples with > 126 Vienna Units (VIEU)/mL as positive; values between 63 and 126 VIEU/mL were classified as inconclusive. A sensitivity of 97% and a specificity of 99% were assumed in accordance with the product information. The inconclusive and positive samples were confirmed with a serum neutralisation assay as recently described, and antibody titres of ≥ 1:40 were counted as positive [Antibodies against lsewhere , 17, 18.a et al. . A S/P  using the Rogan-Gladen estimator . The preAnaplasma spp., C. burnetii and TBEV at animal level (p < 0.05). Most sheep had antibodies against Anaplasma spp. (47.2%), followed by TBEV (4.7%) and C. burnetii (3.7%). Significantly more flocks with Anaplasma spp. seropositive sheep were detected  compared to flocks being seropositive for TBEV  and C. burnetii  (p < 0.05), but there was no significant different between flocks which contained TBEV and C. burnetii seropositive sheep. Seropositivity against at least two pathogens was detected in 66 (4.7%) sheep from 20 flocks (55.6%). The flocks with co-exposed sheep were widely distributed throughout southern Germany. Details of sheep and flocks with antibodies against more than one of the pathogens are presented in Table C. burnetii and TBEV antibodies. The exposure to TBEV reduced the probability of identifying C. burnetii antibodies in sheep by half  seems to be on the increase in southern Germany [A. phagocytophilum should be investigated in lambs naturally infected with TBEV. Despite the fact that a detrimental influence was not confirmed after experimental infection with TBEV, the artificially infected lambs did not develop clinical signs of TBE [The significant differences among antibody rates of tophilum , whereastophilum , and C. s so far , 26. Infely rare . Signifiin sheep , 28, 29.in ticks –32. Consin ticks , who dem Germany , and thes of TBE . TherefoAnaplasma spp. ELISA, and a possibly reduced specificity of the C. burnetii ELISA [Anaplasma spp., C. burnetii and TBEV seems to be sporadic among grazing sheep flocks in southern Germany. The further spread of TBEV and the emerging onset of A. ovis in Germany might increase cases of co-infection [A. phagocytophilum influences the immune response and disease progression of concurrent flavivirus infections [The authors are aware of the limitations of the present study. The antibody response against the three tick-borne pathogens is the result of a natural exposure in the field in the past. Therefore, it is impossible to determine the exact time of infection and the possibly related clinical impact. Moreover, we cannot rule out that false-seropositive sheep were included in the evaluation because of an estimated specificity of 86.9%  of the Aii ELISA . Only thii ELISA . This minfection , 4. Morefections , 16. In fections . Therefo