Streptococcus canis causes deep pyoderma in canines, which raises concerns about the risk of isolates from lesions acquiring an antibiotic-resistant phenotype. It is necessary to identify effective antibiotics and the characteristics of the pathogenic cluster for S. canis-associated deep pyoderma.S. canis isolated from deep pyoderma lesions (27 strains) and oral cavities (26 strains) were analyzed. Older dogs tended to have S. canis-associated deep pyoderma (15 of 27 dogs over 10 years old). Veterinarians chose quinolones for 10/16 cases (63%), even though the rate of quinolone-resistant strains of S. canis is 38–59%. Although 70% of the strains showed resistance to three or more antibiotic classes (37/53), 94% (50/53) strains showed sensitivity for penicillins. We also identified β-lactamase activity among penicillin-resistant strains of S. canis. Clonal complex 13 (CC13) was detected only in lesions and formed independent clusters in the phylogenetic tree. One strain of CC13 was resistant to the anti-methicillin-resistant Staphylococcus aureus drugs, vancomycin and linezolid.The signalment, molecular typing, and antibiotic-resistant status of S. canis are isolated at a high rate, they can currently be treated with β-lactamase-inhibiting penicillins. CC13 may be a pathogenic cluster with high levels of antibiotics resistance.Although antibiotic-resistant strains of The online version contains supplementary material available at 10.1186/s12917-022-03482-3. Canine deep pyoderma is a bacterial infection that affects tissues deeper than the epidermis, such as cellulitis and furunculosis . The devStreptococcus canis is known to be one of the causative gram-positive cocci of deep pyoderma in dogs and humans [S. canis-associated deep pyoderma sometimes causes a potentially fatal prognosis [S. canis puts their owners at risk of developing deep pyoderma [S. canis-associated deep pyoderma has been reported to date, and it is not known which dogs are more likely to develop the disease.d humans , 4. S. crognosis . It is apyoderma . HoweverS. canis is a β-hemolytic Lancefield group G streptococcus, which colonizes the skin, the oral cavity, the upper respiratory tract, and the reproductive tract of dogs [S. canis-associated deep pyoderma is thought to be an opportunistic infection, with resident bacteria infecting the destroyed skin [s, and S. canis is endemic [ of dogs , 8. S. cyed skin , 9. Stre endemic , 11. BecS. canis has been reported to be resistant to quinolones  and tetracyclines , although there have been no reports of resistance to β-lactam drugs [S. canis in dogs were a mixture of strains of various origins, including earwax-derived, oral-derived, and lesion-derived strains, and to our knowledge, no detailed information on the antibiotic resistance status of S. canis from canine deep pyoderma is available to date.Unlike staphylococci, most streptococci are not penicillin-resistant; therefore, penicillins are used as first-line therapy in animals and humans , 13. Howam drugs , 16–18. S. canis isolated from deep pyoderma lesions and the oral cavity. We compared the molecular typing and antibiotic resistance of the strains to determine the essential clinical features of S. canis-associated deep pyoderma and clarify whether it is an opportunistic infection. We also aimed to clarify which antibiotic agents should be used for patients with deep pyoderma associated with S. canis.In the present study, we analyzed the signalment of S. canis. Among the samples in which S. canis was detected, other organisms were detected in six specimens. Therefore, 27/546 (5%) of the gram-positive cocci-associated deep pyoderma detected were caused by S. canis.Gram-positive cocci were detected from lesion contents from 546 of 719 cases. One-hundred-two isolates were grown in Columbia agar with 5% sheep blood , but did not grow in staphylococcus selective agar  or enterococcus selective agar . Forty-five of the 102 isolates were negative for the catalase test. Twenty-seven of the 45 isolates were identified as S. canis-associated deep pyoderma is provided in Table S. canis-associated deep pyoderma, the samples were taken from abscesses in 10, followed by cellulitis in three. No preferred breed or sex of the affected dogs was found. By contrast, 15 of the 27 affected dogs were older than 10 years, with a mean ± standard deviation (SD) of 9.67 ± 4.58 years for the affected dogs. This age distribution is significantly older than the mean ± SD age distribution of dogs kept in Japan  [Information on the dogs with < 0.001) . Of the S. canis were detected in samples from the oral cavity of 82 dogs with no clinical signs. Information about the dogs is provided in Supplementary Table S. canis was isolated (p > 0.05).Twenty-six strains of S. canis. Among the 26 distinct biochemical profiles that were found with this system, 8  corresponded to S. canis with a confidence of ≥99% according to the manufacturer’s instructions. All strains were detected following PCR amplification of the S. canis ISR and SodA genes.All isolates were β-hemolytic and carried the Lancefield group G polysaccharide. The API 20 Strep system identified all as S. canis, 27 strains derived from dogs with deep pyoderma and 26 oral-derived controls. MLST analysis revealed a diverse collection, with nine CCs and two independent sequence typings (STs) (Table S. canis from specimens in which multiple organisms were detected simultaneously were often CC2 and CC9 (5/6 specimens). We found no significant differences in the age, sex, API phenotype, or breed of diseased animals from which CC2, CC9, and CC13 were isolated.Multilocus sequence typing (MLST) analysis was performed on 53 strains of The seven sequence sequences used in the MLST analysis were combined for phylogenetic tree analysis Fig. . A groupp > 0.05)  drugs (linezolid (LZD), vancomycin (VCM)), which are not commonly used in veterinary medicine, were found in 2–6%. The number of strains resistant to three or more antibiotic classes  was 37/53 (70%).From disk diffusion testing, among the isolates, the percentage of strains resistant to each antibiotic agent was not significantly different between strains from lesional or oral origin (05) Fig. . We founThe phylogenetic tree and the distribution of antibiotic resistance phenotypes showed that resistance phenotype was evenly distributed in all clusters. The distribution of multidrug-resistant strains did not differ significantly among clusters (60–85%). Notably, the P03 strain resistant to penicillins, vancomycin, and linezolid, which formed lesion-specific clusters was classified as a CC13 strain in the phylogenetic tree . By contrast, P03 possessed the same level of β-lactamase activity as penicillin-sensitive P02 and P05 (p > 0.05).The five strains for measured MICs were also examined for β-lactamase activity Fig. . Of the S. canis that causes canine deep pyoderma. Although S. canis showed aspects of an opportunistic infection, it may be that a highly pathogenic cluster with respect to CC13 has evolved. Penicillins are the most effective antibiotic agents for S. canis, but β-lactamase inhibitor-formulated penicillin should be used because some strains harbor β-lactamase.To our knowledge, our study is the first to analyze the antibiotic-resistant status and molecular typing of S. canis-associated deep pyoderma was more likely to occur in older dogs. In humans, deep pyoderma caused by group G streptococci, including S. canis, has been shown to occur more frequently in the elderly with chronic diseases [S. canis-associated deep pyoderma in this study, further analysis is required because immunocompromised hosts may be involved in the pathogenesis of deep pyoderma caused by S. canis. Erysipelas, a type of deep pyoderma, is known to show a bimodal pattern in young and old individuals [S. canis [S. canis-associated deep pyoderma is warranted.diseases . Althougividuals . YoungerS. canis , 22. We S. canis-associated canine deep pyoderma. Similarly, the overuse of inappropriate antibiotics may induce drug resistance in S. canis in canine deep pyoderma. At present, Japanese veterinarians choose quinolones for 10/16 cases at first (62.5%), even though many quinolone-resistant strains were detected. In human medicine, multidrug-resistant Streptococcus pyogenes have increasingly been isolated, which is thought to be the result of the emergence of antibiotic-resistant strains from previously susceptible populations because of horizontal gene transmission and chromosomal point mutations caused by overuse of antibiotic agents [Excessive use of inappropriate antibiotic agents may induce antibiotic resistance in c agents , 23.S. canis-associated deep pyoderma, penicillins in combination with β-lactamase inhibitors are considered to be the most effective antibiotic agents [Streptococcus spp. carrying β-lactamases have also been found in other streptococci, and their increasing numbers in recent years are beginning to be a problem in human medicine [S. canis is beginning to show a trend similar to that of the streptococci. Note that the P03 strain showed resistance to penicillins despite that the absence of β-lactamase is likely to have another mechanism of resistance. For most staphylococci, the principal causative agents of deep pyoderma are gram-positive cocci [To treat c agents , 13. Strmedicine , 23, 24.ve cocci . TherefoS. canis from specimens in which multiple organisms were detected simultaneously were often CC2 and CC9 (5/6 specimens). These data suggest that S. canis-associated deep pyoderma is an opportunistic infection, which is consistent with the prevailing theory [S. canis strains reported to date [S. canis causing ulcerative keratitis in dogs found that all were CC13 [S. canis in dogs [S. canis have been reported [S. pyogenes.In the present study, CC2, CC9, and CC13 were isolated mainly from lesions of canine deep pyoderma. CC2 and CC9, which are also known as major isolates from animals and humans , were is to date . An analere CC13 . This su in dogs , 29, fewreported , 31. BecS. canis-associated deep pyoderma, 10 were diagnosed with abscesses, but these diagnoses were made independently by each physician, so it was not a uniform conclusion. In addition, the diagnostic criteria for erysipelas in the veterinary dermatology field are not clear to date [S. canis in clinical practice. Future studies on the usefulness of the Christie–Atkins–Munch-Peterson test and Antistreptolysin O antibody titers might make the detection of S. canis more convenient. Furthermore, the physician in charge of each sample transported it from each hospital in the study. In the case of S. canis-associated pyoderma, multiple bacteria were detected in six specimens, which suggests the possibility of contamination. We hope that future research will fill in the gaps in our analysis.This paper has some limitations. Among 27 dogs with S. canis observed in this study was associated with the presence of the van gene from enterococci or with other mechanisms [S. canis infects dogs and humans. Routine surveillance and the analysis of the resistant mechanisms should be strengthened.It is also a severe problem that the P03 strain, included in CC13, is resistant to the anti-MRSA drugs vancomycin and linezolid. Linezolid and vancomycin are among the last-resort antibiotic agents for treating multidrug-resistant gram-positive bacterial infections . In entechanisms . Becausechanisms , the potS. canis, they can currently be treated with β-lactamase-inhibiting penicillins, CC13 may be a highly pathogenic cluster, and highly drug-resistant strains also exist. Further research on S. canis from both the veterinary clinical and bacteriological aspects will be necessary to identify these strains.Although antibiotic-resistant strains were isolated at a high rate among 2 at 35 °C for 48 h. We also incubated samples aerobically at 37 °C for 48 h on mannitol salt agar (Becton Dickinson) and enterococcosel agar (Becton Dickinson). One representative colony was isolated from each sample, and other pure cultures were cultured twice on Columbia agar with 5% sheep blood. The colonies were subjected to a Hacker variant Gram staining kit . Strains that did not grow on mannitol salt agar but did grow on Columbia agar with 5% sheep blood were chosen, and the catalase activity of those gram-positive cocci was examined with 3% hydrogen peroxide . The frozen isolates were stored at − 80 °C until used.Bacterial isolates were collected from the lesional skin of dogs in 719 clinical cases of canine deep pyoderma diagnosed by an attending veterinarian in 227 animal hospitals in Japan between August 2016 and March 2020 for diagnostic purposes. Sterile BBL culture swabs samples (Becton Dickinson), needle aspiration samples, and biopsy samples were used in this study. Before sample collection, we recommended physicians to wipe the skin surface once with alcohol-impregnated cotton swabs and wear surgical gloves. All samples were transported to the Vet Derm Tokyo laboratory at 4 °C after being placed in Stuart’s transport medium (Becton Dickinson). The plates were incubated in 5% COInformation about the age, breed, sex, diagnostic name, and antibiotic used in the affected animal up to 3 weeks before sampling was collected from the physician in charge of each veterinary clinic. Some of the missing patient information was obtained by interviewing the veterinarian in charge by telephone.2 at 35 °C for 48 h. One colony that showed β-hemolytic activity was isolated from each sample and cultured twice in pure culture on Columbia agar with 5% sheep blood. The colonies were stained with a Hacker variant Gram staining kit. For gram-positive cocci, catalase activity was examined using 3% hydrogen peroxide. The frozen isolates were stored at − 80 °C until used.From March 1, 2021, to September 24, 2021, the oral cavity of 82 dogs with no clinical symptoms that came to the veterinary clinic for vaccination was swabbed with sterile swabs. The swabs were immediately streaked onto Columbia agar with 5% sheep blood and incubated in 5% COS. canis was identified using a Lancefield latex agglutination test  , and API 20 Strep . Bacterial DNA was extracted using an Easy DNA extraction kit version 2  [S. canis ISR and SodA genes [, Japan)  and puri, Japan)  accordindA genes , 38. DSMhttps://pubmlst.org/organisms/streptococcus-canis, accessed February 28, 2021).MLST analysis was performed on all the isolates included in the present study according to the protocol reported by Pinho et al. . For seqSTs were grouped into CCs, whereby related STs were classified as single locus variants differing in one housekeeping gene only. Based Upon Related Sequence Types analysis was performed on the MLST website to identify genetically related CCs. Trees were constructed and inference was performed using the neighbor-joining method after 500 iterations.Disk diffusion testing was conducted using 21 different antibiotic agents. These were: PCG ; ABPC ; ACV ; CEX ; CFV ; FOM ; ERFX ; MFLX ; LVX ; GFLX ; CLM ; LCM ; DOT ; MNO ; TC ; GM ; SXT ; EM ; CAM ; and the anti-MRSA drugs LZD  and VCM . We classified strains as susceptible, intermediate, and resistant against antibiotic agents using clinical and laboratory standards institute (CLSI) guidelines for Vet-08 and M-100-Ed31 , 40. TheThe MICs were determined by broth microdilution testing using a dry plate kit  according to the manufacturer’s instructions .Staphylococcus pseudintermedius isolated from canine superficial pyoderma samples was used for quality control [ control . Antibio control , and dru control .The β-lactamase activity was determined using a β-lactamase activity assay kit  according to the manufacturer’s instructions . The anaThe present study was conducted in compliance with the ARRIVE guidelines and applicable animal welfare regulations relating to the care and use of animals for scientific purposes. Informed consent was obtained from the owner of each participating dog. This experiment was approved by the Kyoto Veterinary Medical Center’s Animal Ethics Committee as having no ethical issues .S. canis-associated deep pyoderma and the percentage of resistant isolates. Kruskal–Wallis tests were used to compare the β-lactamase activity among five strains. All variable tests set p < 0.05 as statistically significant.All analyses were conducted with Prism 9 . Data were expressed as mean ± SD and range. Chi-square tests were used to analyze the age of animals with Additional file 1: Supplementary Table 1. Signalment information of dogs with S. canis isolated from the oral cavity.Additional file 2: Supplementary Table 2. Comparison of the antimicrobial resistance of strains colonizing either deep pyoderma or oral cavity controls.


Mikania micrantha is an exotic and aggressive species that can reproduce asexually and sexually through its germinative stem and its featherlike seeds. Present weeding methods cannot effectively or economically control the spread of Mikania micrantha. In this article, we propose a method to suppress the growth and spread of Mikania micrantha by applying a high-energy laser beam to penetrate its stem. The threshold penetrating optical intensity is 3.1 W/mm2. To optimize the damage to the inner tissue of the stem, which includes the vascular bundle and medulla for transporting organic nutrients, water, and inorganic salt, the absorption spectrum of the tissue and laser beam size are analyzed. According to the absorption spectrum of the tissue and growth mechanism of Mikania micrantha, a 455 nm blue laser is used as an irradiated light source. A single beam with two different beam sizes or two laser beams with the same beam size is used to optimize the stem damage. By the time the cumulative energy reaches 15 Joules for a single laser beam with dimensions of 0.81 mm × 0.74 mm, the inner tissue will be damaged 97.5%. We perform laser irradiation on the fresh Mikania micrantha grown hydroponically, with the result that all samples withered in 30 days. Therefore, using the method before the flowering season of Mikania micrantha can effectively inhibit its reproduction. Mikania micrantha (M. micrantha) is listed as one of the 100 most invasive species by the International Union for the Conservation of Nature (IUCN)1. M. micrantha is a perennial twining herb with internodes of 7.5–21.5 cm long. It has opposite leaves and 4 flowers per capitula. A single stalk of Mikania micrantha can produce 20,000–40,000 seeds in one season, and can grow vegetatively from its nodes and tiny segments, expanding rapidly3. Young plants grow quickly at about 8–9 cm per day, and rapidly form a dense cover over entangled leafy stems. Figure M. micrantha. Rapid invasion of M. micrantha causes damage and economic losses in natural and agricultural environments, leading to a decrease in soil and food stability and alters the nutrient cycle, severely affecting biodiversity and production pipelines4.Invasive alien plants harm the diversity and environmental stability of native plants, thereby reducing crop growth and productivity. M. micrantha can be proposed. The Consecutive-Cutting Method is applied during at least two consecutive cuts within a 3-week interval before flowering. Three consecutive cuttings result in 91% mortality of the vines. This method reduces the vegetative propagation of M. micrantha and has no potential adverse effects on native species. Unfortunately, this control method is very labor intensive and, therefore, uneconomical. Rapid regrowth of cut stumps undermines this method7. The Flaming Method is a burning technique that helps limit spread and is widely practiced. It does not completely burn the weeds, but effectively suppresses them with heat, destroying the proteins in the plant. Although it is widely used for weeding, it cannot distinguish between weeds and crops. In addition, the weed stock can survive after burning and produce young shoots in a couple of months. A chemical control approach often uses a more effective herbicide because it kills the entire weed. After processing, M. micrantha and other vegetation in the treated area are completely wilted2. However, chemical herbicides also harm other crops and contaminate the soil. In addition, the other negative impact of using herbicides is greenhouse gas (GHG) emissions10. Biological control is supposed to be long-lasting, with no adverse effect on nontarget species, and an eco-friendlier solution to control invasive species. Several countries are exploring effective biological control agents for M. micrantha2. Controversial issues, such as the cost and benefit of the method and the hidden side effects of biological control agents, need to be resolved.Several methods to control 11. Compared to lamps and sunlight, the advantages of lasers for weed control are; high precision direction, weather independence, high customization, and energy variability. Advances in laser technology enable the use of non-diode lasers or diode lasers with a wide wavelength and power range. This opens up new opportunities for photonic weed control. Several groups have controlled the growth in the laboratory using industry laser sources, such as CO2 lasers16, solid-state lasers17, and diode lasers18. They use lasers to irradiate the apical meristem of mono- or dicotyledonous plants to inhibit their growth18. M. micrantha is a vine without the apical meristem. Since it is easy to grow and distribute by stem nodes, a laser light has to irradiate its other tissue to suppress it. Therefore, we propose a method effectively inhibit Mikania micrantha growth and spread by adopting a suitable wavelength and laser energy, and irradiating on a right tissue.Effective weed control methods to date are based on thermal effects, which increase the temperature to rupture the cell structure, followed by tissue desiccation. Thermal energy can be induced by hot matter or radiation. Heat sources with selectivity and directionality, which are exactly pointed at the location of weeds, can be carried out using lamps, concentrated sunlight, or lasersM. micrantha from the land near the College (location is in the south of Taiwan), to grow these samples in a self-build green house. Figure Mikania micrantha. Mikania micrantha is a perennial twining plant with internodes of about 7.5 to 21.5 cm. It has opposite leaves and 4 flowers per capitula, shown in Fig. Mikania micrantha can grow vegetatively from the stem nodes, as in Fig. Mikania micrantha is a shallow-rooted plant, shown in Fig. We collected M. micrantha leaves and flowers cannot inhibit its spread due to its ability to reproduce asexually through its germinative stem. As the vegetative fragments  of M. micrantha can easily survive, re-sprout, and form new individuals; a practical method to stop M. micrantha is weeding before flowering and avoid the stem node from falling to the ground to reproduce asexually. The stem of M. micrantha is the key to maintaining growth. The outer to inner structure of the M. micrantha stem is divided into the epidermis, cortex, vascular bundle, and medulla. The vascular bundle consists of the phloem and the xylem. The function of the phloem and xylem is to transport organic nutrients, water, and inorganic salt, respectively. If the vascular bundle is damaged, water and nutrients can be interrupted and the germination of new shoots can be prevented. So, we damage the vascular bundle and medulla of the stem to instead of cut it off. We used a laser to increase the temperature of the tissue, then damage the protein to inhibit the growth of M. micrantha. To achieve this, we adapted a wavelength and energy density of laser to carefully destroy the phloem and xylem of the plant. This article focuses on optimizing laser energy to the damaged stem area after irradiation. The wavelength selection was based on the absorption spectrum of the stem to perform an effective interaction between light and tissue. To optimize the damaged area, a suitable laser wavelength from the absorption spectrum of the stem, a suitable laser beam size from the optical lens design, and an appropriate illumination method was selected.Injury to M. micrantha stem, as shown in Fig. M. micrantha, it is too close to the UV spectrum. Since most creatures are sensitive to ultraviolet light, we chose a laser with a wavelength of 455 nm as a source to illuminate the stem of M. micrantha.To effectively increasing the cell temperature, the laser wavelength must be well chosen, based on the absorption spectrum of the vascular bundle and medulla of the M. micrantha. An experimental scheme is shown in Fig. M. micrantha samples. The set of lenses consisted of a cylindrical lens and an aspheric condenser lens. The planoconvex cylindrical lens with a focal length of 12 mm, both sides coated with antireflection of wavelengths from 350 to 700 nm, was used to reduce the large divergent angle to collimate the blue laser beam. The aspheric condenser lens with a focal length of 20 mm and a numerical aperture lens of 0.6 was installed to focus the laser beam on the stem of M. micrantha. The laser beam size was measured by the knife-edge method. The minimum beam size is 0.46 mm × 0.42 mm. A continuously variable neutral density filter (ND filter) was placed between two lenses to control the power focused on the weed. A photodetector was used to measure the laser power and the power that passes through the stem. The laser power times the exposure time provided the energy focused on the weed. The photon intensity was calculated in watts per beam area. In addition, a charge-coupled device (CCD) camera was installed to monitor illumination time and the situation of the stem during laser illumination.We conducted the experimental design after considering the convenience and cost of the laser, optimal absorption lines of the stem, and the average stem size of M. micrantha samples in the experiments are 2 mm. To determine the laser energy requirement for inhibiting the growth of M. micrantha, Fig. M. micrantha in the experiments: one is using a single beam to pass through the medulla of the stem, or pass through the vascular bundle of the stem by using two laser beams. After laser illuminating, we cut the irradiated area of the stem by a knife to observe its cross-section. For further observation of the status of the stem during laser irradiation, we removed the leaves. We irradiated the stem 2 mm below the leaves to observe the impact of laser irradiation on the stem. A CCD was on the top position to observe the variation of the cross-section surface during laser irradiation.The diameters of all fresh Mikania micrantha was collected from deserted fields on our Campus in this study.Our study complies with relevant institutional, national, and international guidelines and legislation. The CCD records the process of a laser from irradiating to penetrating the stem. Photos 1 through 6 of Fig. M. micrantha. Laser power divided by the laser beam area is the intensity of the laser. Figure 2 damaged only the surface of the stem. When the laser intensity increased to 1.47 W/mm2 and 2.2 W/mm2, the laser energy penetrated the surface layer and reached the vascular bundle. The white dashed line marked the laser stop location. When the laser intensity increased to 3.1 W/mm2, the stems began to be penetrated. It is worth noticing that the laser passes through the stem with both laser intensities of 3.1 W/mm2 and 5.27 W/mm2. It caused the tissue around the path of the laser to scorch. When the laser intensity rose to 5.8 W/mm2, the laser crossed the stem and left an obvious line. Although a higher laser intensity could easily penetrate the stem, the damage region was relatively small. The stem penetration depth could be approximated by a linear relation to intensities, as shown in Fig. 2. For both piercing the stem and damaging the vascular bundle inside, the laser intensity should be between 3.1 W/mm2 and 5.27 W/mm2.In addition to the laser power, the laser beam area is also an essential parameter for inhibiting the growth of 2 was used to irradiate the stem and damage the medulla. When the energy on the M. micrantha reached 2.7 J, the laser penetrated the stem, as shown in Fig. 2, corresponding to a beam dimension of 0.81 mm × 0.74 mm, accumulated energy of 15 J, resulted in the laser beam passing through the stem. In this condition, the damaged region of the stem grew to 97.5%, which included the medulla and the vascular bundle, as shown in Fig. M. micrantha at lower laser energy, the damaged tissue area was too limited to suppress its growth. Figure M. micrantha. When the photon energy reached 10.7 Joules, 60.9% of the stem was damaged. Both the medulla and the vascular bundle were burned, which damaged their transport water and nutrients and then inhibits their growth. Although a single laser beam with a large beam size can damage more area of the stem than two laser beams, however, the single laser beam required a longer exposure time to achieve enough energy.Once the intensity of the laser has been determined, sufficient damage area must be achieved within the stem to allow the growth of the plant to be controlled. Another factor that contributes to efficient plant growth control is the beam size and illumination method of the laser. Use these two parameters to optimize the destructed region of the stems. Figure 2 beam area to fresh M. micrantha samples grown hydroponically in a green house. M. micrantha samples with a stem diameter of 2 mm were divided into two groups of 10, the control group without laser irradiation and the experimental group irradiated with laser. The statuses were observed and recorded daily, as shown in the S1—Fig. M. micrantha samples varied, as shown in Fig. 21. If too much water is lost, the cells will dry out and die. On the contrast, all samples of the control group, without laser irradiation, remained live.Then, we perform 15 J laser energy with 0.6 mmMikania micrantha is a severe invasive species in large parts of Asia. To effectively limit its spread, we use a laser to damage the inner tissue of Mikania micrantha, the vascular bundle and the medulla, instead of cutting it off. Based on the absorption spectrum of the inner tissue of the stem and cost, a 455 nm blue laser diode was used as the light source. The penetration depth of the stem depends on laser intensities; the laser intensity for stem penetration has to be greater than 3.1 W/mm2. The requirement of damaged energy depends mainly on the efficiency of the energy coupling in the stem of Mikania micrantha. High-efficiency energy coupling is achieved through the size of the beam and the duration of irradiation. A single beam with two different beam sizes or two laser beams with the same beam size is used to optimize the stem damage. Using a single beam to irradiate the stem, enlarging the beam dimension to 0.81 mm × 0.74 mm, with accumulated energy of 15 J, achieves 97.5% damaged area of the stem. In contrast, with two laser beams with a beam dimension of 0.46 mm × 0.42 mm irradiating the stem, the photon energy reaching 10.7 Joules, only 60.9% of the stem is affected. Although a large beam size needs more photoenergy to destroy the inner tissue of the stem, it is useful to depress the growth of M. micrantha. As the xylem and phloem are functionless after laser illumination, the M. micrantha is going to wither due to blockage of water and nutrient transportation. We implemented this method on fresh M. micrantha samples and demonstrated that all fresh samples wilt completely within 30 days. In other words, simply irradiating the stems with a laser one month before flowering can stop their growth and spread. With the combination of an intelligent identification system and a laser, automatic work can be performed in the future.Supplementary Figure 1.


Food production environments in low- and middle-incomecountries(LMICs) are recognized as posing significant and increasing risksto antimicrobial resistance (AMR), one of the greatest threats toglobal public health and food security systems. In order to maximizeand expedite action in mitigating AMR, the World Bank and AMR GlobalLeaders Group have recommended that AMR is integrated into wider sustainabledevelopment strategies. Thus, there is an urgent need for tools tosupport decision makers in unravelling the complex social and environmentalfactors driving AMR in LMIC food-producing environments and in demonstratingmeaningful connectivity with other sustainable development issues.Here, we applied the Driver-Pressure-State-Impact-Response (DPSIR)conceptual framework to an aquaculture case study site in rural Bangladesh,through the analysis of distinct social, microbiological, and metagenomicdata sets. We show how the DPSIR framework supports the integrationof these diverse data sets, first to systematically characterize thecomplex network of societal drivers of AMR in these environments andsecond to delineate the connectivity between AMR and wider sustainabledevelopment issues. Our study illustrates the complexity and challengesof addressing AMR in rural aquaculture environments and supports effortsto implement global policy aimed at mitigating AMR in aquacultureand other rural LMIC food-producing environments. This study provides a framework for decision makers to betterunderstand and mitigate the risks of antimicrobial resistance in aquaticfood-producing environments in low- and middle-income countries. Food production environments are considered to pose a particularlyhigh risk to the emergence and dissemination of AMR, especially inlow- and middle-income countries (LMICs), where traditional extensivefarming systems are moving toward more intensive practices.4 exposing a significant“action gap”, with many countries struggling to translatepolitical will into practice.7 Recent reports by the World Bank7 and the AMR Global Leaders Group3 suggest that the key to reducing this gap, especially inLMICs, is through integrating AMR into wider sustainable developmentpolicies; they call for major efforts in the field of AMR implementationresearch in order to increase our capacity to identify and characterizemeaningful connectivity with wider sustainable development strategies.This would enable the measurement of potential co-benefits acrossa broad array of AMR-sensitive interventions in country-specific contexts,providing a more impactful and cost-effective approach to tacklingAMR in LMIC contexts.7 In order to achieve this, decisionmakers first need to be able to understand the underlying driversof AMR in a given context. This is particularly challenging for foodproduction environments in rural LMICs, where AMR data are scant anddisjointed, and it is recognized that a very wide range of socioeconomicfactors are driving AMR emergence and transmission.6 These issues are not limited to LMIC food-producing environments,however, with studies showing that the environmental sector is poorlyintegrated in the AMR National Action Plans (NAPs) of many countries.9 Thus, tools are urgently needed to support decision makers in betterunderstanding and mitigating AMR in complex environments.Despite considerable motivation and momentum for addressing AMRat the highest international governance levels, global AMR levelsare continuing to rise,10 DPSIR (Driver-Pressure-State-Impact-ResponseFramework)11 ,that result in changes of “States” . Together, these lead to “Impacts”, which require societal and/orpolitical “Responses”. Responses targeted further upstreamof this causal pathway are considered to have the strongest impact11 1 is an et impact1. DPSIR 15 and a strong literature base now evidences that resistant microbesare present in aquaculture land and aquatic environments, as wellas in animals within farm settings and at the point of sale.18 The international community recognizes the unique risks that aquacultureenvironments pose to global AMR;20 however, ofthe top five aquaculture-producing countries, only three of theirexisting AMR national action plans (NAPs) specifically mention aquaculture(Table S1). In the absence of effectiveaquaculture AMR management strategies, the AMR NAP plans that arein place remain predominantly focused on surveillance and monitoringrather than directly addressing the fundamental socioeconomic driversof the issue.21 Here, we show how the DPSIRframework can be used to systematically identify the wider societaldrivers of AMR in a rural aquaculture environmental context. Mappingthe DPSIR outputs then allowed us to illustrate meaningful connectivitybetween AMR and broader sustainable development goals in these environments,providing a foundation for improving the implementation of nationaland global AMR policies.In this study, we explored whether the DPSIR frameworkcould beapplied to AMR in a complex food-producing environment case study,namely, that of a rural aquaculture environment in north-central Bangladesh,in order to understand and delineate the connectivity between AMRand wider sustainable development issues. Aquaculture is the fastestgrowing food sector globally, with industry expansion largely beingfacilitated through the rapid intensification of existing aquaculturefarms in LMICs in Asia. Antibiotic usage in the industry is high andexpected to rise with intensification and escalating disease levels,Figure S1) was chosen as a case studyfor two main reasons: (i) We had three distinct AMR-related data setsavailable to us, allowing us to investigate AMR risk in this region.(ii) This region is considered typical of many other inland traditionalaquaculture areas across Asia. Small-scale rural aquaculture contextsaccount for the majority of global aquaculture production, and theseare the predominant farming systems in the Mymensingh region.22 Aquaculture has been a traditional way of lifein Mymensingh for more than 40 years, and the region has become amajor aquaculture production hub of Bangladesh due to favorable environmentaland geophysical factors. Small scale aquaculture farms differ fromthe more commercial, intensive systems in that they are more closelyinterconnected within the local environment they share with humansand animals. Traditional rural aquaculture farms are typically a seriesof earthen ponds enclosed by dykes and land for crop/vegetable production.Rice and a variety of fish species are often cultivated either together(known as polyculture) or in succession, depending on seasonal andclimatic conditions.2 Species include catfishes), Nile Tilapia (Oreochromis niloticus), variousIndian and Chinese Carp species, perch, and barbs (a term referringto a number of ray-finned fish species). Farmers and their familieslive on site and often employ people from their local community. Theregion is now seeing increasing intensification of practices: in 2010–2011,the region produced 219,000 metric tonnes (MT) of fish, and by the2018–2019 season, this had increased to 370,000 MT and accountedfor 14.87% of national aquaculture production.4 Thereare thought to be around 112,000 aquaculture farmers in the region,5 with more than 11% of the population employeddirectly or indirectly through the aquaculture industry.6The Mymensingh region in north-central Bangladesh(Table S2 for coordinates) on a monthly basis for microbialcommunity profiling, as part of a UKRI-Newton Fund Global ResearchPartnership project investigating aquaculture pond microbiomes entitled Prediction and Mitigation of Diseases Outbreaks in Aquaculture throughLarge Scale Community Engagement.23 Triplicate water samples were collected from the same two or threeponds on each farm site every month over a period of one year. Samplecollection involved passing pond water through a polycarbonate filter to collect the microbialbiomass. The sample volume was determined by the concentration ofsuspended particulates in the water, so the end point was 200 mL orwhen water would no longer pass through the filter, whichever camefirst. Filters were immediately placed in 100% ethanol and kept atambient temperature until they arrived in the laboratory in the UnitedKingdom, where they were stored at −20 °C until processing.Prior to DNA extraction, the ethanol was removed by freeze-dryingat −110 °C (ScanVac CoolSafe Pro), and filters were thenstored at −80 °C. DNA was extracted using an in-houseCTAB/EDTA/chloroform method adapted from Bramwell et al.24 and Lever et al.25 The full protocol is available at https://www.protocols.io/view/ctab-chloroform-dna-extraction-from-ethanol-preser-rm7vz3rjrgx1/v2.In 2017–2018, pond watersamples were collected from six different farms , postmonsoon (October/November 2017), winter (January/February2018), and premonsoon (April/May 2018). Replicate pond DNA extractsfrom each of the six farms and from each of these four time pointswere pooled, totalling 24 samples. Pooled samples were cleaned usingthe Genomic DNA Clean & Concentrator-10 kit (Zymo Research), quantifiedwith the Qubit dsDNA BR kit (ThermoFisher Scientific), and submittedto the Exeter Sequencing Service for PCR-free whole metagenome sequencingon the NovaSeq 6000 with the S1 Reagent Kit (300 cycles). Raw sequencedata were deposited in the European Nucleotide Archive under BioProjectnumber PRJEB53918.https://github.com/ash-bell/AMR-Bangladesh-Fish-Ponds. Using an established BBTools v38.79 pipeline,26 reads were quality trimmed, and adaptors and syntheticartifacts were removed, decontaminated, and error corrected. Readassembly was performed by metaSPAdes v3.15.327 using four k-mer lengths .Resistance genes were identified using the Resistance Gene Identifier(RGI) v5.2.128 with the additional flags–alignment_toolBLAST – exclude_nudge −clean −low_quaility −split_prodigal_job.Plots were constructed in R using the ggplot2 package.29For resistome profiling, metagenomic readsfrom all 24 pondwatersamples were processed identically, with scripts available at 30 and Sickle version 1.3331 to remove adaptor sequences and reads with quality scores less than30 and/or shorter than 60bp. These quality filtered reads were thentaxonomically classified using Kraken 2 v2.1.232 and species abundances reassessed with Bracken 2.6.2.33 Pathogenic species were identified from theKraken/Bracken output using the American Biological Safety Association(ABSA) database, accessed March 2021.34 To validate the identification of the most abundant pathogens fromthe Kraken 2 output, reads from each sampling site were mapped tothe relevant reference genomes of the 10 most abundant (in terms ofnumbers of reads) pathogens identified for that sample. Alignmentsthat had less than 50% coverage were not considered for further inferenceson pathogen presence.For taxonomic and pathogenic identification,raw reads were firstprocessed using a combination of Trimmomatic version 0.40Since October 2019, the BrahmaputraLab of Quality Feed Limited, a pharmaceutical company based in Mymensingh,has been offering disease diagnostic services to farmers to identifythe causative pathogen of disease in order to recommend and sell themost appropriate treatment. In their endeavors to help ensure thattheir antibiotic treatments are effective, they have been testingbacterial pathogens for resistance to a number of different antibioticsand kindly shared these data with us.Table S3A) and incubatedat 35 °C for 36 h. If clinical signs of disease were observed,then diagnosis assumptions were made, and swabs were streaked onlyon media associated with the causative pathogen . If no colonies grew, then additional media weretested. Bacterial genera of isolates were confirmed via morphologicalcharacteristics and a range of biochemical tests, including gram staining,catalase, oxidase, indole, motility, citrate, urease, gas production,Voges–Proskauer, and a number of sugar tests, following thestandard protocols as described by Holt et al.35 A lack of access to reference strains limited efforts forquality control. Following genera identification, resistance of isolateswas assessed using the disc diffusion method according to Hombachet al.36 with some modification. In brief,pure inoculum was inoculated on Mueller–Hinton agar plates(preparation included 25 mL of liquid agar for each 100 mm disc).Commercial paper discs of 6 mm impregnated with antibiotics were placedon agar plates, and the plates were incubated at 35 °C for 24h. The diameters of the zone of inhibition were measured using a slidecalliper and SIRScan automatic reader .Antibacterial susceptibility was defined according to CLSI guidelineinhibition zone diameter values37 (Table S3B). Testing for resistanceto levofloxacin and neomycin did not begin until May 2020. All antibioticstested were classified as either critically (∗∗) orhighly (∗) important for human health, according to the WorldHealth Organization’s List of Critically Important Antimicrobialsfor Human Medicine.38In their work, moribundfish were transported from farms around the Mymensingh region to thelaboratory in pond water. Specific location data are not providedhere in order to maintain farmer anonymity. External lesions, suchas signs of necrotic skin, gill, or other external tissues or ulcers,were swabbed. In case of asymptomatic conditions, fish were dissected,and different organs  were examined macroscopically to identify any abnormalityand swabbed if affected. Due to the company’s aim of providinga rapid diagnostic service, CLSI guidelines were not strictly adheredto. Swabs were streaked directly onto selective media agar plates. Ethical approval for the datacollection was received from the University of Exeter’s BiosciencesEthics Committee on 5 March 2021 (Reference: eCLESBio000401). Farmingpractices among the farmers in the Mymensingh region are very similarin terms of drug and chemical usage,39 and30 representative farms from the Trishal, Phulpur, and Muktagachadistricts were selected for participation. All gave written consent.Socioeconomic data were collected byinterviewing 30 farmers from the Mymensingh region in March 2021 aspart of a United Kingdom Government ODA-funded project From our team’s experience, some farmers do not know whichof the drugs and chemicals they use are antibiotics. Thus, prior tothe interviews, we visited 41 farm shops in the region to gain a comprehensivelist of the antibiotics that were on sale. A photographic inventoryof the antibiotics was prepared so that farmers could identify whichcompounds they used from this list. Similarly, since many of the farmersdo not have formal disease diagnosis training, for questions referringto infectious disease diagnoses of cattle, we sought to confirm etiologies,where possible, by asking for diagnosis reports or prescriptions issuedby competent authorities. If these were not available, we asked forphotos and information on allied clinical signs and symptoms, thenvalidated these through consultation with veterinarians at BangladeshAgricultural University Veterinary Clinic.40 to minimize the emergence and spread of AMR in the environment andits transmission into the community and are further described in Table S5. The underlying “Pressures”and “Drivers” of each “State” were thenconsidered in turn, using our three data sets, existing literature,and our team’s extensive experience of working in this field.These were systematically documented, as shown in Table S10).We defined a rural aquacultureenvironment as encompassing all culture ponds, the land within theaquaculture farm area (upon which crops are often grown), and allhuman/other animal inhabitants within an individual farm environment.The “Impact” was then taken to be the presence of AMRin these environments, thus the “States” were definedas the environmental conditions that together provide the optimalconditions for the development of AMR, namely, (1) presence of infectiousdisease, (2) presence of antimicrobial compounds, and (3) opportunitiesfor human, animal, or plant exposure to infectious agents. For thepurposes of this study, we considered only environmental states whichdirectly lead to AMR emergence and/or transmission and therefore didnot include commensals (nonpathogenic microbes that live on or inorganisms). These states are based upon the framework for AMR environmentalrisk assessment proposed by Berendonk et al.41 the Drivers,Pressures, States, and Impact from Table S6) were imported into Gephi and visualized using node-sized rankedaccording to out-degree.To create the networkmap in Gephi version 0.9.2,Figure S1), for which we had access to threedistinct data sets: (i) a microbiological data set, collected betweenOctober 2019 and September 2020 by Quality Feed Ltd., a feed and pharmaceuticalcompany based within the Mymensingh region, (ii) a metagenomic DNAdata set, in which water from six ponds was sampled and analyzed forfour seasonal time points between April 2017 and February 2018, and(iii) a social data set, collected in February 2021 from 30 farmersthat were representative of the wider aquaculture community in theregion. More details of the case study region, the three data sets,and the application of the framework can be found in We applied the DPSIR frameworkto a case study of aquaculture environmentsin the Mymensingh region of north-central Bangladesh (Tables S7 and S8). Some of the amoxicillinresistance may be attributed to the intrinsic resistance commonlyreported in Aeromonas bacteria, which made up 34%(80 of 235) of the total isolates; however, amoxicillin resistancewas also detected in almost all of the other bacterial genera isolated(Table S8).We began byevidencing the “Impact”, which we defined as the presenceof AMR in our case study aquaculture environment. Systematic AMR surveillanceand monitoring data are not available to assess levels of AMR in theMymensingh region, as is typical for the vast majority of aquacultureenvironments; however, all three of our data sets provided evidenceof AMR. Our microbiological data set was limited to 12 bacterial generaisolated from diseased fish due to the laboratory methods and capacityat Quality Feed Ltd., but nevertheless clearly shows evidence of resistance,with very high levels of resistance in particular to amoxicillin anderythromycin .In our social data set, farmers listed a rangeof antibiotics whichthey had used previously but no longer use, with the main reason givenfor stopping use (93% of responses) being ineffectiveness. These includedamoxicillin, oxytetracycline, ciprofloxacin, and sulfamethoxazole+ trimethoprim  was then considered inturn. We used the three data sets, existing literature, and our team’sextensive experience of working in this field to explore the underlying“Pressures” and “Drivers” of each “State”in turn , with anaverage estimated annual production/profit loss due to disease of21% (range 5%–35%). Best management practices (BMPs) that helpto reduce disease, in particular, fish health management and biosecurity,are generally poor across Bangladesh due to very limited access totraining, education, diagnostics and advice, power, affordable creditfor investment, governance, and many other factors.47 Only 43% of farmers interviewed had accessto a fish disease diagnostic facility. The main symptoms of diseasereported were external skin lesions , and fin, gill, or tail rot, all indicatorsof infectious disease and/or stress (Table S4).Intensificationis being actively promoted by the Bangladesh Government’s Departmentof Fisheries as part of their development strategies48 In July,the monsoon season begins, and by August, high temperatures and heavysurface runoff bring further sediment and pollutants  into aquaculture ponds. In April, temperaturesrise quickly as the season transitions from winter to summer, andthis is also when farmers introduce fingerlings (juvenile fish) intoponds, so disease outbreaks are more likely as higher temperaturesand increasing biomass favor proliferation of many pathogen types.Water quality is an issue throughout the season, due toupstream microbial and chemical pollution from industrial, urban,agricultural, and aquaculture sources, and this is worsening withthe impacts of climate change.49 Similarly, infectious diseasesin other animals within the farm environment were reported by 70%of farmers (Table S4 and Table S4). These are considered poor biosecuritypractices, but are common features of traditional farming lifestyles.Within the rural aquaculture environment, infectious disease isalso relatively common among human and other animal inhabitants, largelyas a result of limited access to healthcare and sanitation/wastewatertreatment (WWT) infrastructure. Ninety-seven percent of farmers reportedthat people on their farm (family or workers) suffer from infectiousdiseases, with the most prevalent ones being gastrointestinal andskin diseases 4. This iTable S9). P. aeruginosa wasthe only fish pathogen identified. It is also a serious human pathogen,as is M. tuberculosis, and these bacteria are establishedas being widely multidrug resistant and common causes of human diseasein Bangladesh.51 Metagenomic analysis of environmentalsamples is only likely to detect pathogens present in relatively highnumbers, due to the high levels of taxonomic diversity within theseenvironments, and five of these pathogens are environmental bacteria.The exception is M. tuberculosis, which is usuallyconsidered an obligate parasite but is known to survive in anthropogenic environments, including humanwastewater.52 The detection of M. tuberculosis in our metagenomic data set could reflectregular human exposure to these aquaculture ponds, or it could bepresent due to upstream anthropogenic pollution of the incoming water.Alternatively, this may be an incorrect annotation, resulting fromthe heavy focus of the pathogen database on human disease and illustratingthe limitations of using metagenomics for environmental AMR characterization:mycobacterial diseases of fish are common in aquaculture, and so itis possible that the pathogen identified is an uncharacterized Mycobacteriumthat is very similar to Mycobacterium tuberculosis.In line with the social data set, our metagenomicdata set alsoidentified a number of pathogens (including fish pathogens) acrossall samples collected, but only six at sufficient genomic coverageto reach our confidence cutoff limit for confirming presence (Table S1), although we understand that a more comprehensive NAP thatincludes aquaculture is soon to be released.53 The Bangladesh Government is also (as of July 2022) preparing anew Guideline for Antimicrobial Consumption Surveillance inBangladesh that outlines a monitoring framework incorporatingboth human and animal health sectors.54 Regulations on antibiotic usage in Bangladesh aquaculture do existbut are very limited. They are poorly implemented due to insufficientmonitoring and surveillance, poor institutional resource capacity(in particular staffing), and poor regulatory control on aggressiveand irrational marketing.56 A 2020 policy fromthe Government’s Ministry of Fisheries and Livestock bannedantibiotic use in food production, with the exception of oxytetracyclineand sulfadiazine which are allowed under prescription.57 In our social data set, only one farmer couldname the antibiotics that are approved for aquaculture use, and noneof the farmers kept records of drug use. There was also poor awarenessand knowledge on AMR in general and much uncertainty over the recommendedwithdrawal period . Antibiotic misuse was common amongthe farmers interviewed in our social data set, with 23% of use reportedas prophylactic (prevention of disease), a practice that is widelydiscouraged and banned in many countries (Table S4). Of the antibiotic treatments reported, 44% were higherthan the recommended dosage given on the label, and 66% of farmersdid not complete the full course of antibiotics. Some farmers reporteduse of two different drugs which contained the same active ingredient,and farmers commonly reported using more than one antibiotic withinthe same time period. In the 12 months prior to interviews, a rangeof antibiotics were used by farmers, and on average, these accountedfor 6% of business costs . Farmers reported that their antibiotic usage was greatestin April and August, which correlates with increasing infectious diseaselevels due to seasonal climate and water quality variations (see above).Antibioticusage is currently poorly governed in the Bangladesh aquaculture sector.As in many LMICs, antibiotics are readily available over the counteracross Bangladesh, without a prescription. Aquaculture is currentlynot addressed in the Bangladesh AMR NAP (Table S4). Commercial aquaculture feedmay itself be another route by which antibiotics enter the pond environment,with reports that feed companies are continuing to add antimicrobialsto aquaculture feed in order to prolong shelf life, a practice thatwas banned in Bangladesh through the 2010 Fish Feed and Animal FeedAct.59 Unused feed and antimicrobial residues canaccumulate in pond sludge,60 and BMPs stipulatethat sludge should be removed at least annually, but all farmers inthis study reported removal less often than this. Ninety-three percentof farmers disposed of their sludge onto the pond dykes surroundingthe pond, so runoff during rainfall may provide another route forthese antimicrobial compounds to re-enter ponds. All farmers sourcedtheir water from underground, which in Bangladesh is widely contaminatedwith heavy metals and other agrochemical compounds that are knownto co-select for AMR.62 Many aquaculture farmers dischargewater into the same local water system, with little or no WWT; thus,pollution from upstream farms (aquaculture and agriculture) can alsointroduce antimicrobials to the rural aquaculture environment duringflooding (Table S4).Antibiotics used in aquaculture are most commonly purchased fromlocal farm supply shops. We catalogued 48 different antibiotics beingsold by farm shops in the Mymensingh region. Of these, 42 gave informationon how to use the drugs on the label, although active ingredient concentrations(and therefore doses) varied between manufacturers, and 6 of the 42labels included terms promoting prophylactic use. All of the farmersapplied antibiotics by mixing with feed (as is recommended), but only17% followed personal safety precautions when handling antibiotics.Antimicrobialscan enter rural aquaculture environments via theiruse by human and other animal inhabitants in order to treat diseaseor  for prophylactic use. Of those reported in our socialdata set, 76% of human disease cases and 100% of animal disease caseswere commonly treated with antibiotics 4. AntibiTable S4). Exposure pathways related to human/animal consumptionof vegetation grown within the farm environment were also identified.For example, 77% of farmers used pond water for agricultural farming,and 97% of farmers discharged pond water to rice fields after harvest.Thirty percent of farmers used pond water for rearing animals  on a daily basis. Dead animals, which are likelyto contain pathogens (and antibiotic residues), were commonly fedto wild animals (reported by 53% of farmers), who may also serve ashosts for pathogen propagation. Some farmers reused dead animals bydrying for fish meal (20%) or use as a vegetation fertilizer (13%).Dead animals were also disposed of by throwing “to and fro”(27%), burying (23%), or releasing directly (13%) or indirectly (10%)into local water channels. Due to the open nature of the farms, allfarmers reported daily access of wild birds, which have been shownto contribute to AMR dissemination in a number of studies67 (Table S4).Traditional farming methods providemany routes for human, animal, and plant exposure to pathogens. Farmersall sold their fish at the local wholesale market, and on average,their household consumed 3% (range 1%–5%) of their production.All farmers reported regular (daily/weekly) human and animal exposureto pond water, for sampling, harvesting, application of drugs andchemicals, observation, and bathing purposes .According to the DPSIR framework1, the stnfluence 5. In ter/drivers 1, and ou/drivers 5 would b71 Effective AMR strategies therefore need to move upstream from surveillanceand stewardship of antibiotic usage on a local (farm) level and takea greater focus on a more proactive, preventative approach. This hasbeen shown to be an issue for AMR strategies in general, with globalAMR commitments from 2015 to 2021 remaining heavily focused on research,surveillance, and stewardship rather than the more upstream, preventativemeasures outlined in the 2015 AMR Global Action Plan.72 This further speaks to the need for AMR-sensitive approachesand the integration of AMR into wider sustainable development strategies.7 A recent study evaluating the implementation of the Bangladesh AMRNAP reported a lack of activities targeting AMR in the environmentalsector, a shortage of a dedicated and trained workforce, and insufficientfunding.73 Integrating AMR into wider sustainabledevelopment policies may also therefore prove to expedite NAP implementationfrom an efficiency perspective, through making use of existing funding,resources, and activities targeting the wider sustainable developmentgoals.This work illustratesthe complexity of the issue of AMR in rural LMIC aquaculture environmentsand supports many other studies on AMR in rural LMIC contexts whichhave consistently shown that antibiotics are often used as “quickfix” solutions to complex social and economic situations.74 Here, we foundthat DPSIR was useful as a conceptual framework to help systematicallystructure our enquiries into the relationships between AMR and thecomplex underlying social and ecological causal factors in a ruralaquaculture environment, but there was considerable fluidity in definingthe drivers and pressures. This is also one of the most common discrepanciesreported by previous studies using DPSIR74 and in our case reflects the intrinsic complexity, interdependency,and synergy between these underlying factors, many of which are sustainabledevelopment issues themselves. Other studies have dealt with thisreal-world complexity by adapting DPSIR to meet their needs, for example,through using a nested DPSIR approach 75 or reframing DPSIR toincorporate the four spheres of the sustainability model .76 Thus, future development of the DPSIR framework for applicationto AMR in LMIC rural food production environments should examine howto most effectively and appropriately structure these aspects of theframework.Since its development in the 1990s, the DPSIR frameworkhas been applied to a very wide variety of issues and contexts, asboth a conceptual and analytical tool.77 In comparison to the more conventionaltop-down AMR policy implementation methods, we believe that this bottom-upapproach, beginning at the community level, offers a more pragmaticroute to identifying potential intervention targets, and we believethis is the first study to demonstrate meaningful connectivity betweenAMR and such a wide range of sustainable development issues. Whenapplying a conceptual framework in this way at a community level,we expect that the value of the outputs will be relative to the qualityof the data, knowledge, and expertise available for input and thereforethat more valuable outputs  will be gained from engaging with a wide range of localstakeholders in order to gain a perspective of the issue that is asbroad and deep as possible. An important challenge here will be howto best ensure objectivity when doing so. Each of these pressures/driversis in itself a complex issue, so more comprehensive analyses shouldalso better establish how far the boundaries of the framework applicationshould be extended in order to facilitate interconnectivity whilecontaining it enough to maintain practicability as a decision-makingtool. Equally, wider discussions on the most appropriate definitionof the states used when applying DPSIR to AMR would be useful, assome would argue that commensal microbes, as vectors for AMR transmission,should be included. Inclusion of commensals would blur the boundarybetween States 1 and 3, by increasing the scope of State 1 (Presenceof infectious disease) to include the presence of all human-, animal-,and plant-associated microorganisms  andexpanding State 3  to includeopportunities for exposure not just to humans, animals, and plantsbut also their commensals. In turn, this would impact on the pressures,drivers, and responses identified.To our knowledge, this is the first study to applya conceptualframework to a rural LMIC food production AMR case study. It is inline with a growing number of reports emphasizing the importance oftailoring implementation strategies to specific contexts by incorporatinglocal knowledge and expertise and integrating evidence from acrossdifferent disciplines.78 This would further aidintegration into wider sustainable development strategies and supportObjective 5 of the AMR Global Action Plan, “to develop theeconomic case for sustainable investment”, by better understandingand valuing the wider sustainable development goal co-benefits.5 We suggest that one or more of the states identifiedthrough the DPSIR application could also be used to measure the relativeimpact of different interventions on AMR, as described in Table S5. This may be a more accessible way ofmeasuring AMR intervention impacts, in the absence of large-scaleAMR surveillance and monitoring, so it may be more applicable to anLMIC environmental context.Overall, this study providesa useful foundation for developinga DPSIR framework that can be applied to AMR in rural LMIC food productionenvironments. Further work is needed to establish the appropriateparameters and categorization of variables within the framework, butonce achieved, this approach will provide novel and valuable opportunitiesto more effectively implement AMR policy and overcome the AMR actiongap. Systems modeling techniques could be employed to incorporatethe weightings of different factors and support quantification ofthe broader socioeconomic costs/benefits of different AMR-sensitiveinterventions.


Danio rerio) in uninfected or V. vulnificus-infected tissues using real-time quantitative PCR (qPCR). Furthermore, lysozymes that might be involved in the defense against V. vulnificus were selected for over-expression, and the antibacterial activity of over-expressed lysozyme proteins were evaluated using V. vulnificus. The results showed that three types of zebrafish lysozyme, i.e., c-type lysozyme (DrLysC), g1-type lysozyme (DrLysG1), and g2-type lysozyme (DrLysG2), were identified, and V. vulnificus infection significantly changed the expression levels of DrLysC and DrLysG1. Then, DrLysC and DrLysG1 were over-expressed in E. coli, and the purified recombinant DrLysC (rDrLysC) showed more potent antibacterial activity against V. vulnificus. This finding lays the foundation for further application of rDrLysC to treat V. vulnificus infection.Lysozyme is a crucial component of the host’s innate immune system. Due to its natural non-toxic and harmless characteristics, lysozyme is considered to be an ideal antibiotic substitute. In this study, we analyzed the expression profiles of lysozymes from zebrafish ( As aquatic vertebrates, fish have a strong innate immune system, which plays an essential role in the early defense against pathogens . LysozymOncorhynchus mykiss) [Epinephelus coioides) [Paralichthys olivaceus), Senegalese sole  [P. olivaceus) [Cyprinus carpio L.) [Siniperca chuatsi) [Larimichthys crocea) [Gadus morhua L) [Scophthalmus maximus) [P. olivaceus), grass carp (Ctenopharyngodon idella), and turbot (S. maximus) [The c-type lysozyme was first isolated from rainbow trout ( mykiss) , and latoioides) , Japanesalensis) , and othivaceus)  and thenrpio L.) , mandarichuatsi) , large y crocea) , Atlantiorhua L) , and turmaximus) . Both c-maximus) . Lysozymmaximus)  since itmaximus) . In fishmaximus) .Oplegnathus fasciatus) g-type lysozyme could inhibit Gram-negative bacteria V. salmonicida and Gram-positive bacteria Listeria monocytogenes [E. coioides) c-type lysozyme could inhibit Gram-positive bacteria Streptococcus iniae and Gram-negative bacteria V. alginolyticus [Antibiotics are used to treat bacterial diseases; however, their continuous use has resulted in a variety of drug-resistant bacteria, the deposition of drug residues, and environmental pollution . Thus, tytogenes , and recolyticus .Vibrio vulnificus is an important zoonotic pathogen, which causes skin ulcers, traumas, gastroenteritis, and primary sepsis in humans and aquatic animals, resulting in a high fatality rate and significant losses in the breeding industry. V. vulnificus FJ03-X2 is highly virulent and pathogenic strain, and it was previously isolated from a diseased European eel (Anguilla anguilla) by our group. We observed that the strain could significantly alter the expression of c-type lysozyme in different zebrafish tissues [V. vulnificus [ tissues , suggestlnificus .Danio rerio) lysozymes, namely DrLysC, g1-type lysozyme (DrLysG1), and g2-type lysozyme (DrLysG2), were identified, and their expression profiles in natural or V. vulnificus-infected tissues were analyzed using real-time quantitative PCR (qPCR) analyses. These two lysozymes, DrLysC and DrLysG1, which might be involved in the defense against V. vulnificus, were selected for over-expression. Furthermore, the antibacterial activity of the purified recombinant lysozyme proteins was evaluated using V. vulnificus. The results indicated that DrLysC was expressed in response to V. vulnificus infection, and the over-expressed rDrLysC showed excellent antibacterial activity against V. vulnificus. This study lays the foundation for further research on fish lysozymes and further application of rDrLysC.Zebrafish c-type lysozyme (DrLysC) was first cloned, which could be detected for expression in zebrafish macrophage cell line . Wang Z Three zebrafish lysozymes, including one c-type (DrLysC) and two g-type (DrLysG1 and DrLysG2), were retrieved from ZFIN . The seqFurthermore, a phylogenetic tree was constructed based on the amino acid sequences of DrLysC, DrLysG1, DrLysG2 and their homologs in fishes . Fish lyTo examine the constitutive expression patterns of DrLysC, DrLysG1, and DrLysG2, tissues of zebrafish were collected for qPCR analysis . The resV. vulnificus-infected zebrafish were collected at 0, 24, and 72 h post infection (p.i.) for qPCR analysis. The results showed that DrLysC and DrLysG1 were up-regulated after V. vulnificus infection  that form four pairs of the disulfide bond, while the number of Cys in the g-type lysozyme is usually uncertain, with no disulfide bonds . FurtherA. japonica), Chinese giant salamander (Andrias davidianus), and Qi river crucian carp (Carassius auratus), and the expression of g-type lysozyme in corresponding tissues was lower than that of c-type lysozyme [Lysozyme is a bactericidal, innate immune effector and one of the important evaluation indicators of host immune function. Both the c-type and g-type lysozymes could be detected in all of the examined tissues of Japanese eel  [External stimulation could change lysozyme expression in fishes. The expression level of both DrLysC and DrLysG1 were up-regulated in V. anguillarum and Flavobacterium sp. [S. maximus) showed strong antibacterial activity against Gram-positive bacteria Micrococcus luteus [The function of lysozyme is determined primarily via its structure. Although fish lysozymes are more potent on Gram-negative bacteria, they also showed antibacterial activity against Gram-positive bacteria. For example, rainbow trout lysozyme had a significant inhibitory effect on Gram-negative bacteria, such as rium sp. , while ts luteus .V. vulnificus is a bacterium that generally exists in the ocean, with a high mortality rate in immunocompromised patients [V. vulnificus infection [V. vulnificus strains might have an adverse impact on public health [V. vulnificus treatment. This study showed that both DrLysC and DrLysG1 play a crucial role in the defense against V. vulnificus. Over-expressed rDrLysC and rDrLysG1 with high purity (>90%) were successfully obtained. Further antimicrobial activity analysis showed that rDrLysC had 66.14% bacteriostatic activity against V. vulnificus but had no antimicrobial activity against Micrococcus luteus. On the contrary, rDrLysG1 showed no bacteriostatic activity against V. vulnificus but had antimicrobial activity against Micrococcus luteus. This suggested that zebrafish lysozymes might play different roles in the defense against different bacteria, and DrLysC might mainly act on Gram-negative bacteria and DrLysG1 mainly on Gram-positive bacteria. Further application study of the over-expressed rDrLysC paves a way for the prevention and treatment of V. vulnificus diseases.patients . Antibionfection . Howeverc health . Hence, V. vulnificus strain FJ03-X2 was isolated from European eel by the Institute of Biotechnology, Fujian Academy of Agricultural Sciences [Sciences , and culWild-type zebrafishes (AB strain) were purchased from China Zebrafish Resource Center (CZRC) and cultivated in an aquatic animal culturing system at 28 °C with 0.05% salinity. Fishes were fed with a commercial pellet feed  twice a day, and the feeding amount was 1% of the body weight.https://zfin.org/, accessed on 10 January 2020), and the conserved functional domains of the encoding proteins were analyzed using CDD Tools . The homologs of zebrafish lysozymes were retrieved from National Center for Biotechnology Information . A neighbor-joining phylogenetic tree was constructed using MEGA 4.0 software with 1000 bootstrap replicates.The sequence of zebrafish lysozymes was retrieved from the Zebrafish Information Network (5 CFU/mL of V. vulnificus FJ03-X2 (LD20), and the other group was intraperitoneally injected with 10 μL of PBS and used as control. Fishes that died and those that showed apparent morbidity were removed and discarded.Adult zebrafish (>6 months) with an average weight of 0.2 g were divided into two groups (120 fishes/group); one group was intraperitoneally injected (I.P.) with 10 μL of 2.94 × 10V. vulnificus FJ03-X2, liver, kidney, gill, and intestine of V. vulnificus-infected fishes were collected at 0, 24, and 72 h post injection. All samples were collected from ten fishes and pooled together with three replicates.To examine constitutive gene expression patterns of zebrafish lysozymes, fishes were euthanized with tricaine methane sulfonate . Tissues including heart, liver, spleen, kidney, gill, intestine, muscle, and skin were collected from healthy fishes. Each sample was collected from 10 tails as a parallel group with three replications. To examine the response of lysozymes against Total RNA was extracted from collected samples using TRIzol reagent . Quality and concentration of the isolated RNA were determined via DeNovix DS-11 Spectrophotometer/Fluorometer and stored at −70 °C until further use. Further, 1 µg of total RNA was taken for cDNA synthesis using HiScri Ⅲ RT SuperMix for qPCR (+gDNA wiper) .−∆∆Ct method, and β-actin was used as the internal control. Relative expression of zebrafish lysozymes in V. vulnificus-infected fishes was calculated using the 2−∆∆Ct method, and data are expressed as fold-change values. Each sample was tested in triplicate, and all data are presented as means ± SD. Statistical analysis was performed using two-way ANOVA for multiple comparisons. p < 0.05 indicated a significant difference.Specific primers were designed for the amplification of DrLysC, DrLysG1, and DrLysG2, and β-actin was used as the reference gene . qPCR waE. coli BL21 (DE3) cells, and protein expression was induced using IPTG. Expression of the recombinant protein was examined using SDS-PAGE, then the protein was purified using Ni-NTA Sefinose™ Resin  and eluted using imidazole. The resulting recombinant DrLysC (rDrLysC) or DrLysG1 (rDrLysG1) was stored at −70 °C for further analysis.The nucleotide sequence of DrLysC (GenBank accession No.: NM_139180.1) and DrLysG1 (GenBank accession No.: NM_001002706.1) were optimized according to their amino acid sequences. DrLysC and DrLysG1 were synthesized and cloned into pET-28a(+) expression vector. The constructed plasmids pET-DrLysC and pET-DrLysG1 were transformed into competent V. vulnificus FJ03-X2 at the mid-logarithmic phase was collected and diluted to 1 × 106 CFU/mL. Then, 50 μL bacterial suspension was taken and incubated with 10 μL (1μg/μL) purified rDrLysC or rDrLysG1 at 28 °C for 2 h, and sterile-water-incubated bacteria was used as control. The bacterial culture was added to a 96-well plate and cultured at 28 °C for 24 h, then OD600 was measured using an xMark™ Microplate Spectrophotometer . The growth of control bacteria was defined as 100%, and the relative antibacterial activity of the purified rDrLysC and rDrLysG1 were calculated. All the samples were tested in triplicate.A bacterial inhibition assay was performed as described previously  with minV. vulnificus strain FJ03-X2 was detected using the agar diffusion method. Briefly, the V. vulnificus was diluted to 106 CFU/mL and coated on Tryptic Soy soybean Agar (TSA) solid plate. The sterile filter paper (5 mm diameter) containing 10 μg of rDrLysC or rDrLysG1, respectively, was evenly pasted on the plate, and sterile water was used as control. The plates were incubated at 28 °C for overnight, and then, the inhibition zone was observed and photographed.The inhibitory effect of the rDrLysC and rDrLysG1 on the growth of V. vulnificus were cloned and successfully expressed in E. coli. The expressed rDrLysC had efficient antibacterial activity against V. vulnificus. These results laid a foundation for further application of rDrLysC to treat V. vulnificus infection.Three lysozymes, namely DrLysC, DrLysG1, and DrLysG2, were identified in zebrafish. These lysozymes showed different expression patterns in all the examined zebrafish tissues. DrLysC and DrLysG1, which might participate in the defense against


Allium mongolicum Regel (AMR) is a traditional Mongolian food. Various drying methods play an important role in foodstuff flavor. However, the effect of different drying methods on AMR is limited. In this study, freeze drying (FD), vacuum drying (VD), and hot-air drying (HAD) were applied to dry fresh AMR to a moisture content of 8% (wet basis); headspace gas chromatography mass spectrometry was adopted to identify volatile compounds in AMR; and principal component analysis and fingerprint similarity analysis based on the Euclidean distance was used to distinguish the fresh and three dried treatments. In total, 113 peaks were detected and 102 volatile compounds were identified. Drying causes significant changes to the amounts of volatile compounds in AMR, and the drying method plays a key role in determining which volatile compounds appear. Compared to FD, VD and HAD were more appropriate for drying AMR because the volatile compounds after VD and HAD were closer to those of fresh AMR. These findings can provide a scientific basis to help to preserve future seasonal functional food and aid in Mongolian medicine production. Allium mongolicum Regel (AMR), a perennial Liliaceae herb native to Mongolia, Kazakhstan, and China  [injiang)  and alsoinjiang) . AMR is AMR is a traditional Mongolian medicinal herb . Its leaAMR is widely distributed on grassland, and it is eaten by both humans and sheep. It is found that sheep meat from AMR-pasture-fed animals tastes better. Zhao et al.  found th4 emission, and the antioxidant capacity of Simmental calves in northwest China [Some research yielded AMR extracts to feed sheep ,6. The wst China .However, fresh AMR is seasonal and perishable because of its high moisture content, making AMR difficult to be enjoyed year round. Overcoming this seasonality is crucial to utilizing AMR. Drying, as an essential processing operation, can reduce moisture to a safe level at which microorganisms and deterioration reactions are inhibited. Freeze drying (FD) has the highest cost, but produces the highest quality food products among all drying methods ,10. Hot-Tricholoma matsutake Singer under different heating temperatures and times [Flavor is mainly determined by volatile components and is a key factor for consumers to accept dried AMR. Headspace gas chromatography mass spectrometry (HS-GC-MS) is a new and reliable method used to identify volatile compounds in foods, such as fermented rose jams , Dezhou nd times , and a tnd times . Their rnd times . Changesnd times . Their rOnce moisture is removed from AMR, it has a potential to be used as a feed supplement and edible spice over a long term. However, dried AMR still has not been widely adopted. Until now, the kinds of drying methods that can be employed to dry AMR and retain flavor to its maximum are still not subject to research. In this study, HS-GC-MS was used to detect volatile components of AMR after processing by HAD, VD, and FD. The effect of these drying methods on the volatile compounds was studied with principal component analysis (PCA) and Euclidean distance analysis. Finally, the knowledge obtained from this study has the potential to be used to improve the industrial processing of AMR traditional feed supplements and edible production.Fresh aerial parts of AMR were purchased from a local market in Baotou, Inner Mongolia, China, in August 2021. These parts were washed and the surface water of the sample was removed by centrifugation. Then, samples were cut into segments of 5 cm in length. Portions of each sample were immediately stored at 4 °C in sealed plastic bags as samples. Before the drying experiment began, the AMR in a sealed plastic bag was taken out and placed indoors until its temperature stabilized. The other samples were placed in a polypropylene tray and frozen at −25 °C for at least 8 h as the frozen samples. The initial moisture content of the sample was 91.93% (wet basis).The AMR samples were dried by using different drying methods until the final moisture content was <8% (wet basis). These methods are described below. After drying, samples were vacuum-packed and stored for 10 days at ambient temperature (25 °C ± 5 °C) for subsequent analyses.The frozen samples (500 g) were dried by using a freeze dryer . The cold trap temperature was set at −60 °C, and the pressure of the drying chamber was set at 2 Pa during the drying process. Drying was completed after 29 h.4 Pa during the drying process. Drying was completed after 36 h.The samples (500 g) were dried by using a vacuum dryer . The drying temperature was set at 50 °C and the pressure of the drying chamber was set at 2.0 × 10The samples (500 g) were dried by using a hot-air dryer . The drying temperature was set at 50 °C. Drying was completed after 46 h.®, Gesellschaft für Analytische Sensorsysteme mbH, Dortmund, Germany) equipped with an autosampler  with a headspace sampling unit and a 1 mL gas-tight syringe . The GC was equipped with an MXT-WAX capillary column (30 m × 0.53 mm × 1 μm).The analyses were performed on a GC-MS . Laboratory Analytical Viewer was used to view the analytical spectrum, where each dot represents a volatile compound. A reporter plugin was directly used to compare the spectral differences between samples . A gallery plot plugin was used to compare fingerprints and visually and quantitatively compare the differences in volatile components among different samples. A dynamic PCA plugin was used for dynamic PCA and clustering analysis of the samples and to quickly determine the types of unknown samples. GC-IMS Library Search is an application software that was used to qualitatively analyze substances based on the NIST2014 Mass Spectral Library and IMS Library.Y-axis represents the retention time of the gas chromatograph, the X-axis represents the ion migration time for identification, and the Z-axis represents the peak height for quantification. As can be seen from The differences in volatile compounds in AMR samples from fresh and different drying methods were analyzed by using HS–GC–IMS. The data are represented by three-dimensional topographical visualizations in The data are represented by two-dimensional topographical visualizations in The difference comparison model was applied to compare the differences of AMR samples. The topographic plot of fresh AMR was selected as a reference, and the topographic plot of dried samples was deducted from the reference. The results are shown in To further compare the specific volatile compounds in each group of samples, all peaks were selected for fingerprint plot comparison, as shown in The analysis of this fingerprint plot clearly illustrates the differences in volatile components of AMR with different drying methods. As shown in 2-Propanone, isopentanol, 2-butanone, butanal, methyl acetate, 1-penten-3-ol, isobutyric acid, ethyl trans-2-butenoate, 2-butanol, 3-methyl-2-butenal, hexanal, 1-penten-3-one, heptanal, 6-methyl-5-hepten-2-one, 3-methyl butanal D, 2-methyl-1-heptene, and 2,6-dimethyl pyrazine, labelled as B, were not detected in fresh AMR samples, but exist in dried AMR samples, so there were generated during drying. Alpha-terpinene M, 1,8-cineole M, isovaleric acid, alpha-phellandrene, 2,3-dihydro-4-hydroxy-2,5-dimethyl-3-furanone, hexane nitrile, 2,3-dimethyl-5-ethyl pyrazine, n-hexanol, 1,8-cineole D, methyl hexanoate, alpha-terpinene D, (Z)-4-heptenal, and 2-methyl-2-pentenal D, labelled as C, were not detected in fresh AMR samples, but only exist in the FD AMR samples, so these compounds can only be formed in FD.The amounts of dimethyl disulfide, methyl propyl disulfide M, methyl propyl disulfide D, amyl acetate, ethyl hexanoate M, dipropyl trisulfide M, ethyl hexanoate D, propyl butyrate D, 2-ethyl furan, 1-butanol, (3E)-hexenol, (E)-2-hexenal, 3-hepten-2-one, and 2-methyl-1-butanol, labelled as D, dramatically decreased once the fresh AMR samples were dried. The amounts of 3-methyl butanal M, 2-hexen-1-ol M, and 2-hexen-1-ol D, labelled as E, were similar in both the fresh and dried AMR samples, so these compounds cannot be significantly damaged by the drying methods used in this study.The amounts of methional, N-nitroso-morpholine, propyl 1-propenyl disulfide M, 2,3-diethyl-5-methyl pyrazine, propyl hexanoate, 2-acetyl furan and diallyl disulfide, labelled as E, decreased in dried AMR samples and their signal intensity was similar among different drying methods. Ethyl sulfide and ethyl acetate, labelled as E, suffered the most serious damage under FD and suffered less damage under VD and HAD. The amount of 2-methyl-2-pentenal M, labelled as E, was not decreased in FD, but almost vanished in VD and HAD, that is, 2-methyl-2-pentenal M in fresh AMR samples can only be preserved well by FD. This means that 2-methyl-2-pentenal M can be protected well by low temperature and sublimation.Volatile components labelled as F can appear after drying. Of these, hexyl butanoate formed in HAD; acetoin formed in VD and HAD; 3-methyl-2-pentanone, methyl 3-methyl butanoate, and 3-methyl-1-pentanol formed in VD; and 2-ethyl-1-hexanol formed in FD and VD.After analyzing AMR samples under different drying methods, we tentatively identified a total of 113 typical target signals by comparing the feature retention and drift times with those of the individual standard ion signals. These were confirmed by using the commercial GC-IMS Library Search and are represented by different numbers (1–113). The identified and unidentified compounds are listed in PCA is a multivariate statistical analysis technique established using signal intensity to highlight the differences in volatile compounds. The PCA results of the volatile compounds in the fresh and dried AMR samples are presented in The PCA results clearly show fresh and different dried AMR samples in a relatively independent space and these would be well distinguished in the distribution map. Dried AMR samples can be well distinguished according to the positive score values of PC1, while fresh samples can be well defined according to the negative scores of PC1, and the difference in different parts from different areas can be separated by combining the scores with the score values of PC2.From In this study, a total of 113 signal peaks from topographic plots were identified in the fresh and dried AMR samples treated by using different drying methods. The drying process changes the amounts of volatile components found in the fresh AMR. Different drying methods significantly affected the volatile components of AMR. A total of 28 new volatile components formed under the operation of FD, VD, and HAD, of which 14 new volatile components can only be formed in FD. Thus, the differences of volatile components between FD samples and fresh samples were much greater than those between fresh samples and VD and HAD samples. That is, FD is unsuitable for attaining dried AMR samples if the maximum amounts of volatile components in dried samples is required. In this study, the volatile component variation under FD, VD, and HAD was discussed in detail, and the basic experimental data were supplied for different requirements based on the choice of volatile components desired.