CD4+ T lymphocyte responses to viruses and virus-relevant stimuli in teleost fish.

Fish diseases caused by viruses are a major threat to aquaculture. Development of disease protection strategies for sustainable fish aquaculture requires a better understanding of the immune mechanisms involved in antiviral defence. The innate and adaptive arms of the vertebrate immune system collaborate to mount an effective defence against viral pathogens. The T lymphocyte components of the adaptive immune system, comprising two major classes (helper T, Th or CD4+ and cytotoxic T lymphocytes, CTLs or CD8+ T cells), are responsible for cell-mediated immune responses. In particular, CD4+ T cells and their different subsets orchestrate the actions of various other immune cells during immune responses, making CD4+ T cells central drivers of responses to pathogens and vaccines. CD4+ T cells are also present in teleost fish. Here we review the literature that reported the use of antibodies against CD4 in a few teleost fish species and transcription profiling of Th cell-relevant genes in the context of viral infections and virus-relevant immunomodulation. Studies reveal massive CD4+ T cell proliferation and expression of key cytokines, transcription factors, and effector molecules that evoke mammalian Th cell responses. We also discuss gaps in the current understanding and evaluation of teleost CD4+ T cell responses and how development and application of novel tools and approaches to interrogate such responses could bridge these gaps. A greater understanding of fish Th cell responses will further illuminate the evolution of vertebrate adaptive immunity, inform strategies to address viral infections in aquaculture, and could further foster fish as model organisms.

Membrane vesicles from antibiotic-resistant Staphylococcus aureus transfer antibiotic-resistance to antibiotic-susceptible Escherichia coli.

AIM: Bacteria naturally produce membrane vesicles (MVs), which have been shown to contribute to the spread of multi-drug resistant bacteria (MDR) by delivering antibiotic-resistant substances to antibiotic-susceptible bacteria. Here, we aim to show that MVs from Gram-positive bacteria are capable of transferring ß-lactam antibiotic-resistant substances to antibiotic-sensitive Gram-negative bacteria. MATERIALS AND METHODS: MVs were collected from a methicillin-resistant strain of Staphylococcus aureus (MRSA) and vesicle-mediated fusion with antimicrobial-sensitive Escherichia coli (RC85). It was performed by exposing the bacteria to the MVs to develop antimicrobial-resistant E. coli (RC85-T). RESULTS: The RC85-T exhibited a higher resistance to ß-lactam antibiotics compared to the parent strain. Although the secretion rates of the MVs from RC85-T and the parent strain were nearly equal, the ß-lactamase activity of the MVs from RC85-T was 12-times higher than that of MVs from the parent strain, based on equivalent protein concentrations. Moreover, MVs secreted by RC85-T were able to protect ß-lactam-susceptible E. coli from ß-lactam antibiotic-induced growth inhibition in a dose-dependent manner. CONCLUSION: MVs play a role in transferring substances from Gram-positive to Gram-negative bacteria, shown by the release of MVs from RC85-T that were able to protect ß-lactam-susceptible bacteria from ß-lactam antibiotics. SIGNIFICANCE AND IMPACT OF STUDY: MVs are involved in the emergence of antibiotic-resistant strains in a mixed bacterial culture, helping us to understand how the spread of multidrug-resistant bacteria could be reduced.

Aerosol Delivery of Synthetic mRNA to Vaginal Mucosa Leads to Durable Expression of Broadly Neutralizing Antibodies against HIV.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

The Importance of Porins and ß-Lactamase in Outer Membrane Vesicles on the Hydrolysis of ß-Lactam Antibiotics.

Gram-negative bacteria have an outer membrane inhibiting the entry of antibiotics. Porins, found within the outer membrane, are involved in regulating the permeability of ß-lactam antibiotics. ß-lactamases are enzymes that are able to inactivate the antibacterial properties of ß-lactam antibiotics. Interestingly, porins and ß-lactamase are found in outer membrane vesicles (OMVs) of ß-lactam-resistant Escherichia coli and may be involved in the survival of susceptible strains of E. coli in the presence of antibiotics, through the hydrolysis of the ß-lactam antibiotic. In this study, OMVs isolated from ß-lactam-resistant E. coli and from mutants, lacking porin or ß-lactamase, were evaluated to establish if the porins or ß-lactamase in OMVs were involved in the degradation of ß-lactam antibiotics. OMVs isolated from E. coli deficient in ß-lactamase did not show any degradation ability against ß-lactam antibiotics, while OMVs lacking OmpC or OmpF showed significantly lower levels of hydrolyzing activity than OMVs from parent E. coli. These data reveal an important role of OMVs in bacterial defense mechanisms demonstrating that the OmpC and OmpF proteins allow permeation of ß-lactam antibiotics into the lumen of OMVs, and antibiotics that enter the OMVs can be degraded by ß-lactamase.

Characterization of a specific monoclonal antibody against immunoglobulin light kappa/L1 chain in olive flounder (Paralichthys olivaceus).

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a+ B cells were more abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.

Phylogenomic network and comparative genomics reveal a diverged member of the ΦKZ-related group, marine vibrio phage ΦJM-2012.

Bacteriophages are the largest reservoir of genetic diversity. Here we describe the novel phage ΦJM-2012. This natural isolate from marine Vibrio cyclitrophicus possesses very few gene contents relevant to other well-studied marine Vibrio phages. To better understand its evolutionary history, we built a mathematical model of pairwise relationships among 1,221 phage genomes, in which the genomes (nodes) are linked by edges representing the normalized number of shared orthologous protein families. This weighted network revealed that ΦJM-2012 was connected to only five members of the Pseudomonas ΦKZ-like phage family in an isolated network, strongly indicating that it belongs to this phage group. However, comparative genomic analyses highlighted an almost complete loss of colinearity with the ΦKZ-related genomes and little conservation of gene order, probably reflecting the action of distinct evolutionary forces on the genome of ΦJM-2012. In this phage, typical conserved core genes, including six RNA polymerase genes, were frequently displaced and the hyperplastic regions were rich in both unique genes and predicted unidirectional promoters with highly correlated orientations. Further, analysis of the ΦJM-2012 genome showed that segments of the conserved N-terminal parts of ΦKZ tail fiber paralogs exhibited evidence of combinatorial assortment, having switched transcriptional orientation, and there was recruitment and/or structural changes among phage endolysins and tail spike protein. Thus, this naturally occurring phage appears to have branched from a common ancestor of the ΦKZ-related groups, showing a distinct genomic architecture and unique genes that most likely reflect adaptation to its chosen host and environment.

Combination treatment against scuticociliatosis by reducing the inhibitor effect of mucus in olive flounder, Paralichthys olivaceus.

The olive flounder, Paralichthys olivaceus, is an economically important food fish in Japan and Korea. Scuticociliatosis is a major parasitic disease, and fatal infection with scuticociliates, or mixed infections with scuticociliates and other pathogenic agents (e.g., Vibrio spp.) cause severe mortalities in farmed olive flounders. To date, however, effective chemotherapeutic treatment of scuticociliatosis has only been reported at the in vitro level. In this study, we employed combination treatment, using benzalkonium chloride (to remove excess mucus from the body surface) and bronopol (to kill the parasites), to overcome the protective effect of mucus by some medicine to the scuticociliates. In the presence of the mucus mixture, the higher dose of bronopol (156 ppm) yielded morphologies and motilities similar to those of ciliates treated with the lower dose of bronopol (80 ppm) in the absence of mucus. We also investigated the in vivo effects of this treatment in field trials involving a total of 15,025 naturally infected flounders. We observed that short-term bath treatments with benzalkonium chloride (50 ppm) followed by bronopol (500 ppm) were effective, assessed by the relative percentage mortality (RPS) value. Thus, this study provides a notable therapeutic strategy by removing the mucus to treat scuticociliatosis in olive flounders at the aquaculture field level.

Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus.

Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.

Complete genome sequence of the bacteriophages ECBP1 and ECBP2 isolated from two different Escherichia coli strains.

Escherichia coli is recognized as one of the most abundant avian bacterial pathogens. In this study, we report the sequencing by the traditional Sanger method of ECBP1 and ECBP2: bacteriophages that infected two different E. coli strains which might be used as therapeutic agents in combination with alternative antibiotics.

Heat shock protein profiles on the protein and gene expression levels in olive flounder kidney infected with Streptococcus parauberis.

Heat shock proteins (HSPs) have been observed in cells exposed to a variety of stresses, including infectious pathogens. This study used a label-free, quantitative proteomic approach and transcriptional gene expression analysis to investigate infection-related HSP proteins and their encoding genes in whole kidneys from olive flounder (Paralichthys olivaceus). During Streptococcus parauberis infection in the flounder, the genes encoding Hsp10, Hsp40A4, Hsp40B6, Hsp40B11, Hsp60, Hsp70, glucose regulated protein 78 (Grp78), Hsp90α, Hsp90ß and Grp94 were induced, and the protein levels of Hsp60, Hsp70, Hsp90α, Hsp90ß and Grp94 were differentially regulated over time. Subsequent results also revealed that Hsp60, Hsp70, Hsp90α, Hsp90ß and Grp94 appear to be the dominant and critical HSPs in olive flounder during bacterial infection. This is the first estimation of the differential involvement of HSPs in the immune response of olive flounder exposed to bacterial infection.

Feeding Behavior Comparison of Bean Bugs, Riptortus pedestris and Halyomorpha halys on Different Soybean Cultivars.

Riptortus pedestris (Fabricius) and Halyomorpha halys (Stål) are the major pests that feed on soybean pods, seeds, and fruits. Higher populations and damage occur during the soybean maturity stages (podding to harvest). To compare the feeding behavior of R. pedestris and H. halys, we used the six most cultivated cultivars (Daepung-2ho, Daechan, Pungsannamul, Daewon, Seonpung, and Seoritae) in Korea using the electropenetrography (EPG) technique. Both R. pedestris and H. halys, the NP (non-penetration), a non-probing waveform, was the shortest in the Pungsannamul (298 and 268 min) and the longest in the Daepung-2ho (334 and 339 min), respectively. The feeding waveforms Pb (phloem feeding: E1-Salivation and E2-Sap feeding) and G (xylem feeding) were the longest in Pungsannamul and the shortest in Daepung-2ho. In addition, as a result of investigating the damage rate by planting six cultivars of beans in the field, as expected, the proportions of damage types B and C were highest in Pungsannamul and lowest in Daepung-2ho. These results reveal that both bug species ingest xylem sap from leaflets and stems using a salivary sheath strategy to acquire water and nutrients from soybean pods/seeds with cell-rupture tactics. This study provides perceptive information to understand the feeding behavior, field occurrence, and damage patterns of R. pedestris and H. halys, which may have key implications for the management of hemipteran pests by determining the specificity and susceptibility of host plants.

Pathogenesis of and strategies for preventing Edwardsiella tarda infection in fish.

Edwardsiella tarda is one of the serious fish pathogens, infecting both cultured and wild fish species. Research on edwardsiellosis has revealed that E. tarda has a broad host range and geographic distribution, and contains important virulence factors that enhance bacterial survival and pathogenesis in hosts. Although recent progress in edwardsiellosis research has enabled the development of numerous, highly effective vaccine candidates, these efforts have not been translated into a commercialized vaccine. The present review aims to provide an overview of the identification, pathology, diagnosis and virulence factors of E. tarda in fish, and describe recent strategies for developing vaccines against edwardsiellosis. The hope is that this presentation will be useful not only from the standpoint of understanding the pathogenesis of E. tarda, but also from the perspective of facilitating the development of effective vaccines.

Recombinant interferon-γ activates immune responses against Edwardsiella tarda infection in the olive flounder, Paralichthys olivaceus.

Interferon gamma (IFN-γ) is a cytokine that plays a very important role in defining Th1 immune response in all vertebrates. In this study, recombinant IFN-γ (rIFN-γ) from the olive flounder (Paralichthys olivaceus) was produced in an Escherichia coli system using a pET expression vector. Stimulation of whole kidney leukocytes (immune-related cells) in vitro with the resulting rIFN-γ significantly induced the gene expression of interleukin-1ß (IL-1ß), signal transducer and activator of transcription 1 (STAT1), CXCL13-like chemokine (CXCL13), and IFN-γ. rIFN-γ also weakly induced the expression of IL-1ß, tumor necrosis factor-α (TNF-α), CXCL13, and IFN-γ in olive flounder-derived HINAE (non-immune) cells. The effects of rIFN-γ against Edwardsiella tarda infection in vivo were assessed by intraperitoneally injecting a mixture of rIFN-γ (100 ng) and E. tarda (1 × 10(5) CFU/ml) into the olive flounder. The survival rate in the rIFN-γ-injected group was 60% compared to 0% in the group treated with E. tarda only, demonstrating that olive flounder IFN-γ is effective in reinforcing immune responses and preventing against edwardsiellosis.

Development of Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay To Detect Hemolysin Gene of Vibrio vulnificus in Oysters.

ABSTRACT: Vibrio vulnificus inhabits estuarine waters around the world and can cause severe infections in people who eat contaminated raw or undercooked oysters. Although current detection methods are sensitive and specific, there are continuous demands for the development of rapid and accurate methods without a trained operator and equipment in the field conditions. Herein, we developed a simple and rapid method by detecting the hemolysin (vvh) gene of V. vulnificus by using recombinase polymerase amplification (RPA) combined with a lateral flow dipstick (LFD). The RPA-LFD could detect 100 fg of DNA (P < 0.05) and 20 CFU of V. vulnificus per reaction within 30 min (P < 0.01) and showed the result with incubation temperature ranges from 30 to 45°C (P < 0.001). The test was specific only to V. vulnificus and was not responsive to 10 other closely related Vibrio species and 18 foodborne pathogenic bacteria. Compared with PCR, quantitative PCR, and colony hybridization assays by using naturally contaminated oyster samples, our RPA-LFD showed the same detection ability as quantitative PCR assay. Therefore, the current RPA-LFD would be a valuable tool to detect V. vulnificus in oysters, especially in field conditions.

Molecular Typing, Antibiotic Resistance and Enterotoxin Gene Profiles of Staphylococcus aureus Isolated from Humans in South Korea.

The emergence of antimicrobial-resistant Staphylococcus aureus has become a grave concern worldwide. In this study, 95 strains of S. aureus isolated from stool samples were collected from Busan, South Korea to characterize their antimicrobial susceptibility, enterotoxin genes, and molecular typing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and random amplification of polymorphic DNA (RAPD) assay. Only two strains showed no drug resistance, whereas resistance to three or more antibiotics was observed in 87.4% of strains. Ampicillin resistance was the most common at 90% and all strains were susceptible to vancomycin. The distribution of enterotoxin genes encoded in isolates was sea (32.6%), sec (11.6%), seg (19%), sea & sec (2.1%), and sec & seg (34.7%). Molecular typing using both MALDI-TOF MS and RAPD indicated that S. aureus exhibited diverse clonal lineages and no correlations were observed among the profiling of enterotoxin, MALDI-TOF MS, and RAPD. This investigation provides useful information on foodborne pathogenic S. aureus that has a significant public health impact in South Korea.

Antimicrobial Resistance, Pathogenic, and Molecular Characterization of Escherichia coli from Diarrheal Patients in South Korea.

Diarrheal diseases due to foodborne Escherichia coli are the leading cause of illness in humans. Here, we performed pathogenic typing, molecular typing, and antimicrobial susceptibility tests on seventy-five isolates of E. coli isolated from stool samples of patients suffering from foodborne diseases in Busan, South Korea. All the isolates were identified as E. coli by both biochemical analysis (API 20E system) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The bacteria displayed entero-pathogenic E. coli (EPEC) (47.0%), entero-aggregative E. coli (EAEC) (33.3%), entero-toxigenic E. coli (ETEC) (6.6%), ETEC and EPEC (6.6%), EPEC and EAEC (4%), and ETEC and EAEC (2.7%) characteristics. The E. coli isolates were highly resistant to nalidixic acid (44.0%), tetracycline (41.3%), ampicillin (40%), ticarcillin (38.7%), and trimethoprim/sulfamethoxazole (34.7%); however, they were highly susceptible to imipenem (98.6%), cefotetan (98.6%), cefepime (94.6%), and chloramphenicol (94.6%). Although 52 strains (69.3%) showed resistance against at least 1 of the 16 antibiotics tested, 23 strains (30.7%) were susceptible to all the antibiotics. Nine different serotypes (O166, O8, O20, O25, O119, O159, O28ac, O127a, and O18), five genotypes (I to V, random-amplified polymorphic DNA), and four phenotypes (A to D, MALDI-TOF MS) were identified, showing the high level of heterogeneity between the E. coli isolates recovered from diarrheal patients in South Korea.

Serotype-identifying ions in Listeria monocytogenes using matrix-associated laser desorption ionization-time of flight mass spectrometry.

Listeria monocytogenes is a foodborne pathogen that can cause a potentially life-threatening infection, and almost all cases of human listeriosis are caused by L. monocytogenes isolates in serotypes 1/2a, 1/2b, 1/2c, and 4b. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid, accurate, and high-throughput tool for clinical diagnosis and microbiological research. In the current study, we examined the potential of MALDI-TOF MS for rapid identification of the foodborne pathogen L. monocytogenes and to identify high-risk serotypes. To achieve this, MALDI-TOF MS was applied to 50 L monocytogenes strains. All strains were identified as L. monocytogenes species based on pattern matching against reference spectra for the species. Importantly, 83 specific mass ions were consistently and uniquely found in high-risk L. monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b. These 83 mass ions were also unique to specific combinations of these serotypes, which enabled specific identification of these four serotypes using MALDI Biotyper analysis. Hence, this method shows potential for using MALDI-TOF MS for the rapid identification of L. monocytogenes species and to discriminate high-risk L. monocytogenes serotypes through specific serotype-specific biomarker ions.

Complete genome sequence and immunoproteomic analyses of the bacterial fish pathogen Streptococcus parauberis.

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder (Paralichthys olivaceus) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis, by reaction with an antiserum obtained from S. parauberis-challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae.

Enhanced reliability of avian influenza virus (AIV) and Newcastle disease virus (NDV) identification using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

In-solution enzymatic and nonenzymatic digestion methods have been successfully implemented in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS)-based virus identification, extending to typing/subtyping of deadly influenza viruses. However, these methods are inefficient in obtaining more precise information on surface proteins of myxovirus particles, not only the hemagglutinin and neuraminidase of influenza virus but also the hemagglutinin-neuraminidase of Newcastle disease virus (NDV). Imbalances in viral protein composition cause ion suppression of tryptic fragments from low-abundant target proteins (surface proteins), adversely affecting reproducibility of mass spectra. Additionally, the coexistence of tryptic peptides from several proteins requires sophisticated statistical solutions for precise result interpretations. To circumvent these, we apply detergent-based (gel-free) partitioning of whole viruses into soluble surface proteins and insoluble virus materials, using differential centrifugation. MALDI-TOF or MALDI-TOF/TOF MS was applied to analyze tryptic peptides from separated viral proteins. In this study, we achieved type/subtype of avian influenza virus (AIV) within 5 h, based on 4 major proteins, by significantly reducing ion suppression and signal overlap from various protein sources. Hence, our approach can both yield dependable results and allow Web-based search engines to be directly employed, obviating the need for additional statistical strategy. Additionally, we demonstrate the utility of the method using NDV.

Significant increase in the secretion of extracellular vesicles and antibiotics resistance from methicillin-resistant Staphylococcus aureus induced by ampicillin stress.

Extracellular vesicles (EVs) containing specific cargo molecules from the cell of origin are naturally secreted from bacteria. EVs play significant roles in protecting the bacterium, which can contribute to their survival in the presence of antibiotics. Herein, we isolated EVs from methicillin-resistant Staphylococcus aureus (MRSA) in an environment with or without stressor by adding ampicillin at a lower concentration than the minimum inhibitory concentration (MIC). We investigated whether EVs from MRSA under stress condition or normal condition could defend susceptible bacteria in the presence of several ß-lactam antibiotics, and directly degrade the antibiotics. A comparative proteomic approach was carried out in both types of EVs to investigate ß-lactam resistant determinants. The secretion of EVs from MRSA under antibiotic stressed conditions was increased by 22.4-fold compared with that of EVs without stress. Proteins related to the degradation of ß-lactam antibiotics were abundant in EVs released from the stressed condition. Taken together, the present data reveal that EVs from MRSA play a crucial role in the survival of ß-lactam susceptible bacteria by acting as the first line of defense against ß-lactam antibiotics, and antibiotic stress leads to release EVs with high defense activity.