Mycobacterium avium and Mycobacterium abscessus. The incidence and prevalence of NTM infections are rising globally, especially in developed countries with declining incidence rates of M. tuberculosis infection. Mycobacterium avium, a slow-growing mycobacterium, is associated with Mycobacterium avium complex (MAC) infections that can cause chronic pulmonary disease, disseminated disease, as well as lymphadenitis. M. abscessus infections are considered one of the most antibiotic-resistant mycobacteria and are associated with pulmonary disease, especially cystic fibrosis, as well as contaminated traumatic skin wounds, postsurgical soft tissue infections, and healthcare-associated infections (HAI). Clinical manifestations of diseases depend on the interaction of the host’s immune response and the specific mycobacterial species. This review will give a general overview of the general characteristics, vulnerable populations most at risk, pathogenesis, treatment, and prevention for infections caused by Mycobacterium avium, in the context of MAC, and M. abscessus.Nontuberculous mycobacteria (NTM) are emerging human pathogens, causing a wide range of clinical diseases affecting individuals who are immunocompromised and who have underlying health conditions. NTM are ubiquitous in the environment, with certain species causing opportunistic infection in humans, including  Mycobacterium, which includes Mycobacterium tuberculosis, comprising more than 172 species with many unique virulence characteristics  incidence rate, while NTM disease rose by 94% in almost every geographic area [Nontuberculous mycobacteria (NTM) are a group of bacteria under the genus hic area . AnotherMycobacterium avium in the Mycobacterium avium complex (MAC), the rapid-growing M. abscessus, and M. kansasii. In this review article, we will discuss the vulnerable populations affected by these opportunistic infections, the pathogenesis, treatment, and prevention of infections caused by Mycobacterium avium, with a focus on the Mycobacterium avium complex (MAC), as well as infections caused by Mycobacterium abscessus.NTM infection mainly presents four distinct clinical diseases in humans: (1) chronic pulmonary disease; (2) disseminated disease in severely immunocompromised patients; (3) skin and soft tissue infection; and (4) superficial lymphadenitis, especially cervical lymphadenitis in children. Pulmonary disease accounts for 80–90% of NTM-associated diseases . The mosMycobacterium avium complex (MAC) infections are caused by a group of mycobacteria consisting of M. avium, M. intracellulare, and the newly described mycobacterium species, Mycobacterium chimaera, which are all acid-fast slow-growing mycobacteria, classified as non-chromogens in group III of the Runyon classification of NTM. MAC initially included only M. avium and M. intracellulare, but with new genetic sequencing technology, phenotypic and genotypic tests have identified additional species. To date, MAC contains nine species of slow-growing mycobacteria: M. avium, M. intracellulare, M. chimaera, M. colombiense, M. marseillense, M. timonense, M. boucherdurhonense, M. vulneris, M. arosiense, and a small group of unclassified, “MAC others” [M. chimaera was implicated in worldwide outbreaks in five countries including the USA, Europe, and Australia, due to contaminated heater–cooler unit water tanks in patients that underwent open-heart surgery, such as coronary artery bypass grafting [M. chimera corresponds to the sequevar MAC-A compared to the M. intracellulare sequevar MIN-A (DSMZ 43223) by 20 nucleotide (nt) mismatches. Additionally, the 16S rRNA gene sequence between the two species only has a 1 nt mismatch. Since the sequence of 16S rDNA is considered the approved standard for identifying NTM species, M. chimaera is usually misreported as M. intracellulare [M. avium, M. intracelllulare, or MAC as a whole since treatment is the same for all MAC species. Detailed genotyping of MAC species was limited to specialized research laboratories that can perform PCR of several genes such as rpo or hsp65 and ITS [M. chimaera from other MAC species [ others” . Recentlgrafting . Investigrafting . The seqellulare ,10. Trad and ITS . However species .Mycobacterium avium, with four distinct subspecies. This group of bacteria ranges from environmental bacteria that can cause opportunistic infections in immunosuppressed individuals to pathogens for birds and other animals. The characteristics of the four distinct species of M. avium are discussed. M. avium subsp. Hominissus (MAH) is an opportunistic environmental pathogen for humans, swine, and other animals that are found in soil and water. Drinking water and tap aerosols are considered the primary sources for infection in humans. MAH is clinically the most relevant organism within MAC for humans and a major pathogen for individuals with deficient T cell immunity. Before the development of antiviral therapy, in the early years of the Acquired Immunodeficiency Syndrome (AIDS) epidemic, 20–50% of severely immunocompromised AIDS patients had disseminated infections with MAC organisms, now identified as MAH infections [M. avium subsp. Paratuberculosis (MAP) is a widely known pathogen that causes paratuberculosis, also known as John’s disease, primarily affecting ruminants, causing chronic progressive infection in the small intestine. Non-ruminants, and non-human primates can be infected with MAP as well. MAP has a worldwide distribution with transmission mainly by the fecal–oral route via pasture, milk, and water contaminated with feces [M. avium subsp. Avium (MAA) mainly causes a tuberculosis-like disease in birds, the main reservoir of MAA. Avian TB is a chronic wasting disease, showing little signs of infection. A variety of mammals, but mostly pigs and cattle, can become infected with MAA [M. avium subsp. Silvaticum (MAS) is taxonomically similar to MAA and is a pathogen to the wood pigeon, causing TB-like disease. Some experimental studies also show that, in mammalian species, MAS can cause chronic enteritis [The most clinically significant organism for human disease within MAC is fections . MAH is fections . In immuth feces ,15. In hth feces . M. aviuwith MAA . Transmiwith MAA . M. aviunteritis .M. avium depends on its ability to colonize intestinal or respiratory mucosa, evade protective barriers, and resist intracellular killing in macrophages. The disease normally does not develop in most people; however, immunocompromised patients or patients with pre-existing conditions are at higher risk of MAC infections. There are three types of MAC infections that can occur: (1) pulmonary MAC infections, (2) disseminated MAC infections, and (3) MAC-associated lymphadenitis [The pathogenesis of adenitis .Pulmonary MAC infections affecting lungs are the most common type. These infections mainly affect patients with chronic lung diseases, such as chronic obstructive pulmonary disease (COPD), chronic bronchitis, bronchiectasis, cystic fibrosis, and lung cancer. Pulmonary infections are likely acquired through inhalation of aerosols from the natural or institutional water systems. The fibrocavitary disease form of MAC is common in older male smokers with chronic pulmonary symptoms. The nodular/bronchiectatic disease form is most common in nonsmoking women over the age of 50 with chronic lung disease . Another3, unlike M. tuberculosis that can infect patients at any stage of AIDS [M. avium was the etiologic agent in >95% of individuals living with HIV with advanced immunosuppression. In recent years, with the availability of antiretroviral therapy (ART), the rate of DMAC has fallen substantially [3 is approximately 20% [Disseminated MAC (DMAC) infections usually occur in patients in advanced stages of HIV/AIDS when blood CD4+ T cell counts are lower than 50/mm of AIDS . MAC is  of AIDS . In the  of AIDS . Before antially . Howevertely 20% .MAC can also cause lymphadenitis, mainly cervical lymphadenitis in immunocompromised children under five years old. However, one study indicated that it primarily affects healthy children under 15 years old with normal immune systems, and is an underrecognized cause of lymphadenitis in young children ,26. TherImmunosuppressive therapies taken by transplant recipients or patients with immune-mediated inflammatory diseases also lead to a higher risk of NTM infections. The use of corticosteroids, TNF-α treatment, or TNF-α inhibitors significantly increase the risk of developing pulmonary or disseminated NTM infections. One study found that immune-mediated inflammatory disease patients receiving anti-TNF-α treatment had elevated rates of mycobacterial disease and that NTM disease was associated with rheumatoid arthritis (RA) . For orgM. avium can resist the acidic pH conditions of the stomach and were able to gain access to the intestinal lumen. M. avium administered orally are isolated from the jejunum, ascending colon, and the majority in the terminal ileum [M. avium preferentially entering the terminal ileum via enterocytes of both Peyer’s patches and outside the Peyer’s region [These mycobacteria are acquired through the respiratory or the intestinal route, able to invade mucosal epithelial cells and translocate across the intestinal and respiratory mucosa to then infect phagocytic cells . Studiesal ileum . Other ss region .M. avium’s ability to evade epithelial cells is their interaction with fibronectin-attachment protein (FAP), allowing them to utilize fibronectin like a “bridge” to attach to integrin receptors found on the cell membrane of mucosal cells [M. avium invasion of epithelial cells, although direct evidence is lacking [Another mechanism associated with al cells . Middletal cells . Hemaggl lacking .M. avium triggers uptake on the apical surface, associated with a cytoskeleton reorganization and the phosphorylation of proteins [M. avium is not associated with an inflammatory response [M. avium may also bypass the mucosa, and then locate to lymph nodes of the mesentery, where it survives until the immune system is compromised, such as with AIDS patients [Once attached to the mucosal cells, proteins . The preresponse . Howeverresponse . Mechaniresponse . M. aviupatients .M. avium respiratory infections usually only affect patients with chronic lung disease, and their establishment of the infection could be due to the development of biofilms, which M. avium is known to establish in municipal water systems [M. avium contacts the alveolar space, it was found they are capable of invading type II alveolar epithelial cells and replicating within these cells [The pathway for infection of the respiratory tract is less well known than the intestinal route, but initiation is most likely through inhalation of aerosols of the bacteria.  systems . Once M.se cells .M. avium and most mycobacteria are in mononuclear phagocytes such as monocytes and macrophages. The bacterium can gain access into monocytes and macrophages by several different receptors: complement receptors, mannose receptors, type A scavenger receptors, and in hosts with mycobacteria-specific antibodies, Fc-gamma receptors [The primary host cells for eceptors . The useeceptors . Once boeceptors ,53,54,55M. avium involves both the nonspecific (innate) and specific (adaptive) immune system working together and secreting cytokines in response to M. avium. The innate immune response contains natural killer (NK) cells, which are critical components of this response, and have been confirmed to play a part in the nonspecific immune response against M. avium in mice and human systems [M. avium infection [M. avium has been shown to be CD4+ T lymphocyte-dependent and based on the generation of IFN-γ. CD8+ T cells roles are not well defined, but studies have shown that CD4+, but not CD8 T cells are required for adaptive immunity against M. avium [The host defense against  systems ,57. Micenfection . The hosM. avium ,60.M. avium. Macrophages stimulated with cytokines TNF-α, IFN-γ, and GM-CSF can control infection [M. avium [M. avium are less known, but preliminary research suggests that the activation of macrophages gives rise to changes in the vacuoles of the intracellular environment [Macrophages are the central players in the defense against nfection  and werenfection . IL-12 infection . NK cellM. avium . When stM. avium . AlthougM. avium . The exaironment .Mycobacterium avium complex (MAC) consists of most of the common pulmonary NTM pathogens that are heavily linked to the causes of human disease in almost all regions of the world [The he world . As prevhe world .The three categories of pulmonary MAC infections are as follows: (1) noncavitary nodular bronchiectatic disease, patients who have bronchiectasis clusters with less than 5-mm nodules; (2) cavitary nodular bronchiectatic disease, patients who also present with lung cavities in addition to bronchiectasis; and (3) fibrocavitary disease, patients with cavities, fibrosis, and/or pleural involvement over adjacent areas.The treatment of pulmonary MAC infections consists of antimicrobial therapy, particularly macrolides, followed by close follow-up monitoring for up to a year. For patients with noncavitary or mild/moderate bronchiectatic disease, the current guidelines recommend a three-drug, macrolide-based regimen (including a macrolide and ethambutol) for macrolide-susceptible MAC pulmonary disease.For example, a three-drug regimen of azithromycin (500 mg 3×/week), rifampin (600 mg 3×/week), and ethambutol (25 mg/kg 3×/week) can be used see . An azitIn more severe cases, such as in cavitary or advanced/severe nodular bronchiectatic disease, instead of 3×/week, a daily regimen is recommended. For example, azithromycin (250 to 500 mg daily), rifampin (600 mg daily), and ethambutol (15 mg/kg daily) can be used along with parenteral amikacin or streptomycin in the initial treatment . It is rFor patients with no positive outcome after 6 months with the guideline-based therapy, an amikacin liposome inhalation suspension (ALIS) treatment should be utilized in addition to their current treatment regimen. ALIS has demonstrated efficacy and safety when added to guideline-based therapy and is currently approved by the United States Food and Drug Administration (FDA) for treatment for refractory MAC pulmonary disease ,70. Surgrrl) gene [rrs) gene [It is also important to note that the Clinical and Laboratory Standards Institute (CLSI) guidelines recommend antimycobacterial susceptibility testing (AST) for all slow-growing NTM infections, including MAC on all clinically significant isolates for proper antibiotic selection for a treatment regimen . AST is rl) gene ,76, whilrs) gene . The brers) gene . Findingrs) gene . The CLSrs) gene .Disseminated MAC infections spread to the rest of the body instead of staying localized to the pulmonary region. Through local multiplication and entry into the bloodstream, disseminated MAC infections may complicate the pulmonary disease by spreading to other organs and tissues. This disease is common in advanced HIV patients, patients with a history of immunosuppressive therapy, and other severely immunocompromised patients ,79  and may take more than 12 months. A dual therapy with a macrolide, azithromycin (500–600 mg daily), or clarithromycin (500 mg twice daily), combined with ethambutol (15 mg/kg daily) is initially used. In patients with failing ART, and when the mycobacterial counts in blood culture are high, a third agent  is added ,81.Nontuberculous mycobacterial (NTM) and MAC-specific lymphadenitis is transmitted through environmental sources and has a high risk of incidence among immunodeficient children ,83. The Mycobacterium abscessus are NTM under Runyon group IV, classified as rapid-growing mycobacteria (RGM). Similar to most NTM species, these organisms are ubiquitous in the environment and have been found in soil, natural and drinking water sources, sewage water, as well as decaying vegetation [M. abscessus [M. abscessus is common among cystic fibrosis (CF) patients, while the disseminated infection is common among immunocompromised patients. Soft tissue infections are often healthcare-associated infections due to open wounds being contaminated with non-sterile medical or surgical equipment or materials. Infection can also occur after a wound is contaminated with soil [getation . They argetation ,87. In Jbscessus . Pulmonaith soil . Thus, pith soil .M. abscessus is one of the most resistant organisms to chemotherapeutic agents and its main threat as a human pathogen is due to its high multi-drug resistance. This bacterium is also associated with biofilm formation, resistance to disinfectants, high temperatures, and acidic environments. Previously, M. abscessus was thought to be acquired exclusively from the environment but the use of whole-genome sequencing (WGS) analysis from worldwide clinical isolates of M. abscessus suggested there was possibly human-to-human transmission of M. abscessus [bscessus .M. abscessus was first isolated in 1953. Since then, M. abscessus has displayed a high degree of genotypic heterogeneity among subspecies; identified through genome sequencing of molecular targets such as hsp65 and rpoB [M. abscessus group is divided into three subspecies: M. abscessus subsp. abscessus; M. abscessus subsp. bolletii and M. abscessus subsp. massiliense, which for the rest of the paper will be referred to as M. abscessus, M. bolletii, and M. massiliense, respectively. Both M. abscessus and M. bolletii have functional isolates of the erythromycin ribosomal methylase (erm)41 gene that gives them inducible macrolide resistance compared to M. massiliense which does not have a functional erm (41) gene. This inducible resistance, therefore, explains the better response to treatments of M. massiliense infections compared to M. abscessus and M. bolletii infections [erm gene in M. massiliense isolates that confuses this previous identification of M. massiliense due to not having a functional erm (41) gene [erm (41) gene has also been found in some isolates of M. abscessus subsp. abscessus due to a C instead of T at the nucleotide position 28 in the erm (41) gene [M. abscessus organisms that contribute to its virulence [and rpoB ,92,93. Tfections . However41) gene ,97. In airulence . Two disirulence . Other NM. abscessus is categorized as a rapidly growing mycobacterium (RGM) within the NTM species, demonstrating disseminated infections in immunocompromised, trauma, and postsurgical patients [M. abscessus isolates found in freshwater rivers and lakes, seawater, and animal drinking troughs, as well as peat-rich potting soils. The survival and growth of pathogenic M. abscessus have been recovered in large quantities within our water distribution systems such as drinking water, household plumbing, and even hospital wastewater [M. abscessus has a hydrophobic outer surface, leading to biofilm formation and resistance to disinfectants and antibiotics [M. abscessus of the host tissue barrier [M. abscessus infections [patients . Similarstewater ,101. M. ibiotics ,101. The barrier . Numerou barrier . Healthc barrier . Patientfections .M. abscessus [M. abscessus by far being the most prevalent [M. abscessus has been correlated with declining lung function among CF patients [M. abscessus infection deteriorated from only suffering from minimal bronchiectasis to requiring a lung transplantation procedure within a period of 29 months [M. abscessus infection in connection with a pulmonary transplant, likely generated from her originally infected lungs [M. abscessus is a pathogen that may induce serious infection risks for CF patients. The dangers of M. abscessus complex infections in hospitals relating to lung transplantations and cystic fibrosis are concerning to medical professionals, and these dangers are compounded by the risk of nosocomial infections that have already occurred in the past [Patients suffering from cystic fibrosis (CF) have an increased chance of colonization and infection from bscessus . Prospecrevalent ,105. Thepatients . For exa9 months . Anothered lungs . These othe past .3; however, disseminated M. abscessus infection is rarer [M. abscessus infections [M. abscessus infection despite no underlying lung disease [M. abscessus demonstrates the potential to be a pathogen in hosts with a tissue barrier function that is compromised, hosts with airway abnormalities, and in immunodeficient patients.People with immunosuppressed diseases such as HIV or people who have an organ implant are also vulnerable. As mentioned earlier, in HIV patients, disseminated infection does not usually develop unless the CD4+ T-lymphocyte count is below 50/mmis rarer . Patientis rarer . Bronchifections ,111. In  disease . OverallM. abscessus is the most virulent RGM and the cause of most pulmonary infections due to RGM. NTM infection involves a complex interaction between the host, pathogen, and environment. Typically, M. abscessus pulmonary infections demand the most challenging treatments out of all NTM infections. It shares many pathophysiological symptoms with M. avium and M. tuberculosis, such as the formation of granulomas and enduring infection; however, the specific pathways in the establishment of infection and the exact routes of transmission are not well understood. There is evidence that colonization of the lung airways is facilitated by bacterial biofilm formation, with glycopeptidolipids playing an important role. In NTM, including M. abscessus, glycopeptidolipids (GPLs) are found in the outer layer of the cell envelope. The expression of GPL is correlated with the smooth colony phenotype, while a lack of GPL results in a rough colony [M. abscessus complex can display both colony morphotypes.h colony . Smooth M. abscessus to transition between a colonizing phenotype and a more virulent and invasive form [M. abscessus to change from smooth to rough variants was associated with the increased severity of clinical symptoms [M. abscessus initially gains entry to the lungs as a GPL-expressing smooth variant, and then the spontaneous loss of GPL expression results in a virulent phenotype causing inflammation and invasive lung disease.Smooth and rough colonies not only have different physical appearances but have different pathophysiological traits. The ability to switch between smooth and rough morphologies allows ive form . Rough vive form . GPL in ive form ,116. It ive form . Studiesive form . The abisymptoms . It has symptoms . All theM. abscessus. GPL-rich smooth bacilli are taken up by macrophages individually and maintained in loner phagosomes [Macrophages can take up both smooth and rough variants of agosomes . In contagosomes . The smoagosomes . The ETZagosomes . Due to agosomes .M. abscessus produce little pro-inflammatory cytokines and control infection, as opposed to macrophages infected with the rough variant, produce copious amounts of pro-inflammatory cytokines, with the bacteria surviving and growing both intracellularly and extracellularly [M. abscessus do not. The TLRs mediate the innate immune response, recognizing conserved motifs expressed by the microorganisms, called pathogen-associated molecular patterns (PAMPs) [M. abscessus have been shown to stimulate the macrophage immune response via TLR2 signaling [M. abscessus rough variants also stimulate the release of TNF-α via interaction with TLR2. The release of IL-8 is also involved in the recruitment of neutrophils to the site of infection in the lung. Neutrophil extracellular trap (NET) formation, reactive oxygen species (ROS) generation, and phagocytosis are some common neutrophil mechanisms activated by both smooth and rough variants [Macrophages infected with smooth llularly ,122,123. (PAMPs) . TLRs arignaling . Stimulaignaling . M. abscvariants . In murivariants .M. abscessus MAB2560-treated DCs. Another finding from the same study suggests that M. abscessus MAB2560 enhanced DC maturation via TLR4 and activated MAPKs’ production of IL-12 [M. abscessus, as genetic deficiencies in TNF-α receptor, TFN-γ, or CD4+ cause increased susceptibility.Several studies report that mitogen-activated protein kinases (MAPKs), specifically ERK1/2 and p38 are key modulators for granuloma formation during an NTM infection . This isof IL-12 . The secof IL-12 . Both IFM. abscessus infections are extremely difficult to treat due to their high antimicrobial drug and disinfectant resistance [M. abscessus complex infections mainly result in skin and soft tissue infections (SSTIs) and pulmonary disease, but can also cause ocular and central nervous system (CNS) infections [Complex sistance . M. abscfections ,130.M. abscessus complex-associated pulmonary disease is one of the most common and well-known NTM infections. There is no standard treatment, but current guidelines recommend a macrolide-containing multidrug treatment regimen with intravenous antimicrobial agents, although this therapy is known to have considerable side effects [M. abscessus and M. bolletii have functional isolates of the erythromycin ribosomal methylase (erm)41 that gives these subspecies inducible macrolide resistance compared to M. massiliense, which has a nonfunctional erm (41) gene. Although this is mainly the case, the nonfunctional erm(41) gene has occurred in some M. abscessus subsp. abscessus due to a C instead of T mutation at the nt position 28 in the erm(41) gene [erm gene in M. massiliense isolates further confuse this previous identification [erm (41) gene; therefore, it is important to conduct in vitro susceptibility testing to detect a functional or nonfunctional erm(41) gene. All three subspecies can also develop constitutive macrolide resistance due to 23S RNA (rrl) gene mutations as well [M. abscessus complex and recommends antimicrobial susceptibility testing (AST) on all clinically significant isolates. Susceptibility panels for M. abscessus need to include at least amikacin, cefoxitin, imipenem, clarithromycin, linezolid, doxycycline, tigecycline, ciprofloxacin, and moxifloxacin [M. abscesses, as well as MAC and other NTM pathogens [ effects . This mu effects . As prev41) gene ,97; recefication . Macrolifloxacin . These gathogens .M. abscessus pulmonary disease, treatment regimens should be developed in collaboration with experts [M. abscessus lung disease. Suppressive therapy, with periodic parenteral antibiotic or orally administered macrolide therapy, can be administered to control the progression and symptoms of the disease. Surgical resection of the infected lung following chemotherapy to lessen the microbial burden is a likely curative therapy for M. abscessus lung disease [M. abscessus lung disease, similar to all NTM lung disease, include a poor response to medical treatment for at least 6 months, if patients have sufficient cardiopulmonary function to withstand and tolerate the surgery. The outcomes have been shown to be successful, but with relatively high complication rates [Based on the guidelines from the American Thoracic Society/Infectious Diseases Society of America in 2007, treatment with current existing antimicrobial agents is limited . Given t experts . Additio disease . Compareon rates .M. abscessus complex can also cause SSTIs, ranging from deep tissue to partial skin infections. The two main mechanisms for developing M. abscessus-associated SSTIs are from (1) direct contact with a traumatic injury or surgical wound, with contaminated water, materials, or environmental exposure and (2) through a secondary infection of the skin and soft tissue associated with disseminated disease [M. abscessus-associated SSTIs [M. abscessus from spas or hot springs can also result in SSTIs [M. massiliense subspecies [M. abscessus complex skin, soft tissue, or bone infections, the initial recommendation for treatment includes a combination of antimicrobial drugs with a macrolide  and intravenous agents for between two weeks and several months after oral macrolide-based therapy [The  disease . It has ed SSTIs . Exposurin SSTIs ,131. It bspecies ,133. ForM. abscessus can also cause CNS infections, with cerebral abscesses and meningitis being the most common. Among HIV-positive patients, MAC accounts for more CNS infections, while M. abscessus is responsible for more CNS infections among HIV-negative patients. HIV-negative patients that acquired M. abscessus-associated CNS infections are often patients with recent neurosurgical procedures or intracranial catheters. Treatment includes at least one year of clarithromycin-based combination therapy and surgical intervention if needed [Although it is rare, f needed . The treM. abscessus complex ocular infections were initially controlled by discontinuation of topical corticosteroids. Currently, optimal treatment methods depend on the location of the ocular infection [M. abscessus-associated ocular infections, while topical therapy with topical amikacin and clarithromycin is used to treat only certain M. abscessus complex ocular infections [Ocular infections caused by NTM have also increased over the past decade. nfection . Systemifections . Surgicafections . TreatmeM. abscessus has an uncertain clinical significance that is further complicated by difficultly treating M. abscessus infections. There is not yet an effective or convenient dual therapy for co-infection [M. abscessus treatment, and the need for parenteral antibiotics that are slow and unpredictable for M. abscessus infections are daunting for clinicians to expose prolonged toxic medication to patients [M. abscessus complex (MABC).Co-infection of MAC and nfection . MAC thepatients ,136. CurM. abscessus lung disease in patients has a very similar pattern in radiographic appearance to the nodular bronchiectatic form of MAC lung disease; additionally, 15% of patients with M. abscessus lung disease will also have MAC species isolated in the same sputum. However, TB disease cannot be ruled out based on radiographic appearance alone; therefore, clinical and microbiological criteria must also be met for the diagnosis and initiation of treatment [Presumptive diagnoses based on clinical and radiographic features are not sufficient for the initiation of therapy; there must be an isolation of NTM species through microbiologic cultures. Additionally, since NTM species are widely found in nature and tap water contamination is highly possible; NTM disease diagnosis requires three positive sputum cultures collected on three separate days. Radiographic tests, when compared to TB disease, predominately show NTM disease to appear as (1) thin-walled cavities with less surrounding parenchymal opacity, (2) less bronchogenic but more contiguous spread of infection, and (3) more pronounced involvement of pleura over the affected areas of the lungs ,137,138.Exposure to NTM can come from household and hospital water systems, hemodialysis centers, and dental clinics . Animals3 and if the patient is not on fully suppressive ART [3 [HIV-positive adults are at an increased risk of disseminated MAC infection and should receive primary prophylaxis for certain indications . If HIV-sive ART . After te ART [3 . Long-tee ART [3 . Rifabute ART [3 ,149. Proe ART [3 . Patiente ART [3 .M. abscessus infection specifically should include improved protocols for the hygiene of medical professionals and patients such as handwashing, the wearing of personal protective equipment (PPE), disinfecting surfaces and equipment, and the sterilization of any tools that are used for invasive procedures. Due to the high resistance of M. abscessus complex to disinfectants, bleach in combination with another disinfectant should be used to disinfect surfaces [M. abscessus complex in these at-risk groups, negative pressure ventilation systems for each inpatient room are recommended, as well as optimizing the air exchange rates of the rooms and communal areas [BCG vaccination may also be a prevention strategy, with one study showing that children with a history of bacillus Calmette–Guerin (BCG) vaccination appear to have a reduced risk of MAC lymphadenitis . The presurfaces . Prevental areas . The impMycobacterium avium, and rapid-growing mycobacteria, Mycobacterium abscessus, are important emerging human pathogens causing disease in vulnerable populations such as severely immunocompromised patients, patients with chronic lung diseases, and others with underlying health conditions. Their incidence is increasing worldwide in industrialized countries and they cause substantial yet underacknowledged burden of disease. Within the context of Mycobacterium avium complex (MAC), M. avium causes progressive pulmonary disease, disseminated infection, and lymphadenitis. M. abscessus is a highly pathogenic and drug-resistant fast-growing mycobacterium and is responsible for most pulmonary infections caused by RGM. The pathogenesis of M. avium involves its ability to colonize and invade intestinal or respiratory mucosa to infect phagocytic cells. The pathogenesis of M. abscessus is related to its ability to form biofilms and transform from a colonizing GPL-expressing smooth phenotype that evades innate immune responses to a virulent GPL-deficient rough phenotype, capable of causing extensive tissue damage. Both infections often require a combination of therapies that can include antimicrobial therapy, particularly macrolides, ART therapy in HIV patients, and sometimes surgical excision. The prevention of NTM infection may include the installation of filters and periodic flushing of water systems, and improved protocols for hygiene and the disinfection of medical devices. These recommendations should be shared with people, especially vulnerable patients with recognized risk factors such as immunocompromised patients or patients who have underlying health conditions.Nontuberculous mycobacterium (NTM), including over 172 distinct species, are a group of ubiquitous environmental bacteria found in soil and water sources. Most NTM species are non-pathogenic; however, a few species cause opportunistic human infections. Slow-growing mycobacteria,


Anastrepha fraterculus sp. 1 is considered a quarantine pest in several American countries. Since chemical control applied in an integrated pest management program is the only strategy utilized against this pest, the development of pesticide-free methods, such as the Sterile Insect Technique, is being considered. The search for genes involved in sex-determination and differentiation, and in metabolic pathways associated with communication and mating behaviour, contributes with key information to the development of genetic control strategies. The aims of this work were to perform a comprehensive analysis of A. fraterculus sp. 1 transcriptome and to obtain an initial evaluation of genes associated with main metabolic pathways by the expression analysis of specific transcripts identified in embryos and adults.serendipity alpha and transformer-2 displayed similar expression levels in the analyzed stages, while heat shock protein 27 is over-expressed in embryos and females in comparison to males. The expression of genes associated with courtship  differed between males and females, independently of their reproductive status . Genes associated with metabolic pathways  showed differential expression between embryos and adults. Furthermore, 14,262 microsatellite motifs were identified, with 11,208 transcripts containing at least one simple sequence repeat, including 48% of di/trinucleotide motifs.Sexually mature adults of both sexes and 72 h embryos were considered for transcriptome analysis. The de novo transcriptome assembly was fairly complete (62.9% complete BUSCO orthologs detected) with a total of 86,925 transcripts assembled and 28,756 GO annotated sequences. Paired-comparisons between libraries showed 319 transcripts differently expressed between embryos and females, 1242 between embryos and males, and 464 between sexes. Using this information and genes searches based on published studies from other tephritid species, we evaluated a set of transcripts involved in development, courtship and metabolic pathways. The qPCR analysis evidenced that the early genes A. fraterculus sp. 1, contributing with a fairly complete transcript database of embryos and adults. The expression analysis of the selected candidate genes, along with a set of microsatellite markers, provides a valuable resource for further genetic characterization of A. fraterculus sp. 1 and supports the development of specific genetic control strategies.Our results significantly expand the available gene space of The online version contains supplementary material available at 10.1186/s12863-020-00943-2. Anastrepha fraterculus Wiedemann (Diptera: Tephritidae), is considered one of the most economically important pest species in several American countries. This species has broad geographical distribution and a wide host range, attacking at least 110 host plant species including major fruit crops , Rhagoletis zephyria [PRJNA331175/321204], and C. capitata [PRJNA201381]  were predicted regardless of BLASTX hits to NR DB. An in silico analysis using Transdecoder software resulted in the detection of 38,933 putative proteins with at least 100 amino acids. We identified ortholog groups by inter-specific pairwise comparisons (OrthoMCL) considering all predicted proteins of three closely related species , 49 withPaired-comparisons between libraries were performed by DE analysis (EdgeR) considering only transcripts with more than 10 counts per million (cpm > 10) in at least two of the three samples. The Biological Coefficient of Variation (bcv) was set at 0.4, a value recommended by EdgeR  for compA. fraterculus sp. 1 transcriptome. We detected transcripts annotated as heat shock proteins , odorant-binding proteins , and transcripts associated to female and male physiology   previously described for A. fraterculus by Campanini et al. . The criteria used for SSR selection were based on the minimum number of repeats as follows: five for dinucleotide, four for trinucleotide, three for tetra, penta, and hexanucleotide motifs.To identify SSRs markers, we ran the software MISA [http://emboss.open-bio.org/wiki/Appdocs). An in silico PCR was performed on all SSR using the assembled transcriptome (in-house script).To design pairs of primers with no genome information available that amplify the corresponding SSR marker in every transcript, we ran an in-house script that uses EPRIMER32 from the EMBOSS package  and total number (right Y-axis) of transcripts in each category are shown. Y-axes are in log (10) scale. WEGO was used to produce the graph.Additional file 2. Gene Ontology (GO) analysis of A. fraterculus sp. 1 transcripts. The table shows main terms at the second and third GO levels involved in biological processes, development, behaviour, and reproduction.Additional file 3. Sequence information of related Tephritidae species available in the NCBI RefSeq_Genomes database.Additional file 4. Prediction of orthologs from closely related species (B. oleae and R. zephyria) and the best-annotated tephritid species (C. capitata).Additional file 5. EdgeR results. Each Excel sheet shows the paired-comparison between libraries . LogFC: logarithm of the fold change for each contrast performed; LogCPM: logarithm of the counts per million for each transcript; P value: statistical P value for the individual test performed; FDR: false discovery rate .Additional file 6. A. List of differentially expressed transcripts obtained in paired-comparisons of libraries. A.1. 72 h embryos vs female ; A.2. 72 h embryos vs male ; and A.3. female vs male . B. Functional annotation of differentially expressed transcripts between libraries. Only transcripts with CPM > 10 and 70–100% complete sequence (related to the best BLAST hit) are shown. Over-expressed transcripts: sequences with FC > 10; Under-expressed transcripts: sequences with FC < 0.1.Additional file 7. Selection of putative transcripts associated with function of interest. A. Transcripts selected according to their differential expression among libraries and predicted function (GO or BLAST hit). B. Transcript selected according to the literature associated to early development, sex determination, and communication.Additional file 8. Correlation between RNA-Seq and qPCR datasets for the nine analyzed transcripts  for each comparison are shown. For the three comparisons, r values were highly significant (P < 0.0001) and demonstrate a high degree of correlation between the two datasets. Data used to create these graphs are detailed in Additional File ts Table  in 72 h Additional file 9. List of transcripts containing microsatellite motifs and description by repeat type classes and number of repeating motif.Additional file 10. List of transcripts containing microsatellite motif and primers designed available to be tested.


Morchella conica Pers. is a highly-prized mushroom for its edible and medical values. In this study, we determined the complete mitochondrial genome of M. conica combining both PacBio and Illumina sequencing technologies. The complete mitochondrial genome is 280,763 bp in length with a GC content of 39.88%. We identified a total of 14 core conserved protein-coding genes, 127 non-conserved open reading frames (ncORFs) and 30 tRNA genes in the M. conica mitogenome. However, no large or small rRNA subunits (rnl or rns) were identified in this mitogenome. In addition, we detected two mitochondrial RNase P (rnpB) genes and one ribosomal protein genes (rps3). Phylogenetic analysis was performed among M. conica and 18 other representative fungi from Ascomycota, Basidiomycota and Mucoromycota. Our results showed that M. conica was most closely related to M. importuna. The availability of the M. conica mitochondrial genome will form the basis of genetic breeding program and enhance our understanding of the evolution of this species. Morchella conica Pers. is an edible mushroom belonging to the Ascomycota phylum , Yunnan Province, China. Fresh fruit bodies were harvested and immediately frozen in liquid nitrogen after collection. Genomic DNA was extracted using a modified CTAB method  . The whole mitogenome sequence data reported here has been deposited in the Genome Warehouse in National Genomics Data Center , 3 cytochrome oxidases , apocytochrome b (cob) and 7 subunits of NAD dehydrogenase . Two mitochondrial RNase P (rnpB) genes and one ribosomal protein genes (rps3) were identified in the mitochondrial genome. Besides, 127 non-conserved open reading frames (ncORFs) were identified to be uniformly dispersed in the M. conica mitogenome. We also detected 30 tRNA genes, but no rRNA genes were found in the genome.The mitochondrial genome of M. conica we aligned 14 conserved protein-coding genes using MAFFT v7.305 (Katoh and Standley M. conica is closely related to M. importuna in the genus Morchella (To investigate the phylogenetic position of orchella .


Prevalence of gambling behaviours and their associations with socioemotional harm among 11–16 year olds in Wales: findings from the School Health Research Network survey G.J. Melendez-Torres, Rebecca E. Anthony, Gillian Hewitt, Simon Murphy, Graham F. Moore.There were several transcription and tabulation errors in Table 2 in the original paper. These have now been corrected and the corrected table appears below.


Mycolicibacter kumamotonensis-pulmonary disease was diagnosed in a 68-year-old immunocompetent woman in Greece; the disease was initially treated as tuberculosis. The patient responded favorably to a new treatment regimen of azithromycin, amikacin, moxifloxacin, and linezolid. Complete symptom resolution and radiologic improvement resulted.Severe  Mycolicibacter (formerly Mycobacterium terrae complex) are considered not pathogenic, with the exception of causing chronic tenosynovitis of the hand  dropped from 46 to 25 mm/h (reference range 0–20 mm/h), and her weight was stable. Computed tomography (CT) scan of her chest showed multiple cavities, bronchiectasis, nodules, and tree-in-bud appearance (2 of 91% breathing room air), and her ESR rose to 59 mm/h. A new bronchoscopy was performed. Acid-fast staining results were negative, whereas results of a culture on MGIT960 automated system (strain GR- 21075)  and Lowenstein-Jensen slants  were positive. No other pathogens were isolated.Five months into treatment, the patient’s condition gradually worsened. She developed productive cough and shortness of breath with hypoxemia (SpOhttps://www.thermofisher.com) using the Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems) and previously described primers (http://www.ncbi.nlm.nih.gov) using BLAST (http://hsp65blast.phsa.ca/blast/blast.html) and deposited them in GenBank (accession nos. MT491187 and MT491188).For molecular identification, we sequenced regions of 927 bp of 16S rDNA gene and of 440 bp of the 65-kDa heat shock protein (hsp65) gene . We then determined the MICs  (The sequence of 16S rDNA and hsp65 genes showed 100% similarity with the type strain of M. kumamotonensis from bronchoalveolar lavage (At the end of antituberculosis treatment, a second CT scan revealed slight improvement of the nodules and the tree-in-bud appearance but persistence of the cavities (M. kumamotonensis has been isolated from respiratory specimens, lymph nodes, and soft tissue all over the world (M. kumamotonensis identification. In their recent report, Iemura-Kashiwagi et al. (M. terrae infection had pulmonary disease. Because M. kumamotonensis and M. arupense are the most frequently isolated species of the complex (M. kumamotonensis. On the other hand, M. kumamotonensis has recently been found in a hospital environment (In an older study, Smith et al. (M. kumamotonensis infection should be included in the differential diagnosis of mycobacterial pulmonary disease with cavity formation in immunocompetent adults with bronchiectasis.The patient responded favorably to the selected regimen even though the strain was resistant to moxifloxacin and of borderline MIC to linezolid. Increase of moxifloxacin dose was not attempted because of fear of QT prolongation in an elderly woman. In conclusion,


Salmonella spp. remain to be the major pathogens causing splenic abscess, with an increasing worldwide frequency of splenic abscess due to non-typhoidal Salmonella infection. Here, we report the case of a 12-year-old boy, who was presumably diagnosed with acute gastroenteritis on admission and eventually diagnosed with a large splenic abscess  caused by non-typhoidal Salmonella. Although splenectomy has been considered in cases of large splenic abscesses, the patient was treated with antibiotics and ultrasonography-guided percutaneous drainage. A detailed physical examination and appropriate imaging studies are necessary for the early diagnosis of extra-intestinal complications of non-typhoidal Salmonella enteritis. For treatment, percutaneous drainage, rather than splenectomy, can be used in large splenic abscesses.Splenic abscess occurs very rarely in healthy children. Although typhoid fever was the leading cause of splenic abscess in the pre-antibiotic era,  Salmonella enterica serovar Typhi frequently predisposed individuals to splenic abscess, although its occurrence decreased with the introduction of antibiotic therapy [S. ser. Typhi still remains a leading cause of splenic abscess [Streptococcus and Staphylococcus being the most common causative agents [Salmonella spp. are still considered major pathogens of splenic abscess, as evidenced by the increasing worldwide frequency of splenic abscess due to non-typhoidal Salmonella (NTS) infection rather than S. ser. Typhi infection [Isolated splenic abscess occurs very rarely in children. In the pre-antibiotic era, typhoid fever caused by  therapy . However abscess . Currente agents . Howevernfection .In the current report, we diagnosed an isolated large splenic abscess caused by NTS infection in a boy who was presumably diagnosed with acute gastroenteritis (AGE) on admission. Although the patient had a large splenic abscess , he was treated with antibiotics and ultrasonography (US)-guided percutaneous drainage rather than splenectomy.The case was a 12-year-old boy who presented with fever, abdominal pain lasting for three days, and diarrhea that developed one day prior. Vomiting occurred during the first day of fever. He had frequently experienced recurrent abdominal pain and vomiting lasting for 1 or 2 days; however, he reported no recent gastrointestinal (GI) symptoms for the previous month. Moreover, he had not recently traveled abroad. On admission, his vital signs were as follows: blood pressure, 120/60 mmHg; body temperature, 39.7 °C; heart rate, 72 beats/min; respiratory rate, 24 breaths/min. He looked acutely ill, and a soft and flat abdomen with tenderness in the epigastric and left upper quadrant areas was noted on physical examination. Laboratory tests revealed white blood cell (WBC) count of 13,000/μL , hemoglobin of 11.8 g/dL, platelet count of 177,000/μL, erythrocyte sedimentation rate of 75 mm/h, and C-reactive protein (CRP) level of 31.00 mg/dL. The chest X-ray showed no abnormality; however, an abdominal X-ray showed a diffusely increased soft-tissue density suggestive of splenomegaly in the left upper quadrant. Based on his symptoms, physical examination, and laboratory results, empirical antibiotic therapy for a presumptive diagnosis of bacterial AGE was initiated (cefotaxime 150 mg/kg/day). On the admission day, abdominal computed tomography (CT) was performed to evaluate suspicious splenomegaly found on the abdominal X-ray and possible intra-abdominal complications of AGE. Results of the CT scan showed a large abscess  in the upper pole of the spleen A. Salmonella spp. group D, which was susceptible to ampicillin and cefotaxime, was detected in the stool and drained splenic abscess fluid samples. The fever disappeared on HD #7. On HD #11, when the amount of drained pus fluid decreased to less than 100 mL/day, an abdominal CT was again performed, indicating that the splenic abscess had decreased in size  8 days after discharge showed a splenic pseudocyst with a maximum diameter of 4.1 cm. Antibiotic therapy was discontinued in the OPD 6 weeks after the diagnosis of splenic abscess. Repeated abdominal US showed a persistent splenic pseudocyst without a change in size at 7 months after the end of the antibiotic therapy.Intravenous metronidazole (40 mg/kg/day) was added to the cefotaxime therapy for possible amoebic abscess. On the second hospital day (HD #2), US-guided percutaneous drainage of the splenic abscess was performed due to an impending rupture. Anchovy paste-colored pus was drained (840 mL), and the laboratory examination of pus revealed a WBC count of 20,800/μL , red blood cell count of 20,750/μL, and protein and glucose levels of 5.5 g/dL and 0 mg/dL, respectively. With antibiotic therapy, diarrhea and abdominal pain resolved on HD #3 and #5, respectively. The blood and urine samples that were collected upon admission were sterile. In contrast,  in size B. MetronSalmonella is divided into two species, S. enterica and S. bongori, and S. enterica is further subdivided into typhoidal Salmonella and NTS [Salmonella, including S. ser. Tyhpi and S. ser. Paratyphi, causes systemic infection (typhoid fever), while NTS causes self-limiting AGE in most cases [S. ser. Typhi was the most common pathogen of splenic abscess in the pre-antibiotic era; however, NTS became a more frequent cause of splenic abscess than S. ser. Typhi after the introduction of antibiotic therapy [The genus  and NTS . Typhoidst cases . Howeverst cases . S. ser. therapy . Neverth therapy . In part therapy . Moreove therapy . TherefoAlthough our patient complained of recurrent abdominal pain and vomiting, his previous GI symptoms improved within 1–2 days, but he denied having any recent GI symptoms accompanying the fever within 1 month prior to admission. Previous reports showed that children and adolescents with large splenic abscesses sized 14–23 cm complained of fever and GI symptoms lasting for only 2–3 days ,8. ThereAlthough splenectomy was traditionally recommended for splenic abscess , successIn conclusion, a 12-year-old boy presenting with symptoms of AGE was eventually diagnosed with an isolated large splenic abscess due to NTS. A detailed physical examination and appropriate imaging studies are necessary for the early diagnosis of extra-intestinal complications of NTS enteritis. For treatment, percutaneous drainage, rather than splenectomy, can be used in large splenic abscesses.


Background: Pancreatic cancer (PC) is an aggressive cancer with worse survival in the world. Emerging evidence suggested that the imbalance of alternative splicing (AS) is a hallmark of cancer and indicated poor prognosis of patients. Genes-derived splicing events can produce neoepitopes for immunotherapy. However, the profound study of splicing profiling in PC is still elusive. We aimed to identification of novel prognostic signature across a comprehensive splicing landscape and reveal their relationship with tumor-infiltrating immune cells in pancreatic cancer microenvironment.Methods: Based on integrated analysis of splicing profiling and clinical data, differentially splicing events were filtered out. Then, stepwise Cox regression analysis was applied to identify survival-related splicing events and construct prognostic signature. Functional enrichment analysis was performed to explore biology function. Kaplan-Meier curves and receiver operating characteristic (ROC) curves were performed to validate the predictive effect of predictive signature. We also verified the clinical value of prognostic signature under the influence of different clinical parameters. For deeper analysis, we evaluated the correlation between prognostic signature and infiltrating immune cells by CIBERSORT.Results: According to systematic analyzing, a final six splicing events were identified and validated the good prognostic capability in entire TCGA dataset, validation set 1 and validation set 2 by Kaplan-Meier curves (P < 0.0001). The area under the curve (AUC) of ROC curves were also confirmed the high predictive efficiency of the prognostic signature in these three cohorts . In order to validate whether prognostic signature highlights a correlation between AS and immune contexture, CIBERSORT was performed to analyze the proportion of tumor-infiltrating immune cells in PC. Based on prognostic signature, we identified survival-related immune cells including CD8 T cells (P = 0.0111), activated CD4 memory T cells (P = 0.0329) and resting mast cells (P = 0.0352).Conclusion: In conclusion, our study contribute to provide a promising prognostic signature based on six splicing events and revealed prognosis-related immune cells which indeed represented novel tumor drivers and provide potential targets for personalized therapeutic. Pancreatic cancer is an aggressive disease with highly malignancy, poor prognosis and immune tolerance and remains one of the deadliest cancers in the world Gene expression profiling can provide evidence to identify survival-related prognostic biomarkers, or suitable therapeutic targets, in cancer The wide studies of RNA-Seq revealed that alternative splicing (AS) is a highly pervasive mechanism which can produce different isoforms and affects more than 90% of human genes In this present study, based on integrated bioinformatics analysis, we build prognostic models which including six AS events. The AUC of the ROC curves and Kaplan-Meier (K-M) survival curves showed the valuable predictive efficacy of the model in PC. Aims to underlying the potential mechanisms between AS and 22 various immune cells, we performed CIBERSORT to analyze the proportion of tumor-infiltrating immune cells for this model in high-risk and low-risk patients. The results indicated that the AS model have strongly differential expression in many immune cells including memory B cells, naive B cells, CD8 T cells, macrophages M1, T cells regulatory (Tregs) and mast cells resting which are associated with survival in PC patients. Therefore, we discuss the relationship between AS and tumor-infiltrating immune cells for broaden the boundary of related cell-based immunotherapy.In conclusion, we identified the prognosis of AS events by integrated bioinformatics analysis and understand in detail the potential relationship between AS and immune cells that regulate PC, which is a key step in developing immunotargeted therapy and improving the prognosis of PC patients.http://bioinformatics.mdanderson.org/TCGASpliceSeq) which provide transcripts with variable splicing https://tcga-data.nci.nih.gov/) https://www.ncbi.nlm.nih.gov/geo/) to serve as validation dataset. The platform of this dataset is GPL6244. These data were normalized by robust multi-array average (RMA) and log2-transformed which were used to validate the result.Alternative splicing data of PC patients (n=176) were collected from SpliceSeq (PSI) value ranging from zero to one was calculated for each detected AS events in a gene to evaluate the mRNA splicing patterns in patients with PC PSI value ≥ 0.75. The overlapped AS events were visualized with Upset plot by using UpSetR package (R software version 3.5.2) which can depict the intersection of more than three sets and rank the intersections In TCGA splice-seq, we analyzed the percent spliced in  and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of these AS events were performed by Bioconductor R package clusterProfler. In general, P ≤ 0.05 were represented significantly enriched pathways. Furthermore, univariate Cox regression analysis and multivariate Cox regression were performed to construct AS related prognostic model in PC patients by survival package in R (n=175). Finally, the prognostic signature, risk score, was calculated as followed:In order to identify differentially AS events between PC and normal tissues, the expression differences were characterized by absolute fold change (FC) over 2 and adjusted n, PSI and βi represented the number, percent-spliced-in value and regression coefficient of splicing events, respectively.In the formula, https://www.proteinatlas.org/). Then, entire TCGA patients with PC (n=175) were randomly separated into two sets . The prognostic signature was identified in entire TCGA data set and validated in both three sets. Based on risk score, patients were ranked into two (high/low) groups by the median cutoff value in three cohorts, respectively. K-M survival curves with Log-Rank test were applied to compare the OS effect of prognostic model in two risky sets. ROC curve was used to evaluate the predictive effect of prognostic marker by calculating 3-year survival with ROC package in R software. Likewise, we used stepwise Cox linear regression analysis to investigate the influence of clinical parameters in the prognostic signature by survival package in R and IBM SPSS 25.0 program.First, we performed differential expressed analysis of six genes in prognostic signature by GEO dataset (GSE28735) and The Human Protein Atlas database , a computational framework which can provide detailed cell type abundance from tumor RNA profiles of intact tissues In order to explore the correlation between prognostic model and infiltrating immune cells, we need to perform CIBERSORT to identify the proportion of 22 different infiltrating immune cells in tumor immune microenvironment. CIBERSORT  was performed and statistically significant P-value was set as ≤ 0.05 with the purpose of ensuring the reliability of the results.In this study, all data were used to determine independent prognostic factors which can predict patients' survival status by the R package (R software version 3.5.2). And the GraphPad Prism 8.0 software was performed to plot graphs containing K-M survival curve. All statistical analysis was used by IBM SPSS 25.0 program. Student The overall workflow in this study was summarized in Figure The integrated profiles of AS genes and events for 175 PC patients were analyzed using RNA-Seq data. The detailed clinical pathological data of patients from TCGA are summarized in Table Furthermore, in order to study the prognostic value of differently AS events, univariate Cox proportional hazards regression analysis were conducted in PC patients. As results, 5452 survival associated AS events were identified in 3207 differently expressed genes. UpSet plot of interactions between genes and OS associated AS events was shown in Figure In addition, it was evident that AS is a RNA processing pathway, which can change the function of protein. For a deeper understanding of the potential biological significant of the survival-related AS events, GO categories (Supplementary Lasso regression (removing genes with high correlation) was applied on differently OS-related AS events after univariate Cox survival analyses and then further multivariate Cox hazards regression analyses was carried out to determine independent prognostic indicators in PC. Finally, we obtained six genes with AS events  could be recognized as an independent prognostic risk scoring system in PC patients  and grade (P=0.028) were independent prognostic indicators with significant differences for PC patients in multivariate survival analysis. Although other clinical indicators in multivariate survival analysis were less powerful, they still have potential value in clinical application. It is notable that risk score (P<0.0001) had the strongest predictive ability among these indicators.Furthermore, we performed univariate and multivariate survival tests in order to prove the predictive ability of the prognostic signature and different clinical pathological parameters including cancer status, history type, pathological stage-T, pathological stage-N, cancer stage, grade, new event and radiation therapy Table . UnivariP<0.05). Immunohistochemical results (Supplementary P<0.0001, Figure P<0.0001, Figure P<0.0001, Figure In order to verify the expression of six genes in prognostic signature is significantly different between normal and patients, we conducted the validation in GSE28735 and HPA database, respectively. As shown in Supplementary P<0.0001, Figure P=0.0018, Figure P=0.0156, Figure P=0.0017, Figure P=0.0340, Figure P=0.0157, Figure P=0.0011, Figure P=0.0039, Figure Based on the previous univariate survival analysis, we know that clinical parameters were effective prognostic predictors in patients with PC. Thus, we performed the K-M survival curves to validate the predictive value of clinical parameters including cancer status (In order to fully understand the clinical value of prognostic signature, stratified survival analysis was performed to validate whether the prognostic model is indeed significant in clinical application. For clinical parameters of cancer status (tumor free or with tumor, Figure P-value to each sample  P ≥ 0.05. As shown in Figure P=0.0137, Figure P=0.0433, Figure P=0.0071, Figure P=0.0392, Figure P=0.0127, Figure P=0.0319, Figure P<0.0001, Figure P=0.0111, Figure P=0.0329, Figure P=0.0352, Figure More studies has been reported, infiltration of immune cells in tumor microenvironment was accompanied by cancer initiation and progression. Further investigation indicated that the presence of infiltrating immune cells can be used as biomarker for immunotherapy response + T cells PC is one of the critical causes of cancer-related deaths worldwide. Its incidence rate and mortality rate increase year by year, which are obliged to attract the attention of scientists Integrated prognostic splicing biomarker identified in the present study is including UBA1, S100A13, SH3KBP1, COPSTA, GSE1 and NISCH. The signature model has robust effect to predict prognosis in PC patients. According to NCBI, Ensemble and SMART database, UBA1 has been understood as the ubiquitin-activating enzyme E1 which involved in protein homeostasis. Inhibitors of UBA1 represent an effective target spot for cancer therapy + T cells and mast cells have prognostic effect in PC. Among them, high densities of mast cells are clearly related to poor prognosis. This view has indeed been reported in the investigation of skin cancers Studies have been reported that AS events have been involved in a variety of tumor processes, including angiogenesis, invasion and immune destruction + or CD4+ T cells, cancer immunotherapy targets will be largely expand. In terms of clinical sense, using the neoantigen peptide to prepare personalized vaccine and inject it into patients to produce T cells responses. This will relieve or eliminate cancer cells and improve patients' prognosis.Immunotherapy based on immune cells can provide effective treatment for some previously untreatable cancers including PC. However, one of the difficulties and urgency in the immunotherapy is to search appropriate target antigens In conclusion, we demonstrated the robust prognostic value of AS events in PC, which can provide basis for clinical application. Furthermore, we indeed provide novel insights into the correlation between AS and immune cells. This result has far-reaching significance for immunotherapy in the future. Moving forward, the profound study of splicing events and immune cells can indeed represent novel tumor drivers and provide potential targets for personalized therapeutic intervention.Supplementary figures and tables.Click here for additional data file.


Staphylococcus aureus: lipoteichoic acid, peptidoglycan) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumoniae: lipopolysaccharide). Intravital microscopy (IVM) was performed to assess immune cell activation and its impact on microvascular blood flow in vivo in the microcirculation of the gut. Plasma inflammatory mediators were measured via multiplex assay, and morphologic change in intestinal tissue was evaluated. DIBI treatment decreased leukocyte (hyper-)activation induced by Gram-positive and Gram-negative toxins. In some cases, it preserved capillary perfusion, reduced plasma inflammatory markers and attenuated tissue damage. These findings support the utility of DIBI as a novel treatment for systemic inflammation, e.g., sepsis.Iron is an essential element for various physiological processes, but its levels must remain tightly regulated to avoid cellular damage. Similarly, iron plays a dual role in systemic inflammation, such as with sepsis. Leukocytes utilize iron to produce reactive oxygen species (ROS) to kill bacteria, but pathologically increased iron-catalyzed ROS production in sepsis can lead to damage of host cells, multi-organ failure and death. Temporary reduction in bioavailable iron represents a potential therapeutic target in sepsis. This study investigates the effect of the novel iron chelator, DIBI, in murine models of systemic (hyper-)inflammation: C57BL/6 mice were challenged with toxins from Gram-positive ( Iron is one of the most abundant metal elements on earth . This biSepsis–the “worst case” of infection—is a global health concern because of its increasing incidence, high mortality, and the absence of an approved treatment for the underlying dysregulated immune response ,5. EssenH) pyridinone (MAHMP) residues per molecule [E. coli—a common preclinical model of sepsis [Staphylococcus aureus) and Gram-negative (LPS from Klebsiella pneumoniae and Escherichia coli).FDA-approved iron chelators include deferoxamine (DFO), deferiprone (DFP) and deferasirox (DFX). They are clinically used for iron overload, e.g., in sickle cell disease, hemochromatosis and thalassemia . DIBI ismolecule . The usemolecule ,16,17,18f sepsis ,20. Howef sepsis . The unip < 0.05) in both collecting and postcapillary venules 2 h after toxin-challenge, with LPSe the most potent Gram-negative toxin, while LTA was the most potent Gram-positive toxin. LPS from K. pneumoniae (LPSk) induced significantly less adhesion in collecting (−78% vs. LPSe) and postcapillary (−69% vs. LPSe) venules, respectively , but not back to control levels (e (57% reduction) in collecting venules.The primary outcome parameter used to assess systemic inflammation was the adhesion of leukocytes in both collecting and postcapillary venules of the intestinal submucosa. All toxins were able to induce inflammation by significantly increasing leukocyte adhesion  of the functional capillary density (FCD) measured 2 h after induction of toxemia in the intestinal muscle layer compared to the control group (e + DIBI group (+45%) and PGN+DIBI group (+19%) but not in the LPSk + DIBI and LTA + DIBI groups  except animals that received PGN (K. pneumoniae (LPSk+DIBI), yet reduced the LTA-induced increases in TNF-α. DIBI did not have any significant effect on the TNF-α plasma level induced by LPS from E. coli.Three hours after toxin administration, a significant increase in TNF-α plasma levels compared to the control group (CON) was observed for all groups  except animals that received PGN .In parallel to increased TNF-α plasma levels, there was a significant increase in IL-6 plasma levels for all groups .Three hours after toxin administration, a significant increase in IL-10 plasma levels relative to the control group (CON) was observed in all groups e,f. MoreE. coli LPS group (LPSe) compared to controls  presented only minor morphological alterations , which trigger various inflammatory pathways. Inflammation was evaluated by intravital microscopy (IVM) of the intestine to measure leukocyte activation and capillary perfusion. The intestine plays a critical role during inflammation. There is a normal physiological balance between the host and its intestinal bacterial colonizers, which make up the gut microbiome. Sepsis pathophysiology, including tissue damage by oxidative stress and hypoxia, disrupts this homeostasis by damaging the intestinal mucosal barrier, producing a “leaky gut”. Intestinal flora is then more able to translocate across the gut into systemic circulation ,24,25. UIn our murine model of acute toxemia, administration of DIBI at a dosage of 10 mg/kg resulted in decreased leukocyte activation observed in the microcirculation by a reduction in the number of leukocytes adhering to the intestinal vascular endothelium and the decrease in sICAM-1 plasma levels.In response to inflammation, leukocytes are typically attracted by endothelial cells, roll on the vascular endothelium, firmly adhere to the vessel wall and then transmigrate to the site of inflammation . QuantifE. coli was more potent than LPS from K. pneumoniae, and LTA was more potent than PGN. According to the literature, LPS from E. coli tends to be more potent than LTA regarding leukocyte adhesion in vivo. When comparing the pro-inflammatory properties of different doses of LPS from E. coli and LTA from S. aureus, LPS was more potent than the same dose of LTA. Moreover, LTA did not necessarily demonstrate a dose-response in terms of leukocyte–endothelial interactions or cytokine secretion when administered at higher doses and assessed over a prolonged duration [S. aureus LTA to Salmonella enteritidis LPS [K. pneumoniae LPS-induced inflammation were surprising. Clinical studies have suggested that K. pneumoniae bacteremia may lead to worsened outcomes when compared to E. coli. [K. pneumoniae infection . Toxic products from bacterial growth, as well as tissue damages due to bacterial destruction, may have made K. pneumoniae more lethal than E. coli. Therefore, more studies are needed to validate our findings that suggest LPS from K. pneumoniae is less potent than both Gram-positive toxins and LPS from E. coli in terms of inflammatory activation. The fact that the PAMPs from Gram-positive and Gram-negative bacteria trigger different inflammatory pathways might also explain the differences in the immune response to the LPS toxins of those species. Gram-negative LPS primarily activates TLR4, while Gram-positive LTA and PG act on TLR2 [E. coli and K. pneumoniae, or LTA and PGN from S. aureus. Changes in amino acid sequence and other structural features give each PAMP a unique ability to induce a specific immune response [Although both Gram-positive and Gram-negative toxins have been shown to induce signs of systemic inflammation, few studies have compared toxins directly . In thisduration ,29. Yippidis LPS . Our resE. coli. . The dif on TLR2 . Variabiresponse .According to the literature, PAMPs increase the expression of adhesion molecules and increase leukocyte–endothelial cell interaction through a process involving reactive oxygen species (ROS) and iron . As prevE. coli and PGN from S. aureus. No significant improvements were seen in the case of LPS from K. pneumoniae or LTA from S. aureus. Other studies corroborate the effects of DIBI in similar E. coli LPS-induced toxemia models. Findings from Thorburn et al. using IVM showed that DIBI restored the LPS-decreased intestinal capillary blood flow back to control levels in endotoxemic mice [K. pneumoniae and LTA from S. aureus, it is important to consider the multifactorial nature of changes in capillary blood flow. Factors, such as capillary leakage, disseminated intravascular coagulation, increased leukocyte–endothelial interaction, endothelial cell dysfunction, systemic redistribution of blood flow and tissue edema, affect capillary blood flow in this context [Microcirculatory dysfunction represents a major issue in the pathophysiological cascade of systemic inflammation and is an independent predictor of mortality in sepsis ,33. IVM mic mice . Arora emic mice . To unde context ,36. ThesE. coli was the only toxin that caused a significant increase across all inflammatory parameters, mimicking the cytokine storm often observed in sepsis [S. aureus PGN challenge. Wang et al. observed elevated levels of cytokines in both plasma and organs following a PGN challenge, which exceeded levels seen in our toxemia model [S. aureus LTA to stimulate human dermal, microvascular endothelial cells showed increased IL-6 production [S. aureus LTA showed extremely low cytokine levels [Quantification of mediators of inflammation allowed for further assessment of the activation of inflammatory pathways in the toxemia model. Plasma levels of inflammatory cytokines and soluble adhesion molecules were measured 3 h after toxin administration. Cytokines act either pro- or anti-inflammatory molecules and are released by several cells, e.g., immune cells and endothelial cells ,38. Cyton sepsis . With reia model . Since toduction . In conte levels . In a coE. coli. A recent study using the iron chelator, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone, identified a dose-dependent reduction in serum levels of IL-1β and TNF-α in a murine model of allergic rhinitis [E. coli or K. pneumoniae. Toxins appear to induce different patterns of cytokine release in experimental models of sepsis depending on toxin dosage, route of administration and model used. Although detrimental side effects from DIBI administration cannot be completely ruled out, an increase in cytokine plasma levels measured at only one timepoint might not reflect a “real” increase in cytokine expression  and from Klebsiella pneumoniae . Lipoteichoic acid  and peptidoglycan  from Staphylococcus aureus were used as Gram-positive toxins. The novel iron chelator, DIBI, was provided by Chelation Partners Inc., Halifax, NS, Canada.All toxins were purchased from Sigma–Aldrich  and used without further purification, as indicated by the supplier. Gram-negative toxins included LPS from This study was carried out on eight to ten-week-old male C57BL/6 mice  housed in the Carleton Animal Care Facility . Animals received a standard diet and water ad libitum and were maintained on a 12 h light/dark cycle in a 22 °C room. Weights ranged from 20 to 30 g. All experiments were approved by the University Committee on Laboratory Animals at Dalhousie University under protocol number #18-057 and were performed following the guidelines and standards of the Canadian Council on Animal Care.n = 5–6/group) were established for the study. Systemic inflammation was induced by 5mg/kg of the toxin with groups including LPSe, LPSk, LTA or PGN. LPSe + DIBI, LPSk + DIBI, LTA + DIBI, PGN + DIBI groups were treated with 10 mg/kg of DIBI. Control animals received the same weight-adjusted amount of vehicle . Pilot experiments had shown no impact of DIBI alone treatment in healthy animals, so DIBI treatment only groups were not included in this study.Nine groups of animals  injection of sodium pentobarbital (90 mg/kg). Depth of anesthesia was assessed by pedal reflex and maintained using sodium pentobarbital bolus administration (9 mg/kg). Mice were maintained at a body temperature of 37 ± 0.5 ℃ and received supplemental oxygen if irregular breathing occurred. Surgery for vascular access was performed, as previously described by Thorburn et al. . In brieIntravital microscopy (IVM) was performed 120 min after toxin challenge. Fluorescent dyes  were administered i.v. 15 min prior to IVM. IVM was performed on the terminal ileum, as described previously ,49. In bBlood samples were centrifuged at 3000 rpm for 10 min to obtain plasma. Plasma levels of selected inflammatory cytokines  were analyzed according to manufacturer protocols using a Procarta Multiplex Cytokine Assay kit  and a Bio-Plex instrument with Bio-Plex software .Samples from the terminal ileum not affected by IVM were collected following euthanasia. Samples were fixed in 10% phosphate-buffered formalin  for 24 h and processed using a Leica Pearl Wax Tissue Processor . Processed tissues were then sectioned (5 mm), and Hematoxylin and Eosin (H&E) stain performed. Tissues were assessed for histological damage using the scoring system developed by Chiu et al. : grade 0p < 0.05 were considered statistically significant.All data were analyzed using the statistical software package Prism 8 . Normal distribution was tested by the Kolmogorov–Smirnov test. One-way ANOVA followed by Newman Keuls’ test for multiple comparisons was used to analyze normally distributed data and post-hoc Kruskal–Wallis analysis for data not normally distributed. Differences at E. coli served as the inflammatory trigger [The present study investigated the impact of the novel iron chelator, DIBI, on systemic inflammation in vivo induced by different Gram-negative and Gram-positive toxins. DIBI had previously been validated using an endotoxemia model, where LPS from  trigger . This st


The present study aimed to investigate the occurrence and species of coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS) in retail pork meat samples collected during nationwide monitoring. The staphylococcal isolates were characterized for antimicrobial and zinc chloride resistance and enterotoxigenic potential.A total of 260 pre-packaged pork meat samples were collected from 35 retail markets in 8 provinces in Korea for isolation of staphylococci. Antimicrobial and zinc chloride resistance phenotypes, and genes associated with the resistance phenotypes were determined on the isolates. Furthermore, the presence and distribution of 19 staphylococcal enterotoxin (SE) genes and enterotoxin-like genes among the pork-associated staphylococci were determined by multiplex polymerase chain reaction-based assays using the specific primer sets.S. saprophyticus , S. sciuri , S. warneri , and S. epidermidis . Among the 29 isolates, four methicillin-resistant CoNS  and one methicillin-resistant CoPS  were identified. In addition, a relatively high level of tetracycline (TET) resistance (52%) was confirmed in CoNS, along with a predominant distribution of tet(K). The most prevalent SEs were sep (45%), and sen (28%), which were carried by 81% of S. saprophyticus.A total of 29 staphylococcal strains  were isolated from samples of retail pork meat, 24 (83%) of which were CoNS. The four CoNS species identified were S. saprophyticus strains, in raw pork meat could be a potential risk factor for staphylococcal food poisoning (SFP), and therefore, requires further investigation to elucidate the role of SEls in SFP and virulence of the pathogen. Our results also suggest that CoNS from raw pork meat may act as a source for transmission of antimicrobial resistance genes such as staphylococcal cassette chromosome mec and tet(K).These findings suggest that CoNS, especially  S. aureus can cause a variety of infections including superficial skin and soft tissue infections, osteomyelitis, and septicemia [S. aureus [Staphylococci are commensal colonizers of the skin and mucous membranes of humans and other warm-blooded animals. Based on their ability to coagulate rabbit plasma, staphylococci have traditionally been grouped into coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS). As a major human and animal pathogen CoPS, pticemia . Althoug. aureus . SFP is . aureus . Althoug. aureus . Further. aureus ,4.mec (SCCmec) containing the mecA gene. An increase in the carriage rate of these SCCmec elements in CoPS and CoNS from various food sources has also been demonstrated in several recent studies [S. aureus (LA-MRSA). Therefore, enterotoxigenic staphylococci with enhanced resistance to antimicrobial drugs in food, especially livestock products, would raise significant public health concerns.In addition to enterotoxigenicity, the incidence of SFP cases, especially those involving antimicrobial resistant (AMR) staphylococci such as methicillin-resistant staphylococci (MRS), has been increasing in recent years . Methici studies . Wendlan studies  also sugIn swine production chains in European countries and Korea, one of the most frequent MRSA clones are CC398 (ST398 and ST541) LA-MRSA, which seem to be associated with the overuse of antibiotics in pig farms ,8. In adThe present study aimed to investigate the occurrence and species of CoPS and CoNS in retail pork meat samples collected during nationwide monitoring. In addition, antimicrobial resistance profiles, carriage of major antimicrobial resistance genes, and prevalence and distribution of SE genes and enterotoxin-like genes were determined in the pork-associated staphylococci. Moreover, susceptibilities to zinc chloride, urease activity, hemolytic activity, and proteolytic activity were examined to characterize the virulence phenotypes of the isolates.A total of 260 pre-packaged pork meat samples were collected from 35 retail markets in 8 provinces in Korea from July 2017 to May 2018: Gyeonggi (58 samples), Gangwon (42 samples), Chungcheong (55 samples), Jeolla (54 samples), and Gyeongsang (51 samples). Four or five retail markets from each province were randomly selected based on regional distribution. At least 3 to 4 different raw pork products including pork belly, loin, leg, blade shoulder, tenderloin, and arm shoulder were collected from each retail market. All meat samples were stored below 4°C in a container with ice packs and transported to the laboratory for isolation of staphylococci within 24 h of purchase.S. aureus with black colonies with a clear halo and/or staphylococcal colonies with gray/black color were all selected and up to 3 colonies of staphylococci from each sample were subcultured on BPA for further confirmation. Staphylococcal species identification was performed using both 16S RNA sequencing and the Vitek 2 system . Genomic DNA samples of staphylococci were prepared using a Genemed kit  according to the manufacturer’s recommendations. All the selected colonies were subjected to antimicrobial susceptibility assays and antimicrobial resistance gene detection assays.Isolation of staphylococci from pork meat samples was performed as described previously with minor modification ,9. BriefAntimicrobial susceptibility tests were performed on all the study strains according to the guidelines of Clinical and Laboratory Standards Institute . Briefly, minimum inhibitory concentrations (MICs) to oxacillin and tetracycline (TET) were determined using a broth micro-dilution method or a standard E-test . Susceptibilities to ampicillin 10 μg), cefoxitin (30 μg), gentamicin (30 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), clindamycin (2 μg), erythromycin (15 μg), sulfamethoxazole-trimethoprim (23.73 to 1.25 μg), quinopristin-dalfopristin (15 μg), rifampin (5 μg), fusidic acid (50 μg), and mupirocin (200 μg) were determined using a disc diffusion method on Mueller-Hinton agar plates . All the antimicrobial discs were purchased from BD. In addition to the antimicrobial susceptibility assays, MICs to zinc chloride (ranging from 0.125 to 32 mM) were determined using a standard agar dilution method on Mueller-Hinton agar (pH-adjusted to 5.5) as previously described [ μg, cefomecA and SCCmec types were determined as previously described [mec types were determined according to the combination of chromosomal cassette recombinase (ccr) and methicillin resistance (mec) regions. TET resistant strains were tested for the presence of tet(K), tet(L), tet(M), tet(O), and tet(S) as previously described [Staphylococcal strains displaying resistance to oxacillin, cefoxitin, and TET were examined for the presence of resistance genes. For methicillin-resistant strains, presence of escribed . SCCmec escribed .Urease activity was measured for 24 h with shaking (200 rpm). Urease activity was scored and classified into four groups according to intensity of color change: none (yellow), weak (orange), moderate (pink), or strong (purple).Hemolytic activity was examined on 5% sheep blood agar after 24 h growth at 37°C. Presence of alpha-, beta-, or gamma-hemolytic activity was determined as previously described .Proteolytic activity was assessed on skim milk agar (SMA) plates containing 10% SMA solution and 1.5% Bacto agar  . Brieflysea, seb, sec, sed, and see) and 13 newer SEs  was examined using multiplex polymerase chain reaction (PCR) assays according to previous publications [tst1 gene) was assessed using specific PCR primers [S. aureus strains  were used as positive controls for each multiplex PCR [Carriage of SE genes in the 29 staphylococcal isolates was determined as previously described . The preications . In addi primers . GenomicS. saprophyticus, comprising 55% of the staphylococci isolated from the samples. As shown in S. hyicus and S. aureus) were detected in the samples, four different species of CoNS  were identified.A total of 29 staphylococcal strains  were isolated from 260 pork meat samples collected in Korea over the 11-month study period . The mosS. saprophyticus strains were isolated from all provinces, indicating that this is the most widespread staphylococcal species in pork meat samples in Korea. All the 29 staphylococci used in this study were isolated from different meat samples.The prevalence of staphylococci in the pork meat samples collected from different provinces is presented in mecA and displayed an OXA-resistant phenotype. SCCmec typing revealed that S. aureus (SA1), S. epidermidis (SE2), and S. sciuri (SS3) contained SCCmec II, SCCmec IV, and SCCmec V, respectively. Although two other S. sciuri strains (SS1 and SS2) were also positive for mecA, the SCCmec type remains undesignated because the ccr genes were not identified. The methicillin-resistant S. aureus strains were of sequence type (ST) 5, agr type II, and spa type t2460.Five MRS were identified among the 29 isolates (17%) collected from the pork meat samples . These ftet genes with TET MICs of ≥32 μg/mL. Of note, 14/15 TET-resistant isolates (93%) carried the tet(K) resistance gene.All the 29 isolates were susceptible to chloramphenicol, sulfamethoxazole-trimethoprim, and rifampin . A multiS. aureus of animal origin [Zinc chloride resistance has been proposed to be associated with methicillin resistance in specific clones of l origin ,16,17. Hsep  and sen  were most frequently detected among the 29 staphylococci  carried nine different SE genes and tst1, six of which are located within an enterotoxin gene cluster . The presence of 6 enterotoxin genes  identified in CoNS strains by PCR analyses were confirmed by sequencing PCR products (data not shown).SEs are pyrogenic toxins and are involved in a significant number of human food poisoning diseases worldwide . As showylococci . In partS. hyicus, three S. warneri, and one S. epidermidis were alpha-hemolytic and all others were gamma-hemolytic  was much lower than that reported for retail pork meat samples in the USA (16% to 67%) [S. saprophyticus was detected with the highest incidence . In contrast, a predominance of S. epidermidis in raw pork meat was reported in a previous study from Nigeria [In the present study, we investigated the prevalence of CoPS and CoNS in retail pork meat samples collected from July 2017 to May 2018 in Korea. The overall prevalence of staphylococci in the 260 pork meat samples was 11% (29 isolates/260 samples) and prevalence rates in five of the eight provinces ranged from 7% to 16%. Previous studies conducted in Nigeria, Poland, and Egypt reported prevalence rates of 40% to 91% for staphylococci in raw meats and meat products . In part to 67%) . The pre Nigeria . These dS. aureus (MRSA), likely originated from CoNS by acquisition of SCCmec [mec V has frequently been associated with livestock, particularly pigs [mec II. While one strain of S. epidermidis (SE2) and a strain of S. sciuri (SS3) was determined to harbor SCCmec IV and SCCmec V, two strains of mecA-positive S. sciuri (SS1 and SS2) could not be typed for SCCmec. Several studies have reported heterogeneity among SCCmec elements in MR-CoNS isolates [S. sciuri, MR-S. sciuri carrying a non-typeable ccr gene was isolated from raw meat [ccr genes and novel ccr types is present in S. sciuri isolates.MRS, especially methicillin-resistant f SCCmec . Methicif SCCmec ,4,23. Corly pigs , one strisolates . In pararaw meat , indicatS. saprophyticus isolates . TET resistance in staphylococci is mediated by acquisition of mobile tet genes, of which tet(K) has been most frequently associated with TET resistance in many CoNS of animal origin [S. saprophyticus and S. sciuri isolates, carried tet(K).In addition to methicillin resistance, recent studies demonstrated that staphylococcal isolates from raw meat samples showed a TET resistance rate of 67% . Similarl origin ,18. Consl origin , the preczrC within the SCCmec V element is typically associated with a high incidence of swine associated ST398 MRSA in European countries [czrC-mediated zinc-resistance has also been reported in S. aureus strains isolated from meat products [aureus staphylococci (NAS) or CoNS are limited despite the possible co-selection of MRS using zinc in pig farms. However, the zinc-resistant gene czrC was identified in methicillin-resistant S. hyicus strains [czrC in methicillin resistant CoNS strains, such as S. epidermidis, S. haemolyticus, S. hominis, and S. lentus has been reported regardless of their SCCmec type [A zinc resistance phenotype mediated by ountries ,28. Receproducts ,30. Unli strains . Moreovemec type . SimilarS. aureus and are known to be responsible for SFP in humans. In addition to the 5 classical SE genes , 19 newer SEs, and enterotoxin-like (SEl) peptides have been identified [ls are mainly associated with CoPS S. aureus-associated SFP worldwide [S. epidermidis, S. chromogenes, S. hyicus, and S. haemolyticus isolates of human and animal origin carried SE genes [ls rather than the classical SEs. Moreover, our data showed that sep and sen were the most common SEs among the CoNS, especially in S. saprophyticus isolates. In contrast to the high prevalence of sep and sen in our CoNS, previous studies reported that sec was the most prevalent SE gene in CoNS isolates [sea, seb, and sec) were identified as the most frequent enterotoxins among CoNS isolated from bovine milk [ls in outbreaks of SFP is still controversial, the predominant presence of enterotoxigenic CoNS in raw pork meat could represent a potential food safety hazard. The SEs are synthesized and secreted by staphylococci throughout the exponential growth phase and during the transition from exponential to stationary growth phases [B), SarA, Agr quorum-sensing system, and SaeRS two-component regulatory system [S. aureus. Therefore, further studies are necessary to characterize expression profiles of enterotoxin genes found in the CoNS, especially sep and sen genes in S. saprophyticus strains.SEs were originally identified in entified . These Sorldwide . HoweverSE genes ,15. In tisolates . More reine milk . Although phases ,35. Usuah phases ,34,35. Ty system ,34. UnliPseudomonas spp. and psychrotrophic bacteria [S. hyicus and S. sciuri strains showed strong protease activity at 37°C. In addition, strong urease activity, which is often associated with acute urinary tract infections, was only observed in CoNS species of S. saprophyticus, S. warneri, and S. epidermidis.Staphylococci can produce secretary proteins such as pro teases and lipases that may have a negative effect on food preservation, causing a deterioration in quality and food spoilage . Althougbacteria , pork meIt should be recognized that the current study has several limitations. Our results were generated from a rather limited number of samples and staphylococcal isolates, which warrants future investigations with a larger number of various meat samples. In addition, complete genotypic characterization of the CoPS and CoNS for antimicrobial resistance, virulence, and synthesis of SEs in culture supernatants was not included in the current investigation.S. saprophyticus species, are present in retail pork meat; ii) while the occurrence of MDR in pork-associated CoNS was low, a high level of TET resistance among CoNS was observed, along with a predominant possession of tet(K); and iii) a frequent presence of newly defined SEls such as sep and sen were confirmed in CoNS species, especially in S. saprophyticus strains isolated from pork meat. Thus, in addition to CoPS, our results suggest that CoNS from raw pork meat could serve as a potential reservoir for antimicrobial resistance as well as enterotoxins typically associated with SFP. Future studies are also necessary to investigate prevalence and characteristics of CoPS and CoNS in other foods of animal origin such as milk, chicken, and beef meats.Our results demonstrate the following: i) a relatively high level of CoNS, especially


Mucinous peritoneal metastases (PM) generally respond poorly to systemic treatment, and there is a clear unmet need for new treatment strategies to improve survival and quality of life for patients with PM. In this work, the growth inhibitory effect of five drugs , irinotecan , cabazitaxel , regorafenib , and capecitabine  was investigated in three orthotopic patient-derived xenograft models that mimic mucinous PM.KRAS, GNAS, and BRAF oncogenes), supporting the representativeness of the models. The results presented in this work support the continued exploration of i.p. treatment protocols for PM, with OXA remaining and CBZ emerging as particularly interesting candidates for further studies.Drugs were administered intraperitoneally (i.p.) as monotherapy weekly for 4 weeks , as one single i.p. injection (CBZ), or orally  daily 5 of 7 days per week for four weeks, and i.p. tumor growth and survival were monitored and compared between treatment groups. The i.p. administered drugs  had the strongest growth inhibitory effect, with OXA being most efficacious, completely inhibiting tumor growth in the majority of the animals. CBZ and IRI also strongly inhibited tumor growth, but with more variation in efficacy between the models. A moderate reduction in tumor growth was observed in all models treated with REG, while CAP had little to no growth inhibitory effect. Targeted next-generation-sequencing has identified mutational profiles typically associated with PM (mutations in  Peritoneal metastases represent an important therapeutic challenge, since patients generally respond poorly to systemic chemotherapy and targeted treatments . A possiWe have generated orthotopic patient-derived xenograft models that mimic mucinous PM, and that were shown to closely resemble the original disease with respect to growth pattern and protein expression ,11. We pnu were bred at Department of Comparative Medicine, Oslo University Hospital, and kept in a specific pathogen-free environment at constant temperature (22 ± 1 °C) and humidity (62 ± 5%), 15 air changes/hour and a 12-hour light/dark cycle. Food and water were supplied ad libitum, and the mice were given paper and card board houses for environmental stimulation. A maximum of 10 mice were housed in each cage. The model establishment was previously described ). This work was also supported by funding from the 10.13039/100001260National Organization for Rare Disorders (NORD) through Appendix Cancer Pseudomyxoma Peritonei (ACPMP) Research Foundation.KGF and CL-A are postdoctoral fellows of the Bayer AG provided regorafenib and research support for the study.KGF: Investigation, formal analysis, visualization, writing original draft. CLA: Formal analysis, investigation. SW: Investigation. TWA: Resources. YM: Resources. KB: Conceptualization. AT: Formal analysis, investigation. KF: Conceptualization, methodology, writing reviewing and editing, supervision, funding acquisition. All authors approved the manuscript.