Listeria monocytogenes are Gram-positive rods, which are the etiological factor of listeriosis. L. monocytogenes quickly adapts to changing environmental conditions. Since the main source of rods is food, its elimination from the production line is a priority. The study aimed to evaluate the influence of selected stress factors on the growth and survival of L. monocytogenes strains isolated from food products and clinical material.L. monocytogenes (PFGE method). Sixty-two percent of the tested strains represented 1/2a-3a serogroup. Sixty percent of the rods possessed ten examined virulence genes . Listeria Pathogenicity Island 1 (LIPI-1) was demonstrated among 38 (76.0%) strains. Majority (92.0%) of strains (46) were sensitive to all examined antibiotics. The most effective concentration of bacteriophage  was 5 × 108 PFU. In turn, the concentration of 8% of NaCl was enough to inhibit the growth of 31 strains (62.0%). The clinical strain tolerated the broadest pH range (3 to 10). Five strains survived the 60-min exposure to 70˚C, whereas all were alive at each time stage of the cold stress experiment. During the stress of cyclic freezing-defrosting, an increase in the number of bacteria was shown after the first cycle, and a decrease was only observed after cycle 3. The least sensitive to low nutrients content were strains isolated from frozen food. The high BHI concentration promoted the growth of all groups.We distinguished fifty genetically different strains of Data on survival in stress conditions can form the basis for one of the hypotheses explaining the formation of persistent strains. Such studies are also helpful for planning appropriate hygiene strategies within the food industry. Listeria monocytogenes are Gram-positive, facultatively anaerobic, non-spore-forming rods. Bacteria settle many niches due to their tolerance to changing environmental conditions  [L. monocytogenes infections (listeriosis) are characterized by a high mortality rate (up to 30%) [listeriosis related to various food products  [ronment) . Major s to 30%) . There haloupes) .L. monocytogenes experiences many unfavorable conditions, including low and high temperatures, variable pH, osmotic stress, nutrient deficiency, and contact with disinfectants. The most commonly encountered stresses within the food industry for L. monocytogenes are acidic and osmotic stresses , temperature  [L. monocytogenes to adapt to low pH environments, e.g., during the passage through the stomach and in the phagosome, is relevant for the host invasion [L. monocytogenes is indispensable for the survival in the food environment and may affect bacterial pathogenicity [B, σC, σH and σL, which σB plays a key role [L. monocytogenes, these factors control more than 300 genes, including stress and virulence genes. The use of sublethal doses of stress factors in the food processing environment (FPE) may increase resistance to antibiotics or stimulate resistance to other stress conditions (cross-resistance) and higher doses of the same stress (stress adaptation) [During food processing, ization) . The abiinvasion . The adagenicity . One of key role . In L. mptation) .In recent years a growing concern has focused on the identification of strains that survive in the food processing environment –12. ReseMicrobiological typing  is a tool that has been used for years to identify organisms within a species , 17.TherL. monocytogenes isolated from food products and clinical material.The study aimed to evaluate the impact of selected stress factors , salinity (0–20%), availability of nutrients) on the growth and survival (screening) of L. monocytogenes .The most studied strains represented the serogroup 1/2a-3a (31 (62.0%)), followed by the serogroup 4b-4d-4e (14 (28.0%)). Most strains of seogroup 1/2a-3a were of clinical origin, while 4b-4d-4e strains derived from frozen food  among 38 (76.0%) tested strains. We did not identify the mpl gene in 5 strains and the fbpA gene in 8 strains. Seven tested strains representing all groups of isolates possessed 8 virulence genes (profile IV)  Table . There wp = 0.180).We distinguished three antibiotic sensitivity profiles. The most numerous profile (A) included 46 (92.0%) tested strains sensitive to all tested antibiotics Table . Resista4 to 5 × 105 PFU (Plaque Forming Units) did not inhibit the growth of the tested L. monocytogees strains . The concentration of 5 × 106 PFU inhibited the growth of one strain , while 5 × 107 PFU—5 strains (10.0%) (no observed turbidity). The most effective concentration of bacteriophage inhibiting the growth of the largest number of strains  was 5 × 108 PFU. The concentration of 5 × 1010 PFU inhibited the growth of 11 strains (22.0%). One strain isolated from salmon and two obtained from frozen food were insensitive to all studied concentrations of PhageGuard L (visible turbidity)  value of PhageGuard L was – 5 × 10The lowest concentration inhibiting the growth of the tested strains was 7% NaCl, at this concentration growth inhibition was observed for 5 out of 50 strains (10.0%). The concentration of 8% NaCl was enough to inhibit the growth of strains 31 62.0%). Then, the concentration of 9% NaCl inhibited the growth of 6 more examined strains (12.0%), and 11% NaCl—5 (10.0%) strains. The concentration of 13% NaCl inhibited the growth of one strain isolated from cold cuts (459CC), and 14% NaCl—one strain isolated from the clinical material (50C)  was 8% Fig. . The higThe study assessed the pH in the range of 3 to 10. The analysis of the results included “the growth-inhibiting pH value < 7” and “the growth-inhibiting pH value > 7”. When growth  was found at pH 10 and pH 3, there were arbitrarily assinged values 11 and 2 as growth-inhibiting, respectively.The median for "the growth-inhibiting pH value < 7" was 4.5, 5, 4 and 3.5 for strains isolated from clinical matrerial, cold cuts, frozen food and salmon, respectively. In contrast, the median in “the growth-inhbiting pH value > 7” variant was 11, 9, 9, and 9 for strains isolated from the clinical matrial, cold cuts, frozen food and salmon, respectively  strains. Further lowering of pH resulted in the inhibition of additional 19 38.0%) strains – pH 5, 12 (24.0%) strains – pH 4, and 11 (22.0%) strains – pH 3. At pH 7, the growth of all tested strains was observed. In "the growth-inhbiting pH value > 7" variant, pH 8 inhibited the growth of 6 (12.0%) strains, pH 9 – additional 20 (40.0%) strains, and pH 10 – additional 6 (12.0%) strains. Importantly, 18 strains (36.0%), including 12 strains isolated from the clinical material, survived the pH 10 . In turn, strains with growth inhibition at pH 3, were able to multiply at maximum of pH 8 Fig. . Two strThe clinical strain  tolerated the widest pH range , while three strains were able to grow only in the very narrow range of pH  . The lowest growth-inhibiting temperature in the study was 65˚C, the exposure time was 30 min. In this variant, we did not demonstrate survival of 10 strains, including 8 frozen food , one salmon (no. 20S) and one cold cuts (no. 463CC) strains. The 60-min exposureof 65˚C inhibited the growth of 16 tested strains (8 from cold cuts and 8 from frozen food). Five strains, including 4 isolated from clinical material  and one isolated from salmon (no. 17S)  and 65˚C . Exposure to 55˚C for 60 min did not inhibit the growth of the tested All strains survived all stages of the cold stress experiment. In the control variant, without the stress factor, the median number of bacteria was as follows: clinical – 6.97 log CFU/ml; salmon—6.68 log CFU/ml; cold cuts—6.34 log CFU/ml; frozen food—6.79 log CFU/ml. After 24 h, the number of CFU increased, median for clinical—7.78 log CFU/ml; salmon – 8.13 log CFU/ml; cold cuts—7.99 log CFU/ml; frozen food – 8.01 log CFU/ml. After 30 days of the experiment, a significant increase in the number of CFUs of bacteria was visible, median for clinical—11.02 log CFU/ml; salmon—10.42 log CFU/ml; cold cuts—10.12 log CFU/ml; frozen food—10.12 log CFU/ml.In order to compare the response to cold stress, the difference between the number of bacteria after 30 days and the number of control bacteria  was examined for each group; however, there were no significant differences Fig. .Fig. 6ChThe median number of bacteria (without the stress factor) was 7.40 log CFU/ml. Thereafter, the median number of bacteria was 7.55, 7.49, 7.50 and 7.30 log CFU/ml after 1, 2, 3, 4 freezing-defrosting cycles, respectively. In the variant with defrosting only after the 4th cycle, the median number of bacteria was 7.44 log CFU/ml  in the bacteria number between the variant of 4 cycles of freezing-defrosting and the variant of defrosting after the fourth cycle.The Wilcoxon test showed significant differences between the cycles 0 and 1 (a visible increase in the number of bacteria), and also between cycles 4 and 1, 2, 3 (after the fourth cycle the number of bacteria decreased) Fig. . There wTo comapre the groups of strains, we divided the results according to the nutrient content: low (10–40%) and high (130–200%) BHI content. The results are presented as the change (relative to control) in the number of bacteria (an increase in the number of bacteria with an increase in nutrient content).L. monocytogenes growth. In each experiment variant, the growth of the tested strains was observed. The median bacterial count for the 10% nutrient availability variant, was: 6.11, 6.18, 5.59, 6.65 log CFU/ml for clinical, salmon, cold cuts and frozen food strains, respectively. In turn, in the variant with the highest availability of nutrients (200%), the median number of bacteria was: 13.30, 13.51, 13.23, 12.26 log CFU/ml, for clinical, salmon, cold cuts and frozen food strains, respectively.We assessed the impact of low and high availability of nutrients on In the low BHI (Brain Heart Infusion) (10–40%) variant, the median number of bacteria was 0.23, 0.21, 0.22 and 1.0 log CFU/ml for strains isolated from clinical material, cold cuts, salmon and frozen food, respectively. The least sensitive to low BHI content were strains isolated from frozen food. However, we did not observe no significant differences between the groups of strains. In the high BHI concentration (130–200%) variant, the median number of bacteria was 5.27, 4.48, 3.35 and 4.99 log CFU/ml for strains isolated from the clinical material, cold cuts, frozen food and salmon, respectively  and clinical material (including cases of systemic infections).In 2020 of 13.0% . High moreatment . Many reThe PFGE method, referred to as the "gold standard", was used to assess the genetic similarity of the tested isolates. Currently the PFGE and WGS methods are most popular methods used for typing of microorganisms. The PFGE method is widely apllied for, e.g., outbreaks' investigation , food saL. monocytogenes 4b, 1/2b and 1/2c serotypes are most often responsible for listeriosis (98% of documented cases) [L. monocytogenes infection cycle mainly involves genes located on LIPI-1 [The initial characterization of the strains included the determination of serogroups belonging, the presence of virulence genes and the assessment of drug susceptibility. We have shown that most studied strains represented the serogroup 1/2a-3a (31 (62.0%)), followed by the serogroup 4b-4d-4e (14 (28.0%)). d cases) . In contd cases) , 44. In n LIPI-1 . Due to n LIPI-1 , antibioL. monocytogenes. The concentration of bacteriophage inhibiting the growth of the largest number  of the strains was 5 × 108 PFU. In turn, Reinhard et al. [L. monocytogenes from 1.27 to 3.33, 1.17 to 2.76, 1.19 to 1.76 log10 CFU/cm2 on the surface of stainless steel, polyurethane thermoplastic tape and on the epoxy resin floor, respectively. Additionally, Reinhard et al. [8 PFU/cm2) and longer contact time (3 h) resulted in a higher reduction of L. monocytogenes. Also Truchado et al. [9 PFU/ml) against L. monocytogenes. They have observed maximum reduction of L. monocytogenes (by about 3.5 log units) on freshly cut endives after 3 days of storage [L. monocytogenes on food products or food contact surfaces. However, more research on its safety and effectiveness is required.Scientists are constantly searching for new methods of microbiological food control. The study assessed the effect of the bacteriophage in PhageGuard L on d et al.  showed td et al.  confirmeo et al.  have sho storage . Based oL. monocytogenes to many stress factors, including heat and cold shock, acids, high salt content and preservatives [The stress adaptive response of bacteria is one of the greatest challenges in controlling pathogens within the food industry. A relevant is balance between the microbiological safety of food and maintaining the freshness of products . To datervatives –54. Our L. monocytogenes [L. monocytogenes. The main mechanism of osmotic stress response in L. monocytogenes relies on osmoprotectants  [L. monocytogenes is able to grow at high salt concentrations. Rods can withstand up to 10% of NaCl [L. monocytogenes strains tested were resistant to saturated NaCl (about 7 M (40% w/v)) for a long time (20 h and possibly longer). In turn, Magalhães et al. [L. monocytogenes was able to survive in soybean tryptose broth containing 25.5% (w/v) NaCl for 4 days at 37°C, and lowering the experimental temperature to 22°C extended the survival time to 24 days and up to over 132 days at 4˚C. We conducted experiment at 37˚C and did not observe relevant differences in salt stress resistance between strains of diiferent origin and serogroup. Also, Hingston et al. [L. monocytogenes to high concentrations of NaCl varies and may depend on the time-exposure, previous contact with the stressor (including the source of isolation), the availability of osmocrotectants in the environment, temperature or individual strain. It is also important to remember that exposure of L. monocytogenes to osmotic shock can contribute to cross-protection against other stresses such as heat and acid [Since salt is a common food preservative, especially in meat and fish products, osmotic stress is one challenge in the food processing environment for ytogenes . The mosrnitine) . L. mono of NaCl  and 12%  of NaCl . In our  of NaCl  showed ts et al.  observeds et al.  showed ts et al. . The tems et al. , 58, 62.s et al.  found thn et al.  noted noand acid .L. monocytogenes in the food industry are exposed to changing environmental pH conditions. Since the use of acids  is a routine food preservation method, acid stress is a common phenomenon. Also, during host invasion L. monocytogenes encounters low pH, i.e., gastric juice and bile salts [L. monocytogenes include acid tolerance response (ATR), glutamate decarboxylase (GAD), arginine deiminase (ADI), and F1F0-ATPase [L. monocytogenes [L. monocytogenes LO28 and EGD strains, respectively. Metselaar et al. [L. monocytogenes invasiveness after exposure to acid stress. Researchers have shown [L. monocytogenes is possibly associated with the synthesis of appropriate proteins that change the structure of the cell membrane. As a result bacterial ability to maintain intracellular pH increases. It suggests that to different pathways for H+ entry and exit through the cell membrane. In response to lethal acidic conditions bacteria synthesize additional stress proteins [L. monocytogenes than strong inorganic acids such as HCl [L. monocytogenes during passage through the simulated gastrointestinal tract. They have observed that clinical isolates were more resistant than food isolates recovered from various food products. On the other hand, Lundẻn et al. [L. monocytogenes cells. Such atypical cells may be associated with increased survival of L. monocytogenes in unfavorable environments [L. monocytogenes cells survived for at least 6 days in tryptose phosphate broth at pH 9.0, 10.0, and 11.0 at 4°C or 21°C. L. monocytogenes may also experience alkaline stress in the food processing environment. Alkaline solutions are generally used in detergents to remove charred sediment, oil or grease, facilitate protein denaturation, saponification of fats. Interestingly, our own study showed that 12 strains isolated from clinical material multiplied at pH 10. Many factors have an impact on the ability to survive and grow at different pH, including the individual characteristics of the strain.le salts . The mai0-ATPase . In our ytogenes , 68, 69.ytogenes  revealedr et al.  concluder et al. –75. Addir et al. , 76. Metr et al.  distingur et al. . Cataldor et al.  demonstrve shown  that resproteins , 78, 79.h as HCl . In the h as HCl  found loh as HCl  showed th as HCl  and Ramah as HCl  have assn et al.  have notn et al.  have docronments . Taorminronments  have repL. monocytogenes have the ability to grow in a wide range of temperatures (0–45˚C) [L. monocytogenes strains subjected to heat stress (temperature 58˚C). Hingston et al. [L. monocytogenes lag phase delay in trypic soy broth supplemented with 0.6% yeast extract at room temperature for 3 to 5 h compared to non-stressed control cells [Application of low and high temperatures is a well-known and widely used method of eliminating microorganisms. 0–45˚C) . We obse(0–45˚C) , after h˚C . We on et al.  found non et al.  divided ol cells . The strol cells . TherefoL. monocytogenes to grow under refrigerated conditions, transport or refrigerated food storage by the consumer increases the risk of infection or epidemics. A number of researchers have studied the cold stress response of L. monocytogenes [L. monocytogenes at 4˚C. After 30 days of incubation at 4˚C clinical strains reached the highest number (increase by 4.05 log CFU). The final total bacteria numbers were 10.12, 10.42 and 10.12 log CFU for strains isolated from frozen food, salmon and cold cuts, respectively. However, the differences between groups were not significant. Arguedas-Villa et al. [The ability of ytogenes –92. Our a et al.  have dema et al.  have fouL. monocytogenes [L. monocytogenes for 5 days resulted in an extension of the lag time and a slowdown in the specific growth rate. In turn, Miladi et al. [L. monocytogenes cells stored for 10 months at -20˚C. The researchers found no changes in the metabolism of L. monocytogenes [L. monocytogenes after six months of freezing (-20˚C) on cheese slices. Jiang et al. [Listeria coatings, reducing the population of L. monocytogenes on roasted turkey. The research conducted so far, has focused on the survival of L. monocytogenes mostly under freezing conditions. Our study assessed the impact of cyclic freezing and defrosting, which is essential from the consumer's point of view. In our study, we demonstarted that the bacteria number after one cycle of freezing was significantly higher than the control group (not subjected to freezing). Strains isolated from clinical material, cold cuts and frozen food increased their number up to the third freezing-defrosting cycle. After the fourth cycle, all bacteria significantly decreased their number. In the defrosting variant, only after the 4th cycle we noticed an increase in log CFU compared to the control. In contrast, Azizoglu et al. [L. monocytogenes cells grown at 37°C, with a decrease of < 1 log after 18 freeze–thaw cycles. Conversely, freeze–thaw tolerance was significantly reduced when bacteria were grown at 4 or 25°C (2 to > 4 log). On the other hand, Simpson Beauchamp et al. [L. monocytogenes populations (< 0.5 log CFU/cm (2)). In turn, Kang et al. [L. monocytogenes number at 7°C. Due to a significant gap in the data on the effect of freezing–thawing on L. monocytogenes further studies are recommended.Freezing food is a process that extends the shelf life of a product and is also readily used by consumers. Freezing and thawing are big challenges for bacteria, including ytogenes . Yoon etytogenes  have foui et al.  have obsytogenes . Also Beytogenes  have notg et al.  showed tu et al.  have shop et al.  have docg et al.  have obsL. monocytogenes can grow and multiply in environments with varying nutrient availability. In our study, in conditions of low nutrient content, strains isolated from frozen food performed best (visible increase in log CFU number), which may be associated with previous freezing. High BHI content favored the growth of strains of all groups. In the environment of insufficient nutrients availability L. monocytogenes initiates the starvation survival response (SSR) [L. monocytogenes EGD can be induced by glucose and other nutrients restriction but not amino acid restriction. Surviving cells show reduced cell size and increased cross-protection to several environmental stresses [L. monocytogenes during multicomponent starvation are still not fully understood.The availability of nutrients in the environment varies. The food processing environment is rich in waste, such as blood, fruit juices and food fragments. The availability of nutrients decreases significantly after disinfection procedure. se (SSR) . Severalse (SSR) , 104 havstresses , 104. Atstresses  suggest L. monocytogenes found in food products exhibit full pathogenic potential. High adaptability to unfavorable stress conditions may increase microbial virulence [L. monocytogenes is associated with a higher expression of virulence genes  [In our ranking of the stress response, four clinical strains and one isolated from salmon got the highest score. Clinical strains were more resistant to the stress factors used in the study. Clinical strains derived from, e.g., blood or cerebrospinal fluid, which may indicate their high virulent potential. Nevertheless, the response of food strains was heterogeneous. Strains isolated from cold cuts received the lowest number of points in the ranking (103.5 points). It may support Dykes and Moorhead  suggestiirulence . Kazmierirulence  have repernalin) .L. monocytogenes is one of the most dangerous foodborne pathogen. Its ability to survive and grow under various stressful conditions in the food production environment, is of great concern. Knowledge about the influence of stress factors on L. monocytogenes will allow to design appropriate methods of control and production line disinfection. Adaptation to stress factors is an individual feature of a given strain, and so far no clear link between stress response and the strains' origin has been described. We included, in our study, clinical and food strains to have a more diverse population of L. monocytogenes. We expect that some strains could have experienced earlier adverse conditions. Nonetheless, evaluation of the impact of stress factors on phenotypic traits and virulence of L. monocytogenes merits further investigation. Our study aimed to screen a stress response to factors most commonly encoutered in the food production environment in large population. Future studies should concentrate on the molecular bases of the observed resistance to stress factors.L. monocytogenes isolates from the collection of the Department of Microbiology, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Toruń, Poland, previously isolated from clinical material  and food , CC – cold cuts (n = 20), FF – frozen food (n = 20))  with 15.0% glycerol (Avantor) at − 80°C until the beginning of the research.The research material consisted of 80 L. monocytogenes were used for further studies.Tested isolates were plated on Columbia Agar supplemented with 5.0% sheep blood  and incubated . Then, the grown colonies were cultured under the same conditions. The grown strains of L. monocytogenes isolates was determined with PFGE (“gold standard”). The procedure for genotyping was performed in accordance with the standard operating procedure for PulseNet PFGE of Listeria monocytogenes [After confirming the species identity, the genetic similarity of the selected ytogenes . To deteL. monocytogenes strains included the assessment of belonging to serological groups, the presence of specific virulence genes and sensitivity to selected antibiotics. The presence of LIPI-1 which contains virulence genes involved in the intracellular infection cycle of L. monocytogenes and locusInlA-InlB was also assessed.The initial characterization of DNA was isolated from overnight cultures grown on Tryptic Soy Agar . A single colony was resuspended in 100 µl of 1 × Tris–EDTA buffer (Sigma-Aldrich), heated at 90°C  for 10 min, cooled on ice (2 min), and centrifuged .L. monocytogenes serogroups  was performed as described by Doumith et al. [L. monocytogenes strains described earlier by Wałecka-Zacharska et al. [Multiplex PCR for the identification of the main h et al. . The foua et al.  were useL. monocytogenes. Three separate PCR reactions were prepared: I , II , and III . Primer sequences and reaction conditions were previously presented by Skowron et al. [L. monocytogenes ATCC 19111 strain, possessing all detected genes, was used as the reference.The multiplex PCR technique was used to determine the frequency of 10 selected virulence genes occurrence among n et al. . The mulThe evaluation was performed using the disk-diffusion method. Bacterial cultures (24 h) were diluted to 0.5 McF in 0.9% saline solution (Avantor). The prepared suspensions were plated on MHF medium  and then antibiotic discs were added. The susceptibility of isolates to penicillin (1 IU), ampicillin (2 μg), meropenem (10 μg), erythromycin (15 μg), and cotrimoxazole (1.25–23.75 μg) was evaluated. The prepared antibiograms were incubated at 35°C for 20 h. Then, growth inhibition zones around the antibiotic discs were measured. The results were analyzed in accordance with the EUCAST  v. 12.0 recommendations .5 CFU/mL) [4 to 5 × 1010 PFU). The negative control was 100 µl of bacteriophage of appropriate concentration and 100 µl of sterile TSB. The positive control consisted of 200 µl diluted bacterial suspensions. Incubation was carried out in a moist chamber . Next, the turbidity was assessed visually. Turbidity indicated the growth, and no turbidity marked the growth inhibition of L. monocytogenes . For the identified MIC value concentration and the three above concentrations of bacteriophage suspension, bacteria were streaked on the TSA medium to determine the MBC value. After 24-h incubation at 37°C the growth was assessed . No growth on the medium after the incubation period was taken as the MBC value.The evaluation was carried out on the basis of Vipra et al. , with so CFU/mL) . Then, 1L. monocytogenes.The final NaCl concentration used for the experiment ranged from 0 to 20% (variation every 1%). Suspensions of the tested strains in Mueller Hinton Broth  (0.5 McF) were mixed with an equal volume of MHB with the appropriate NaCl concentration (in triplicate). The test included a positive control (suspension of the given strain) and a negative control (sterile MHB). After the incubation period  in a moist chamber, the presence of turbidity was assessed visually (screenig method). Turbidity indicated the growth, and no turbidity marked the growth inhibition of L. monocytogenes.The pH tested ranged from 3 to10 (variation every 1 degree). Suspensions of the tested strains in Mueller Hinton Broth  (0.5 McF) were mixed with an equal volume of MHB with an equal volume of MHB at the appropriate pH (using HCl (POCH) or NaOH (Chempur)) (in triplicate). The test included a positive control (suspension of the given strain) and a negative control (sterile MHB). After the incubation period  in a moist chamber, the presence of turbidity was assessed visually (screening method). Turbidity indicated the growth, and no turbidity marked the growth inhibition of L. monocytogenes were assessed. A suspension of strains in Phosphate Buffered Saline  (0.5 McF) was subjected to selected temperatures for an appropriate time . Then, a serial tenfold dilutions in PBS (BTL) were prepared and plated (100 µl) on sheep blood agar . After the incubation period , grown colonies were counted and presented as log CFU/ml. The negative control was sterile PBS, and the positive control was a suspension of the tested strains.The impact of high temperatures, the so-called heat shock  and low temperature, the so-called cold shock (4˚C), on the survival rate of A suspension made in Tryptic Soy Broth  (0.5 McF) was placed at -20°C for one day. Next, the samples were defrosted at 23°C for three hours (complete liquefaction) and serial tenfold dilutions were plated (100 µl) on CAB (Graso) medium (in duplicate). After the incubation period , the grown colonies were counted and presented as log CFU/ml. The sample was then placed back in the freezer. The procedure was repeated three times. A sample not subjected to cyclic freeze-defrosting  was also prepared.A broth with a weight of loose substrate ranging from 20 to 400% recommended by the manufacturer was used (variation every 20%). Strain suspensions in Brain Heart Infusion (BHI (Becton Dckinson) (0.5 McF) were diluted 500-fold. Then 100 µl of such suspension was mixed with 100 µl of BHI medium (Becton Dickinson), obtaining 10—200% availability of nutrients. A positive control (suspension of the given strain) and a negative control (sterile BHI) were prepared. After the incubation period  serial tenfold dilutions were made and 100 µl was plated onto CAB (Graso) (in duplicate). After the incubation period , grown colonies were counted and presented as log CFU/ml.All statistical analyses were performed using R software . Due to (a) osmotic stress – based on saline concentration that inhibited the growth of the strain (the higher concentration – the higher rank);(b) pH range – based on range at which the growth of the strain was not inhibited (the higher range – the higher rank);(c) heat stress—based on values of log CFU/ml (expressed as change of the number of bacteria after stress exposure); since there were multiple conditions (four time points and four temperatures), mean ranks for each time point/temperature combination were calculated to obtain the final rank;(d) high or (e) low nutrient access – also based on values of log CFU/ml; mean ranks for each nutrient availability level  were calculated to obtain final rank.In order to select the most resistant strains, a scoring system based on non-parametric rank approach was used. The strains were ranked based on their susceptibility to each of the five stresses in a following manner:The strains could score a maximum of 50 points for each stress , therefore, in total, there were 250 points to score. In the case of ties, the average score was calculated. Strains with the highest scores were considered the most resistant. A limitation of this approach is that some of the variables were expressed in a discrete scale, resulting in multiple ties.


Deguelia nitidula (Benth.) A.M.G.Azevedo & R.A.Camargo (Fabaceae) is an herbaceous plant distributed in the Brazilian Amazon, and it is called “raiz do sol” (sun roots). On Marajó Island, quilombola communities use its prepared roots to treat skin diseases commonly caused by fungi, viruses, and bacteria. Thus, in this study, the extract, and its fractions from D. nitidula roots were used to perform in vitro cytotoxic and antibacterial assays against Staphylococcus aureus strains. Thereafter, liquid chromatography–mass spectrometry (LC–MS) was used for the metabolite annotation process. The ethanolic extract of D. nitidula roots show significant bactericidal activity against S. aureus with IC50 82 μg.mL−1 and a selectivity index (SI) of 21.35. Furthermore, the SREFr2 and SREFr3 fractions show a potent bactericidal activity, i.e., MIC of 46.8 μg.mL−1 for both, and MBC of 375 and 93.7 μg.mL−1, respectively. As showcased, SREFr3 shows safe and effective antibacterial activity mainly in respect to the excellent selectivity index (SI = 82.06). On the other hand, SREFr2 shows low selectivity (SI = 6.8), which characterizes it as not safe for therapeutic use. Otherwise, due to a limited amount of reference MS2 spectra in public libraries, up to now, it was not possible to perform a complete metabolite annotation. Despite that, our antibacterial results for SREFr3 and correlated substructures of amino acid derivatives show that the roots of D. nitidula are a natural source of specialized metabolites, which can be isolated in the future, and then used as a support for further bio-guided research, as well as natural drug development. In the last two decades, the increasing number of bacterial infections has been one of the main focuses of the World Health Organization . OverallStaphylococcus aureus, has shown a high level of dissemination because it is usually present in the skin and mucous. However, when it is neglected, it can progress to endocarditis, meningitis, pneumonia, and central nervous system diseases [S. aureus are responsible for high morbidity and mortality in the world due to socioeconomic factors such as low-income, overcrowded and low levels of schooling, and difficulty in accessing multi-professional teams, which, in consequence, increased costs to the health system [This is why the medical sciences already consider that we live in a post-antibiotic era and that the super bacterial infections need to be treated with a level of urgency , since cdiseases ,8. In adh system .D. nitidula (Benth.) A.M.G. Azevedo & R.A. Camargo were reported among some plants cited to treat skin diseases [Rhipicephalus (Boophilus) microplus  [For this reason, the bioprospecting of new natural antimicrobial herbal medicines has become rather common, mainly in traditional populations, in which, due to the unavailability of basic health care, many people use medicinal plants to treat many diseases . From thdiseases . This spdiseases  have beei, 1887)  and acari, 1887)  were repDeguelia is mainly known for the biosynthesis of flavones and isoflavones, which are promising classes of compounds in the race for the discovery and development of new antimicrobial agents [Complementarily, the genus l agents ,16,17. Hl agents ,19, whicl agents .S. aureus for the ethanolic extract and its fractions from the roots of D. nitidula.Hence, bioactive extracts and fractions can have their molecular profile revealed in a few weeks or months by metabolite annotation (levels 2 and 3) , as estaD. nitidula were collected in a remaining quilombola community located in the municipality of Salvaterra, Marajó, Pará (0°45′21″ S 48°45′54″ W) in January 2019. Identification of the botanical material was performed at the Empresa Brasileira de Pesquisa Agropecuária da Amazônia Oriental (EMBRAPA) and an exsiccate was deposited at the Herbarium IAN (voucher 199349). The authorization to access the traditional knowledge was registered in SISGEN (ID SISGEN A8CC64B). The roots were dried in an oven at 45 °C followed by the grinding process, where 1.0 kg of dried material was extracted with 4 L of 99.9% (v/v) ethanol. After extraction, 398 g were recovered (39.8% yield). A total of 320 g of ethanolic extract (SREE) was then fractionated using chromatography in column and silica gel as stationary phase. For this, a glass column  filled with the G60 silica gel adsorbent containing particles ranging from 60–200 µm (70–230 mesh) purchased from Siliciclo  in a ratio of 1:10 (sample/silica) was used. During fractionation process, four fractions were collected using hexane, dichloromethane, ethyl acetate and methanol as mobile phase. All solvents HPLC/Spectro grade were purchase from Tedia Company . Thereafter, the organic solvents were then removed using a rotative evaporator Buchi . Lastly, four resulting fractions were named SREFr1 (70.5 g), SREFr2 (20.28 g), SREFr3 (60.54 g), and SREFr4 (20.94 g), respectively.The roots of −1) was analyzed in the UPLC system, using gradient elution from 5% to 95% solvent B, for 11 min. Leucine-enkephalin  was used as a reference mass. The source temperature was set up to 120 °C with a cone gas flow of 30 L/h. The desolvation gas flow was set up to 600 L/h at a temperature of 250 °C. The capillary sampling was set up to 3.5 kV with the cone voltage of 40 V. A DDA method data was used in positive mode (top5 most intense ions), mass range from m/z 50 to 1200 Da (MS1 and MS2) with a scan time of 0.1 s. A profile of normalized collision energy (10–40 eV) was applied and MassLynx software  was used to control the system and data acquisition.Liquid chromatography–mass spectrometry (LC–MS/MS) analyses were performed using liquid chromatography coupled to a Xevo G2-S Q-Tof mass spectrometer  equipped with an electrospray source. The samples were analyzed in a BEH C18 column  using ultra-pure water (solvent A) and acetonitrile (solvent B) as mobile phases, both acidified with 0.1% formic acid. The column temperature was set at 40 °C and a flow rate of 0.3 mL/min. A total of 2 μL of extract (1 mg.mLA processed quantification table (CSV) and MGF files from MZmine 3.0  containiFor in vitro toxicity test, human venous blood was collected from healthy volunteers who were abstainers of alcohol and tobacco  that signed the informed consent form (ICF). This study was approved by the Institutional Committee of Ethics in Research involving human beings from the health sciences sector of UFPA . For the ethnopharmacological study, five remaining quilombola communities belonging to the city of Salvaterra, Marajó, Pará, were interviewed and this study was registered at CEPS/ICS/UFPA sector, under code CAAE: 22886619.5.0000.0018.−1), 10% (v/v) fetal bovine serum , 2% (v/v) glutamine (Sigma-Aldrich), and, lastly, the resultant solution was incubated with RPMI 1640 Complete Medium supplemented to evaluate cell viability.PBMC were isolated from whole blood using Histopaque-1077 . Thereafter, the whole blood sample was diluted to 1:1 volume ratio with saline solution (0.9%) and centrifugated for 20 min (2000RPM). Posteriorly, PBMC viability was determined by trypan blue exclusion and the viability was always >95%. Then, the cells were washed and suspended in Roswell Park Memorial Institute-1640 medium , and then it was supplemented with sodium bicarbonate (2 g.LD. nitidula roots ethanolic extract (SREE), the cell viability of peripheral PBMC were performed by 3--2,5-bromide-diphenyl-tetrazolium (MTT) assays [5 mL−1 cells concentration using the RPMI 1640 Complete Medium supplemented and incubated with 100 μL of control (RPMI), solutions, extract, and fractions at concentration levels of 1500, 750, 375, 187.5, and 93.75 μg.mL−1, negative control used untreated cells and positive control used 1% Triton-X-100 (v/v). The plate was incubated for 24 h at 37 °C with 5% CO2, and then the medium was withdrawn to add 100 μL of the MTT solution (0.5 mg.mL−1) in all microtitration wells. Thereafter, the MTT solution was withdrawn and 100 μL of HCl (6 N) with isopropanol was added to all wells, and the plate reading was read and performed at an absorbance of 550 nm. For the cytotoxic concentration (CC50) determination, a linear regression curve was used, and the value was expressed based on absorbance in the Graphpad Prism 8 software .To evaluate the cytotoxicity of the ethanolic extract and its fractions from ) assays . Hence, Staphylococcus aureus (ATCC 6538). It was obtained from INCQS/FIOCRUZ . Strains were previously seeded in Petri dishes containing Mueller–Hinton agar  and incubated at 37 °C for 24 h. For the preparation of the bacterial inoculum, the strains growing were performed in Mueller–Hinton (MHB) broth  at 37 °C for 24 h and adjusted by dilution of fresh cultures to a turbidity equivalent to 0.5 scales of McFarland (around 2 × 108 CFU.mL−1). and then diluted to 1 × 103 CFU.mL−1 as described in the literature [Antibacterial properties were evaluated against Gram-positive strains of terature  and accoterature . Minimumterature . The MICterature .v/v) and distilled water at the highest concentration of 1500 µg.mL−1 to the lowest of 46.8 µg.mL−1 by serial dilution. A total of 100 μL of the extract and fractions were placed in each microtitration well together with 100 μL of the bacterial inoculum (1 × 103 CFU.mL−1). After incubation, the development of a pinkish-purple color was considered indicative of bacterial growth. Therefore, the MIC reading was set up to the lowest concentration of the extract where the pink–purple color was not observed. To determine the CBM, 10 µL of broth was taken from each well and incubated in Mueller–Hinton agar at 37 °C for 24 h for each bacterium. MBC was defined as the lowest concentration of extract that resulted in a colony count of fewer than three colonies per mL (90.0% kill) or no bacterial growth [−1) was used as a positive control for Gram-positive bacteria.To determine the MIC, extracts were dissolved in DMSO 10% (l growth . The ass50), a literature-adapted method [D. nitidula at concentration levels of 1500, 750, 375, 187.5, 93.75, and 46.8 μg.mL−1 were transferred to separate microplate wells (100 μL/well) with 100 µL aliquot of S. aureus inoculum (1 × 103 CFU.mL−1). As a negative control, Mueller–Hinton broth and 50 µg.mL−1 of chloramphenicol were used against S. aureus and incubated for 24 h at 37 °C. After 24 h of incubation, the absorbance was measured in an ELISA reader (BioTek Synergy HTX Multimode Reader) at 600 nm with gentle agitation for 10 s. The IC50 was calculated using the linear relation between the MIC and concentration logarithm. The selectivity index (SI) was calculated with the ratio of IC50 (against S. aureus) and CC50 (PBMC).To evaluate 50% inhibition concentration (ICd method  was appl−1) were performed for SREE and its fractions . Thus, PBMC were used, and the CC50 was defined as how efficiently the extract, or its fractions could reduce 50% of the cell viability. The SREE shows a high cytotoxicity level (1500 μg.mL−1: 48.66 ± 11.57%) with a CC50 of 1751.12 μg.mL−1, unlike other concentrations that show cell viability. SREFr1 shows a cytotoxic profile  with a CC50 of 571.6 μg.mL−1 (−1 = 93.59 ± 11.09%). Similar results are observed for SREFr3, i.e., the lowest concentration (93.75 μg.mL−1) shows lower cytotoxicity, and excitedly high cell viability (83.84 ± 4.12%)  see D with CC. SREFr2 C reducesD. nitidula roots (SREE) and hexane fraction (SREFr1), dichloromethane fraction (SREFr2), ethyl acetate fraction (SREFr3), and methanol fraction (SREFr4) were evaluated at different concentrations against S. aureus to determine the MIC, MBC, and the MBC/MIC ratio in order to determine whether the antimicrobial activity have an bactericidal and bacteriostatic effect.The ethanolic extract of 50 of the extract and its fractions against S. aureus were evaluated. SREFr3 shows the lowest IC50 (11 μg.mL−1), which is able to inhibit 50% of bacterial growth, while SREFr2 shows the highest IC50 (128 μg.mL−1), as shown in 50 and IC50 was calculated  [2O (30 Da) from the pyrano. Excitedly, suitable similarities using modified cosine >0.50 were observed for those metabolites.Overall, the main metabolites detected in the ethanolic extract (SREE), fractions SREFr2, were putatively annotated as flavonoid derivatives, and they are illustrated in lavanone B3 showedon (HRF)  in the CD. nitidula roots, we carried out a PCA. Thus, processed LC–MS data containing features with the mass-to-charge ratio (m/z) and retention time were used to obtain relative quantification information of the extract and its fractions. From the results of this PCA, the extract (SREE) tends to have a significant separation from its fractions, as shown in To obtain a general overview of the molecular profile from m/z 281 and m/z 111  was performed to understand the absolute value of change in the bioactive profile between the extract and SREFr3 fraction c. Hence, m/z 111 .−1) and the antimicrobial activity against S. aureus, a Gram-positive bacteria. According to a previous report [−1; otherwise, if the values are between ≥100 μg.mL−1 and ≤2048 μg.mL−1, the extract can have moderate activity, and lastly, MIC > 2048 μg.mL−1 weak activities, while MIC > 10,000 μg.mL−1 is attributed as no antimicrobial action. The same cutoff point was determined. The SI allows measuring observed previously by [S. aureus , and the SREFr1 and SREFr4 fractions show moderate antibacterial activity .Herein, an in vitro screening of the extract and its fractions was performed to assess the cytotoxicity. For this, PBMC assays were performed using different concentrations  [The antimicrobial activity of SREE can be justified by the presence of antimicrobial components such as the flavonoid derivatives described in .aureus) . That coS. aureus can develop resistance to antibiotics through the expression of efflux pumps to pump out antibiotics from the bacteria, for example [This mechanism is important because Gram-positive bacteria such as  example . In this example ,44.−1) when compared to SREFr3 (11 μg.mL−1). With that, this fraction shows a low selectivity index (SI = 6.8), which characterizes SREFr2 as not safe for therapeutic use. On the other hand, the SREFr3 fraction stood out in all assays, as it proved to be safe against human cells and with high antimicrobial activity, with CC50 values of 902.76 and MIC of 46.8 μg.mL−1, thus, obtaining a high value of SI against S. aureus (SI = 82.06), showing a promising fraction for future studies.Although SREFr2 shows high antibacterial activity similar to SREFr3, the IC50 of SREFr2 is significantly higher , which shows interesting antibacterial properties. Excitedly, ethyl acetate fraction (SREFr3) shows potent antibacterial properties, despite it not being possible to define putative precursor metabolites from this fraction. This activity could be explained by the in silico indicative presence of amino acid derivatives from asparagine. In addition, this chemical class, which is reported for the first time in this species, is well-known for its antibacterial properties. On the other hand, it was not possible to calculate the SI of SREFr1, SREFr2 showed significant toxicity, and SREFr4 had no activity against S. aureus. Hence, we show that the roots are a renewable antibacterial source, and SREE and SREFr3 can serve as a pillar for future antimicrobial studies.This study shows the ethanolic extract and ethyl acetate fraction from


In this study, the dissipation behavior and dietary exposure risk of eight pesticides in Brussels sprout were evaluated under greenhouse conditions. Brussels sprout samples were collected 0, 7, 14, and 21 days after the last pesticide treatment. Ultra-high performance liquid chromatography with tandem mass spectrometry was used for sample analysis. Recovery rates at different concentrations of pesticides (0.01 and 0.1 mg/kg) were in the range of 70.2–104.5%, and the relative standard deviations were ≤ 10.6%. The pesticide residues in Brussels sprouts were determined for each treatment. For acephate, etofenprox, imidacloprid, indoxacarb, alpha-cypermethrin, zeta-cypermethrin, fludioxonil, and oxytetracycline, the half-lives were, respectively, 11.3, 9.8, 11.3, 15.8, 10.6, 13, 9.1, and 8.2 d and the dietary intake rates were, respectively, 2.90%, 0.81%, 0.7%, 1.19%, 0.06%, 0.24%, 0.05%, and 0.36% of the acceptable daily intake. The findings of this study provide important insights into the establishment of maximum residue limits in the Republic of Korea and pesticide control measures for Brussels sprout. Pesticide residues may remain on crops; however, pesticide retention poses potential risks to human health and the environment4. A positive list system has been in place in the Republic of Korea (ROK) since 20195, which regulates the limits for pesticides (0.01 mg/kg) applied to agricultural products for which there is no maximum residue limit (MRL). Although agricultural products must be safer for consumers6, maintaining these strict standards is challenging for producers. Pesticide manufacturers have focused on registering their pesticide products for major crops over minor crops7 because of the economic importance of the former. Hence, minor crops with cultivation areas of < 1000 ha8 have garnered less attention.Pesticides are widely used to protect crops against pests and diseases and, thus, increase agricultural productivity9. Brussels sprout contains several compounds that are beneficial to human health, such as vitamins, minerals, enzymes, and amino acids13. Brussels sprout exports totaled USD 218.1 million in 2020, representing a 6% increase from the value noted in 201914.Brussels sprout is a minor crop that belongs to the cruciferous group similar to broccoli, cauliflower, and cabbage. The consumption of these vegetables has been associated with a lower occurrence of cancer in humans15. However, in the ROK, the MRLs established for cabbage are used for Brussels sprout as well; these MRLs were established without considering the latter species16. The MRLs for Brussels sprout (EU) and cabbage (ROK) are 0.01 and 5 mg/kg (acephate), 0.01 and 0.2 mg/kg (etofenprox), 0.5 and 0.5 mg/kg (imidacloprid), 0.06and 0.2 mg/kg (indoxacarb), 1 and 1 mg/kg , 1 and 1 mg/kg (zeta-cypermethrin), and 0.01 and 2 mg/kg (fludioxonil), respectively. An MRL for oxytetracycline has not been established for Brussels sprout in either location.In the European Union (EU), MRLs have been established for several pesticides used for Brussels sprout17, green chili fruit18, and mango19. In the case of etofenprox, there are studies on pesticides’ dissipation patterns in spring onion20, tomato21, and squash22. The dissipation behavior of imidacloprid has been investigated in mango23 and grape24. Some previous studies have assessed the dissipation behavior of indoxacarb in cabbage25, tomato26, and green chili fruit27. Bae et al.28 and Hwang et al.29 conducted studies on the half-life of fludioxonil in mandarin and Chinese cabbage, respectively. However, to the best of our knowledge, no studies have assessed the dissipation behavior of the aforementioned eight pesticide residues in Brussels sprout yet. To fill this knowledge gap, the study evaluated the residue patterns, half-lives, MRLs, and dietary risks of pesticide residues in Brussels sprout in greenhouse condition. The findings are expected to contribute toward the establishment of a set of MRLs for pesticide use in Brussels sprout in the ROK.The dissipation patterns of pesticides have been studied in many crops. Previous studies have examined the dissipation of acephate residues in brinjalAcephate, methamidophos, etofenprox, fludioxonil, indoxacarb, and imidacloprid were purchased from Kemidas Co. . Alpha-cypermethrin and zeta-cypermethrin > 99.91% purity) were obtained from HPC Standards GmbH . Oxytetracycline was provided by ChemScene LLC . The physicochemical properties of the pesticides are presented in Supplementary Table % purity 30.The test field was located in Eumseong-gun . The total field size for each pesticide treatment was 30 m (length) × 5.5 m (width). To eliminate the variation in plant growth and climate change, each field was divided into four plots, and samples on each field were harvest on the same day Table . All fieFor use in liquid chromatography with tandem mass spectrometry (LC–MS/MS), a standard stock solution of oxytetracycline was prepared at a concentration of 1000 mg/L with methanol. The working solutions were prepared via serial dilution of the stock solution with methanol. Standard stock solutions of the other pesticides were prepared at a concentration of 1,000 mg/L with acetonitrile. The working solutions were prepared via serial dilution of the stock solution with acetonitrile. Each of the standard solutions was mixed with an extract of Brussels sprout .All plant samples were rapidly transferred to the laboratory after harvest. Brussels sprout samples were homogenized with dry ice and then stored at − 20 °C in polyethylene bags.Homogenized Brussels sprout samples (10 g) were weighed in 50-ml falcon tubes. Of the mixture containing 50:50:0.9 volume methanol:water: formic acid, 10 mL was added to the falcon tube. The tube was shaken (1200 rpm) for 1 min. After centrifugation , 1 ml of the supernatant was filtered using a 0.45-μm polytetrafluoroethylene syringe filter.31. Homogenized Brussels sprout (10 g) was weighed in a 50-mL falcon tube. A total of 10 mL of acetonitrile was added to the tube. The QuEChERS kit components  were added to the falcon tube. The tube was shaken (1200 rpm) for 1 min. After centrifugation , 1 ml of the supernatant was filtered using a 0.2-μm polytetrafluoroethylene syringe filter.The EN-QuEChERS kit was used to extract the pesticidesAll samples were analyzed using Shimadzu LC–MS-8045 with UHPLC Nexera X2 . Chromatographic separation was performed using a Kinetex C18 column  maintained at 40 °C. Information regarding the mobile phase, gradient program, and injection volume for all analytes are presented in Supplementary Tables The method was validated in terms of linearity, accuracy, precision, and limit of quantitation (LOQ). Linearity was assessed using a pure standard solution and homogenized pesticide-free Brussels sprout based on the data obtained using five concentrations: 5, 10, 20, 50, and 100 ng/mL. The lowest concentration among the chromatograms that produced a signal-to-noise ratio of > 10 was selected as the LOQ. To determine the recovery rate, the pesticide standards were added to the homogenized pesticide-free samples at two different concentrations: 0.01 and 0.1 mg/kg. Each test was performed in triplicate.16. The parent substance only was evaluated except for acephate residues. Acephate residue in plants is the sum of acephate and methamidophos, which is a metabolite of acephate. The molecular weights of acephate and methamidophos are 183.2 and 141.1, respectively. To evaluate the total acephate in Brussels sprout, the sum of acephate residue concentration was calculated as follows:The individual compounds were analyzed for eight pesticides. The information regarding the residue definitions was provided by ministry of drug and food safetyThe dissipation rate was calculated based on the initial residue concentration  and that measured 21 day after pesticide treatment. The dissipation rate was calculated as follows:32:C0is the initial residue concentration (mg/kg) in plot 4, Ct is the residue concentration (mg/kg) in plots 1, 2, and 3, t is the days after pesticide treatment, and k is the rate constant of dissipation.The dissipation patterns of the eight pesticide residues in Brussels sprout over time were expressed using the following first-order kinetics equation Eq. , and theThe initial residues were calculated using the MRLs for Brussels sprout in the EU and those for cabbage in the ROK, which is applied to Brussels sprout.To estimate the human health risk from the pesticide residues present in Brussels sprout, hazard quotient (HQ)was calculated based on the estimated daily intake and ADI using the following Eqs. –7:5\docu33.In case of the acephate and its metabolite (methamidophos), because their ADIs were different, HQs were calculated separately. The daily dietary intake for Brussels sprout was 7.17 g, which is the daily food intake for vegetables, and the average body weight in the ROK is 59 kgStandard curves of all pesticides showed good linearity in samples of Brussels sprout 18, and mango (5 days)19. The half-life of etofenprox was 9.8 days in Brussels sprout, which is similar to that noted in spring onion (9.5 days)20 and higher than that noted in tomato (2.15 days)21 and squash (3.5 days)22; the differences are attributable to the variations in crop properties and enzymes. Imidacloprid’s half-life (11.3 days) was longer in Brussels sprout than in mango (3.06 days)23 and shorter in the former than in grape (16.6 days)24. Indoxacarb’s half-life was 15.8 days and decreased slowly compared with indoxacarb’s half-life in cabbage (2.88 days)25, tomato (3.21 days)26, and green chili fruit (3.85 days)27. Hypertrophy—fast crop growth in a short period—is reportedly a major factor in the half-life of pesticides38. Although cabbage and Brussels sprout have a similar wrapped-over form, the half-lives are different because of the hypertrophy of cabbage. The half-life of fludioxonil in Brussels sprout (9.1 days) was similar to that noted in mandarin (8.7 days)29 and longer than that noted in Chinese cabbage (4.0 days)28. Mandarins have a bumpy surface, and pesticides are, therefore, likely to adsorb to the surface. The half-lives of alpha-cypermethrin, zeta-cypermethrin, and oxytetracycline were 10.6, 13, and 8.2 days, respectively. In the aspect of chemical class of pesticides, dissipation half-lives range from 0.9 to 22.8 days for organophophates, 0.8–10.6 days for carbamates, 3.1–9.3 days for neonicotinoids, and 1.1–5 days for pyrethroids. Except acephate, most of dissipation half-lives were higher than previous reports36. In the aspect of plant species, dissipation half-lives of pesticides were in the range of 0.9–13 days which is similar with this study. In this study, there was less correlation between dissipation half-lives and volatilization (vapor pressure).The half-lives of the eight pesticides studied vary across crops and are influenced by many factors such as class of target and chemical, plant species, field conditions, chemical and microbial decomposition, dilution by plant growth, volatilization, acidity, photodecomposition, temperature, surface washoff, and spatial variabilityThe initial residues (mg/kg) of the eight pesticides were compared with the domestic MRLs for cabbage and that in the EU for Brussels sprout. The initial acephate residue was 143% of the domestic MRL (5 mg/kg) and 71,600% of the EU MRL (0.01 mg/kg). In the case of etofenprox, the initial residue was 1.99 mg/kg, which was 995% of the domestic MRL (0.2 mg/kg) and 19,900% of the EU MRL (0.01 mg/kg). The initial imidacloprid residue (0.34 mg/kg) was 68% of the EU and domestic MRLs (0.5 mg/kg for both). The initial indoxacarb residue (0.98 mg/kg) was 490% of the domestic MRL (0.2 mg/kg) and 1633% of the EU MRL (0.06 mg/kg). The initial residues of alpha-cypermethrin and zeta-cypermethrin were 0.23 and 0.40 mg/kg, respectively; these were 23% and 40% of both MRLs (1 mg/kg). The initial fludioxonil residue (1.74 mg/kg) was 87% of the domestic MRL (2 mg/kg) and 17,400% of the EU MRL (0.01 mg/kg). For oxytetracycline, the initial residue was 0.88 mg/kg but no MRLs have been established in the EU or ROK. The residual amount of oxytetracycline may be used as basic data for the establishment of an MRL. There are no differences between domestic and EU MRLs of imidacloprid, alpha-cypermethrin, and zeta-cypermethrin; however, considerable differences were noted between the EU and ROK MRLs for acephate, etofenprox, indoxacarb, and fludioxonil. The differences highlight the variability in residue patterns with growing conditions, climate, and crop species.In Brussels sprout, residues for all eight pesticides of the %ADI values for ROK average intake were < 3% , 9.8 (etofenprox), 11.3 (imidacloprid), 15.8 (indoxacarb), 10.6 , 13 (zeta-cypermethrin), 9.1 (fludioxonil), and 8.2 (oxytetracycline) days. Pesticide residue is affected by various factors such as vapor pressure, pesticides’ physicochemical properties, minor substances, formulation types, environment conditions, crop species, and growth dilution factors. Based on the %ADI values, it can be concluded that the intake of pesticide residues from Brussels sprout does not pose a significant health risk. These findings provide useful information for the establishment of MRLs in the ROK and pesticide control measures for Brussels sprout.Supplementary Information.


High selenium (Se) and low cadmium (Cd) and arsenic (As) contents in rice grains were good for human health. The genetic basis and relationship of Se, Cd and As concentrations in rice grains are still largely unknown. In the present study, large variations were observed in Se, Cd and As concentrations in brown and milled rice in normal and Se treatment conditions in 307 rice accessions from 3K Rice Genomes Project. Se fertilizer treatment greatly increased Se concentrations but had no obvious changes in concentrations of Cd and As both in brown and milled rice. Total of 237 QTL were identified for Se, Cd and As concentrations in brown and milled rice in normal and Se treatment conditions as well as ratio of concentrations under Se treatment to normal conditions. Only 19 QTL (13.4%) were mapped for concentrations of Se and Cd, Se and As, and Se, Cd and As in the same or adjacent regions, indicating that most Se concentration QTL are independent of Cd and As concentration QTL. Forty-three favorable alleles were identified for 40 candidate genes by gene-based association study and haplotype analysis in 14 important QTL regions. Se-enriched rice variety will be developed by pyramiding favorable alleles at different Se QTL and excluding undesirable alleles at Cd and As QTL, or combining favorable alleles at Se QTL with the alleles at Se-sensitive QTL by marker-assisted selection. Se defi−1 . In some−1 , and the−1 . On the −1 , two hea−1 . They ar−1 . Because−1 .Rice is one of the most important staple crops, and provides food for two-thirds of world’s population, especially for Chinese people , thus it−1, 0.002–0.475 mg kg−1, and 0.010–0.750 mg kg−1 in rice germplasms, respectively  was used for identifying QTL for rice complex traits. It is based on rice germplasm and linkage disequilibrium (LD). Using this strategy, some QTL for Se, Cd and As have been detected, and some candidate genes were identified. In the present study, Se, Cd and As concentrations in brown and milled rice were measured under normal and Se fertilizer treatment conditions, in a diverse panel of 307 accessions from 3K RGP . Based oIn our previous study, a total of 1,016 rice accessions from 3K RAP  were ide−1 in the 2 years. Field experiment was carried out using a randomized complete design. Each accession was planted in three rows and ten plants per row with the density of 17 cm between plants and 20 cm between rows for two replications in two conditions, i.e., normal condition where field management was followed as local farmers’ practices, and Se treatment condition where Se fertilizer in diluent with 3.30 g Na2SeO3 dissolved in 40 kg water per 666.7 m2 was sprayed to rice plants at booting stage and other field management was completely same as the normal condition. At mature stage, seeds were bulk-harvested from each plot, and air-dried and stored for more than 3 months at room temperature before assessing of concentrations of selenium (Se), cadmium (Cd) and arsenic (As) in brown and milled rice.All accessions were grown in the summer seasons of 2019 and 2020 at the Experimental Farm of College of Agriculture, Yangtze University, Jingzhou City , Hubei province, China. The Se, Cd and As concentrations in the soil were averagely 0.33, 0.27 and 7.15 mg kgSeeds of each plot were de-hulled into brown rice, and then polished into milled rice according to the method described previously . Brown ahttp://snp-seek.irri.org) (The 4.8 M SNP dataset of 3K RGP was download from the Rice SNP-Seek Database (rri.org) . For strrri.org) . The parrri.org) . For GWAp < 1.0E-05. Since the LD decay of 3K RGP was about 200 kb . Secondly, all SNPs located on these genes were retrieved from 32 M SNPs data generated from 3K RGP in the RFGB v2.0 database (10(P) > 5.0 (p < 1.0E-05). Thirdly, for each QTL region, all significant SNPs located in promoter region, 5′ UTR region, non-synonymous SNPs in the exon regions, splice region in intron and 3’ UTR region of each candidate gene were used for identifying haplotypes. Candidate genes were assigned by testing significant difference for relevant traits among major haplotypes (samples more than 8) for each important QTL by ANOVA with p < 0.01. When QTL containing cloned genes for element-related traits, the cloned genes were considered as only candidate gene (s) of the target QTL.Gene-based association mapping and haplotype analysis were conducted to detect candidate genes and favorable alleles in the important QTL regions. QTL regions identified for the same trait in both years and/or for more than two different traits were considered as important QTL. QTL with peak SNP having lowest database , and theA wide range of concentrations of Se, Cd and As in both brown and milled rice under normal, treatment and ratio of treatment to normal conditions reflected the substantial genotypic variations associated with concentrations of three elements in the panel of accessions . Phenoty−1 SMN, 0.005 mg kg−1 CMN and 0.161 mg kg−1 AMN; IRIS_313–8856 averagely had 0.108 mg kg−1 SMN, 0.004 mg kg−1 CMN and 0.189 mg kg−1 AMN; and IRIS_313–11968 averagely had 0.044 mg kg−1 SMN, 0.004 mg kg−1 CMN and 0.130 mg kg−1 AMN. IRIS_313–10083 had high Se concentration, and low Cd and As concentrations in milled rice in Se treatment condition in 2 years, with average 0.333 mg kg−1 SMT, 0.003 mg kg−1 CMT and 0.139 mg kg−1 AMT. Another four accessions  were consistently identified as high ratio of Se concentration and low ratios of Cd and As concentrations in milled rice  were consistently identified to have high Se concentration, and low Cd and As concentrations in milled rice in normal condition in 2 years . Specifiled rice .We analyzed correlations of three element concentrations in different parts under different treatments . For thexian , geng , aus (32). In addition, five accessions belong to admix. Using 405,150 high quality of 307 accessions, a total of 237 QTL were identified for 16 traits, ranging from four for AMT to 23 for RCB and RAB. Among them, three QTL were identified in both years  were stably detected in both years. For As concentration, nine QTL for each of ABN and AMN were identified on 12 chromosomes except chromosome 10 in 2019, accounting for phenotypic variation ranging from 1.01% to 23.84%.In normal condition, 87 QTL were detected for six traits . For Se A total of 55 QTL were identified for concentrations of three elements in treatment condition . Ten SBTFor ratio of concentrations of three elements under Se treatment to normal conditions, a total of 95 QTL were identified for RSB, RSM, RCB, RCM, RAB and RAM . NineteeqSBN2.2 and qSMN2.1, qSBN2.3 and qSMN2.2, qSBN3.1 and qSMN3.1, qSBN4.2 and qSMN4.2, qSBN5.2 and qSMN5.2, qSBN6 and qSMN6, qSBN7.2 and qSMN7, qSBN8.2 and qSMN8, qSBN10 and qSMN10, and qSBN11.2 and qSMN11.2 for Se concentration in brown and milled rice under normal conditions, and one harboring qAMN2 and qABN2 for As concentration in brown and milled rice under normal condition. It was indicated that pleiotropy is most probably responsible for association of Se and As concentrations between brown and milled rice. Seventeen regions were identified for concentrations of two or three elements, including three harboring qSMN1.2 and qCBN1.2, qSBN8.2, qSMN8 and qCBN8.2, and qSBT12 and qCBT12 affecting Se and Cd concentrations in normal or treatment conditions, 10 harboring qCBN2, qABN2 and qAMN2, qCBT2.1 and qABT2.1, qCBN3, qCBT3.1 and qABT3.2, qCBT3.2 and qABT3.3, qCBT4.1, qABT4.1 and qAMT4, qCBT7 and qABT7, qCBT8.2 and qABT8.3, qCBT9.1 and qABT9.1, qCBT10 and qABT10.1, and qCBN11 and qAMN11 for Cd and As concentrations under normal or treatment condition, and four harboring qSBT1.2, qCBT1.2 and qABT1.2, qSBT5, qCBT5 and qABT5, qSBT8, qCMN8 and qABT8.1, and qSBT9, qCBT9.2 and qABT9.2 affecting Se, Cd and As concentrations in brown rice under treatment or normal condition. Twenty regions were identified for QTL overlap between concentrations in normal or treatment conditions and concentration ratio of the three elements. It was indicated that partial genetic overlaps exist due to pleiotropy or tightly linkage for the three elements, especially Cd and As under treatment condition.Comparison of QTL located in the same or adjacent regions can reveal genetic relationships of the concentration between brown and milled rice and among three elements. Total of 46 regions harboring QTL for different traits were detected . Among tAmong 237 QTL for concentrations of three elements, 14 important QTL were identified in both years and/or had effect on more than two different traits. A total of 40 candidate genes and 43 favorable alleles were identified by gene-based association analysis and haplotype analysis .qRSB1.1 in 1.95–2.15 Mb on chromosome 1, 1,598 SNPs were found in 34 genes. Among them, 14 genes were identified. Significant differences were identified in RSB and RCM in 2019 between different haplotypes for two candidate genes (LOC_Os01g04580 and LOC_Os01g04590). Also, there was significant difference in SBT in 2019 between two haplotypes in LOC_Os01g04590. No haplotype was found for the rest 12 genes.For qABT1.2 in the region of 24.33–24.53 Mb on chromosome 1, 449 SNPs were found in 25 genes, and five candidate genes were identified. Significant differences were detected in ABT and CBT in 2019 between different haplotypes in candidate genes LOC_Os01g42870, LOC_Os01g42909 and LOC_Os01g42950. Besides, there was significant difference in SBT in 2019 between two haplotypes in LOC_Os01g42870. No haplotype was found for the other two genes (LOC_Os01g42920 and LOC_Os01g42934).For qRSB1.4 in 42.31–42.51 Mb on chromosome 1, 1,137 SNPs were found in 34 genes, and 13 candidate genes were identified. Significant differences in RSB, SBT and RCM in 2019 were detected between two haplotypes in LOC_Os01g73040, LOC_Os01g73080, LOC_Os01g73110, LOC_Os01g73120, LOC_Os01g73130, LOC_Os01g73140, LOC_Os01g73150 and LOC_Os01g73170. Besides, there was significant difference in RCM in 2019 between two haplotypes in LOC_Os01g73250. No haplotype was found for LOC_Os01g72980, LOC_Os01g73100, LOC_Os01g73160 and LOC_Os01g73310.For qRAB3.1 in the region of 13.20–13.40 Mb on chromosome 3, 305 SNPs were found in 26 genes, and 14 candidate genes were identified. Significant differences were detected in RAB, ABT and RCB in 2019 between different haplotypes in LOC_Os03g22840. There was no significant difference in any trait between different haplotypes or no haplotype available in the rest 13 genes.For qRSB4.1 in the region of 5.22–5.42 Mb on chromosome 4, 818 SNPs were found in 15 genes, and five candidate genes were identified. Significant differences were detected in RSB and RCM in 2019 between two haplotypes in LOC_Os04g09880. There was no significant difference in any trait between different haplotypes or no haplotype identified in the rest four genes.For qABT5 in the region of 1.69–1.89 Mb on chromosome 5, 1,086 SNPs were found in 29 genes, and 11 candidate genes were identified. Significant differences were detected in ABT and CBT in 2019 between different haplotypes in LOC_Os05g03934, LOC_Os05g03972, LOC_Os05g04000 and LOC_Os05g04020. Besides, significant differences in RCB and RAB in 2019 were found between two haplotypes of each of LOC_Os05g03972 and LOC_Os05g04000, and there was also significant difference in RAB in 2019 between two haplotypes in LOC_Os05g04020. There was no significant difference in any trait between different haplotypes or no haplotype available in the rest seven genes.For qRAB5.2 in the region of 4.11–4.31 Mb on chromosome 5, 1,508 SNPs were found in 26 genes, and nine candidate genes were identified. Significant difference was detected in RAB in 2019 between two haplotypes in LOC_Os05g07810. No haplotype was found in the rest eight genes.For qCBN5 in the region of 8.95–9.15 Mb on chromosome 5, 596 SNPs were found in 28 genes, and only one candidate gene (LOC_Os05g15960) was identified. Significant difference was detected in CBN in 2019 between the two haplotypes.For qCBT7 in the region of 21.02–21.22 Mb on chromosome 7, 1,245 SNPs were found in 26 genes, and two candidate genes (LOC_Os07g35290 and LOC_Os07g35310) were identified. Significant differences were detected in CBT, ABT and RAB in 2019 between two haplotypes in both the two genes. Besides, there was significant difference in RCB in 2019 between two haplotypes in LOC_Os07g35310.For qSMN8 in the region of 24.64–24.84 Mb on chromosome 8, 629 SNPs were found in 25 genes, and seven candidate genes were identified. Significant differences were detected in SMN and SBN in 2020 between two haplotypes in LOC_Os08g39120, LOC_Os08g39174 and LOC_Os08g39180. No haplotype was found in LOC_Os08g39150, LOC_Os08g39160, LOC_Os08g39170 and LOC_Os08g39280.For qABT8.3 in the region of 25.50–25.70 Mb on chromosome 8, 620 SNPs were found in 22 genes, and three candidate genes  were identified. Significant differences were detected in ABT and CBT in 2019 between two haplotypes in LOC_Os08g40510 and LOC_Os08g40570. Besides, significant difference was also found in RCB in 2019 between two haplotypes in LOC_Os08g40570. No haplotype was found in LOC_Os08g40470.For qRAB9.2 in the region of 20.08–20.28 Mb on chromosome 9, 1,162 SNPs were found in 32 genes, and 15 candidate genes were identified. Significant differences were detected in RAB, ABT, CBT and RCB in 2019 between different haplotypes in LOC_Os09g34200, LOC_Os09g34214, LOC_Os09g34230, LOC_Os09g34250 and LOC_Os09g34270. There was no significant difference in any trait between different haplotypes or no haplotype available in the rest 10 genes.For qSMN10 in the region of 15.95–16.15 Mb on chromosome 10, 807 SNPs were found in 21 genes, and two candidate genes (LOC_Os10g30790 and LOC_Os10g30800) were identified. Significant differences were detected in SBN and SMN in 2020, and RAB in 2019 between two haplotypes in both the two genes.For qSMN11.2 in the region of 25.94–26.14 Mb on chromosome 11, 376 SNPs were found in 16 genes, and four candidate genes  were identified. Significant differences were detected in SMN and SBN in 2020 between two haplotypes in all four candidate genes.For Most Chinese people are likely to consume white rice or polished rice which is produced from brown rice. Although rice bran only accounts for 6–10% of the total weight of rice , it cont−1 in different regions of China, which results in the Se deficiency of inhabitants in China because the rice Se is the main source of Se in the Chinese diet  with CBS domain involved in heavy metal tolerance (LOC_Os01g73140 encodes precursor of UFM1 (ubiquitin-fold modifier 1) and is highly expressed in the milk stage of grain and filling stage of endosperm .In China, the general population in 72% of the total land area is facing a Se deficiency problem . Therefoectively . MeanwhiLOC_Os01g04580 at qRSB1.1/qRCM1.1 and LOC_Os04g09880 at qRSB4.1/qRCM4.1, and Hap2 of LOC_Os01g04590 at qRSB1.1/qRCM1.1, LOC_Os01g73040, LOC_Os01g73130 and LOC_Os01g73140 at qRSB1.4/qRCM1.2 could enhance about 5.1–31.6 times of the Se concentration in brown rice and 5.4–10.5 times of Cd concentrations in milled rice after Se treatment. So, introgressing and pyramiding favorable genes for above three QTL could much improve Se concentration in grains after Se fertilizer treatment although Cd concentration could be increased to some extent due to synergistic effect of Se at these RSB QTL. Four accessions  with high RSM and low RCM and RAM carried all favorable alleles at above QTL (qRSB1.1/qRCM1.1, qRSB1.4/qRCM1.2 and qRSB4.1/qRCM4.1 by MAS. It is worth noting that qRSB3.2/qRSM3.1 for both RSB and RSM is only one locus which is independent of QTL for ratio of Cd and As concentrations. So, after mining favorable alleles at this locus using more diverse germplasms, the favorable alleles can be used for further enhancing Se concentrations in both brown and milled rice after Se treatment without interference of Cd and As in rice breeding for Se biofortification.In most cases, Se deficiency can be corrected by the application of Se fertilizer into the soils or on rice leaf, which is termed as agronomic biofortification . As indibove QTL . TherefoFinally, another efficient strategy to enhance Se concentration in rice grains is probably to pyramid favorable alleles of different kinds of QTL, i.e., QTL for Se enrichment identified in normal condition and QTL for Se sensitivity identified under Se fertilizer treatment by MAS. Thus, new variety pyramiding above two kinds of QTL will show high Se concentration in normal condition and more higher Se concentration after Se fertilizer application in some Se-deficient areas.Large variations in concentrations of Se, Cd and As in grains existed in the panel of 307 rice accessions in normal and Se treatment conditions. Se fertilizer treatment greatly improved Se concentrations but had no obvious changes in concentrations of Cd and As both in brown and milled rice. A total of 237 QTL were identified for Se, Cd and As concentrations in normal, Se treatment and ratio of treatment to normal conditions by GWAS. Most QTL for Se concentration is independent of those for Cd and As concentrations in view of only 13.4% QTL commonly detected in the same or adjacent regions. Forty-three favorable alleles were identified for 40 candidate genes in 14 important QTL regions. Pyramiding of favorable alleles at Se QTL and excluding undesirable alleles at Cd and As QTL, or combining favorable alleles at Se QTL detected in normal condition with the alleles at Se-sensitive QTL detected under Se treatment by MAS will facilitate development of rice variety with Se enrichment and minimum concentrations of Cd and As.


Limosilactobacillus reuteri plays an important role in regulating intestinal functions and maintaining barrier integrity in animals. In this study, Limosilactobacillus reuteri strain SLZX19-12 was isolated from the fecal microbiota of Tibetan pigs, and it was found that this strain is sensitive to common antibiotics and has strong resistance to stress. Upon being administered by gavage at different doses, including low, medium, and high doses, for 14 days, Limosilactobacillus reuteri SLZX19-12 may enhance the intestinal barrier. After administration of a high dose of SLZX19-12, mice were challenged with Salmonella enterica serovar Typhimurium SL1344. Infection with Salmonella Typhimurium SL1344 led to disordered colonic microbiotas, colonic inflammation through the S100A8/S100A9–NF-κB pathway and potential apoptosis, and translocation of pathogens to parenteral visceral organs in mice. However, the mice pretreated with Limosilactobacillus reuteri SLZX19-12 showed lower loads of Salmonella in visceral organs, less colonic inflammation, and higher barrier integrity. More importantly, the administration of strain SLZX19-12 resulted in a more stable microbiota structure of the colon, in which the abundance of Alloprevotella was greatly enhanced. Therefore, this study suggests that Limosilactobacillus reuteri SLZX19-12 can protect the colon from infection by enhancing the stability of gut microbiota and barrier integrity and reducing inflammation.IMPORTANCE The use of antibiotics to treat bacterial infections leads to a series of side effects. As an alternative method, the biocontrol strategy, which uses probiotics to suppress pathogens, is considered a potential way to deal with bacterial infections in gut. However, there are few probiotics that are currently safe and can protect against infection. In this study, Limosilactobacillus reuteri strain SLZX19-12 was obtained from Tibetan pigs, which have higher resistance to infection. This strain is sensitive to conventional antibiotics, secretes a wide spectrum of enzymes, and also promotes the intestinal barrier function in mice. In addition, Limosilactobacillus reuteri SLZX19-12 can promote the stability of the gut microbiota to avoid or alleviate the occurrence or development of foodborne infections. Antibiotics have played an important role in preventing and treating bacterial infections in humans and farm animals. Long-term use of antibiotics has caused gastrointestinal microorganism disorders, emergence of drug resistance in bacteria, and environmental pollution. These outcomes have increased the occurrence of infections with antibiotic-resistant bacteria, diarrhea, and enteritis –3. These–The gut is the first protective barrier for animals. Altered permeability of the intestinal barrier can lead to introduction of multiple antigens into the systemic circulation, thereby inducing systemic inflammation and damage to parenteral organs . Damage Salmonella enterica serovar Typhimurium, can cause acute infection and sepsis, resulting in animal death. In humans, pigs, mice, and other mammals, Lactobacillus can secrete antimicrobial substances and regulate immune defense functions and the gut microbiota to prevent infectious diseases , we discovered a high abundance of L. reuteri SLZX19-12 . L. reuteri SLZX19-12 was sensitive to common antibiotics, including ampicillin (β-lactam antibiotic), neomycin (aminoglycoside antibiotic), and minocycline (tetracycline antibiotic) . In the tolerance test, L. reuteri SLZX19-12 showed about 50% survival in a pH 2 environment for 1 h  and a >10% survival rate in 0.1 g/100 mL bile salt for 4 h . According to the biochemical identification test, L. reuteri SLZX19-12 was found to hydrolyze cellobiose, maltose, sucrose, raffinose, and lactose but not aescinate, mannitol, salicin, sorbitol, inulin, and sodium hippurate . L. reuteri SLZX19-12 can produce a wide spectrum of enzymes, including esterase C4, lipase-like C8, albumin arylaminase, acid phospholipase, naphthol AS-BI-phosphate hydrolase, α-galactosidase, β-galactosidase, and α-glucosidase .Based on enrichment culture and screening of bacteria producing acids, we successfully isolated the desired strain, LZX19-12 . This stL. reuteri SLZX19-12 for 14 days, there was no significant difference in body weight of mice  are shown in L. reuteri and H-L. reuteri groups, the gene expression level of EpCAM in the colon significantly increased compared to that in the Con group  showed less weight loss  agar medium. Similar to the morphology, loads of Salmonella in the livers  staining. From the representative images, the colon interstitium of the SL1344 group exhibited irregular and damaged epithelium, larger and fewer goblet cells, and more inflammatory cell infiltration than that of the Con group . Compareiltrates . Histoloiltrates . However44 group .L. reuteri SLZX19-12 prevents the invasion of the colon by S. Typhimurium SL1344, we detected expression levels of key genes or proteins involved in autophagy, apoptosis, and the inflammatory process. Light chain 3 (LC3) is a marker of autophagy whose function is mainly involved in the formation of autophagosomes  gene was significantly increased in the SL1344 group compared to the Con group . Howeverof IFN-γ . Transfoof IFN-γ –27. Howef IFN-γ –. In the f IFN-γ – and F. TN-γ gene f IFN-γ –. Meanwhif IFN-γ –. Howeverf IFN-γ –. In cont44 group  and H.H_L. reuteri+SL1344 group , was used to challenge the mice.Thirty-seven SPF male C57BL/6J mice (3 weeks old) with similar body weights of about 11.5 g were purchased from Beijing HFK Bioscience Co., Ltd. All the trials involving mice complied with the guidelines of and were approved by the Institutional Animal Care and Use Committee of China Agricultural University (AW72011202-1-1). L. reuteri, 1 g feces was diluted in 10 mL phosphate-buffered saline (PBS) and homogenized. One milliliter of homogenized mixture was added to 50 mL MRS liquid medium with a pH of 4.0 and was cultured under anaerobic conditions for 24 h to enrich L. reuteri. The MRS liquid medium contained 10 g/L soluble starch, 10 g/L peptone, 4 g/L yeast extract powder, 5 g/L sodium acetate, 2 g/L KH2PO4, 2 g/L ammonium citrate, 0.2 g/L MgSO4·7H2O, 0.05 g/L MnSO4·7H2O, and 1 mL/L Tween 80. Subsequently, 50, 100, 150, and 200 μL cultured bacterial solution were spread evenly on MRS agar plates with added bromocresol blue by shaking with sterile glass beads. Monoclonal bacterial colonies of acid-producing bacteria with different morphologies were selected for purification by scribing and anaerobic culture on plates. Monoclonal bacterial colonies were selected for culture, and bacterial DNA was extracted for PCR to acquire full-length 16S rRNA. The PCR products  were sequenced with a 3730xl DNA analyzer. The identities of the bacterial strains were confirmed by conducting a BLAST search with the nucleotide collection (nr/nt) of the NCBI database and multiple biochemical tests.To isolate L. reuteri SLZX19-12 was plotted by reading the optical density at 600 nm (OD600) at different time points, for which the number of bacteria was obtained by counting colonies on MRS agar plates. The acid tolerance and bile salt tolerance tests were performed by adding hydrochloric acid and bile salt, respectively, to the desired pH or concentrations. A biochemical detection kit for lactic acid bacteria  and an API-ZYM enzymatic detection kit  were used to evaluate characteristics of L. reuteri SLZX19-12. All procedures followed the instructions provided by kit manufacturers. Antibiotic resistance was tested with antibiotic-sensitive paper . After the L. reuteri SLZX19-12 solution was spread evenly on MRS agar plates with a sterile spreading rod, the antibiotic susceptibility papers were put on the predetermined area. After 24 h of growth, the presence of inhibition zones was noted and their diameters were measured.The growth curve of L. reuteri SLZX19-12 was inoculated into MRS medium (20 mL) and anaerobically cultured for 12 h. The cultured bacterial solution was centrifuged at 100 × g for 10 min and resuspended with normal saline. The solution was successively diluted to 1 × 107 CFU/mL , 1 × 108 CFU/mL , and 1 × 109 CFU/mL  with normal saline. Each group contained 4 mice. Fifty microliters of sodium bicarbonate (0.05 g/mL) was administered to neutralize gastric acid, and then 150 μL normal saline (Con group) or L. reuteri SLZX19-12 solution was immediately injected into the stomachs of the mice with gastric injection needles. The gavage was given once every 2 days for 14 days until sample collection. To determine the effects of L. reuteri on S. Typhimurium SL1344 infection, 150 μL normal saline (for the Con and SL1344 groups) or L. reuteri SLZX19-12 with 1 × 109 CFU/mL (for the H_L. reuteri+SL1344 group) was administered by gavage to mice to establish colonization. Similarly, the gavage was given once every 2 days for 14 days. Then, 150 μL normal saline (for the Con group) or S. Typhimurium SL1344 solution with 1 × 106 CFU/mL (for the SL1344 and H_L. reuteri+SL1344 groups) was administered by gavage to the mice. Each group contained 7 mice. After 1 week, samples were collected. During the experiment, mice were housed in an environmentally controlled room . Samples collected included serum, feces, chyme, ileum, colon, spleen, and liver. Blood was collected from the eyeball and then centrifuged at 400 × g to acquire the serum. Samples other than serum were collected in Eppendorf tubes, rapidly frozen in liquid nitrogen, and then held at −80°C. Chyme, liver, and spleen were used to determine pathogen loads and were temporarily stored on ice, and detection was carried out as soon as possible. Intestinal tissues for morphological examination were fixed in 4% paraformaldehyde. The relative weights of liver and spleen were calculated as liver weight/body weight and spleen weight/body weight, respectively. The length of the colon was measured with a ruler.Monoclonal Parameters associated with infection, such as weight loss and rectal bleeding, were used to calculate the disease activity index (DAI). Each mouse was evaluated and assigned a DAI based on the average values according to the guidelines presented in Table S1.The tissues from the fixed middle position of the colon were acquired for sectioning and HE staining. The levels of inflammatory cell infiltration, pathological damage to crypt structure, ulceration of epithelium, and edema were evaluated to acquire the histological score. The histological scores in detail are shown in Table S2.3×, 104×, and 105×. One hundred microliters of each dilution was spread evenly on Salmonella-Shigella (SS) agar plates with sterile glass beads. SS agar medium contained 5 g/L beef powder, 5 g/L tryptose, 10 g/L lactose, 8.5 g/L bile salt no. 3, 8.5 g/L sodium citrate, 8.5 g/L sodium thiosulfate, 1 g/L ferric citrate, 0.025 g/L neutral red, 0.33 mg/L brilliant green, and 17.0 g/L agar. After aerobic culture for about 18 h at 37°C, black positive colonies were counted. Viable colony counts ranged from 30 to 300 per plate. Accordingly, loads of Salmonella in the colonic chyme, livers, and spleens were calculated and analyzed.Colonic chyme, livers, and spleens were added to sterile normal saline at ratios of 0.02 g/mL, 0.1 g/mL, and 0.04 g/mL, respectively. After homogenizing with 1-mm sterile steel balls, each mixture was serially diluted to 1×, 10×, 100×, 10The LPS and IL-1β concentrations in serum and colons were detected by a commercial mouse LPS enzyme-linked immunosorbent assay (ELISA) kit  and a mouse IL-1β ELISA kit . All the procedures followed instructions provided by the manufacturers of the ELISA kits.ACTCCTACGGGAGGCAGCAG-3′] and 806R [5′-GGACTACHVGGGTWTCTAAT-3′]) with barcoding to produce amplified fragments of about 500 bp. PCRs were conducted with FastPfu DNA polymerase  and an ABI GeneAmp 9700 PCR amplifier for 30 cycles of predenaturation at 95°C for 3 min, denaturation at 95°C for 20 s, annealing at 58°C for 15 s, and extension at 72°C for 20 s, followed by 1 cycle at 72°C for 5 min. PCR amplification products were detected by 2% agarose gel electrophoresis and then recovered using an AxyPrep DNA gel extraction kit . After product quantification and homogenization, DNA was denatured with sodium hydroxide to produce single-stranded DNA. The constructed MiSeq library was sequenced using the Illumina PE300 platform. The paired-end reads obtained were merged by Flash software (version 1.2.11), and the sequence data were quality controlled and filtered. Clustering of operational taxonomic units (OTU) was performed on the optimized sequence by UPARSE (version 7.1) based on 97% similarity. The representative sequences of OTU were annotated using the Silver database with RDP Classifier .Colon microbial genomes were extracted by a commercial E.Z.N.A. stool DNA kit , and quality was verified by 1% agarose gel electrophoresis. The microbial V3+V4 region of the 16S rRNA gene was amplified by universal primers  with a Bio-Rad Aminex HPX-87H column. The condition of 1.8 mM heptafluorobutyric acid (HFBA) at a column temperature of 42°C was used for elution with a flow rate of 0.6 mL/min. Analysis time was 20 min. Formate, lactate, acetate, propionate, butyrate, isobutyrate, valerate, and isovalerate were all quantified using standard curves based on the appropriate standards.Short-chain fatty acid (SCFA) concentrations in feces were quantified by an ion chromatography method. Briefly, about 25 mg feces from mice was added to 4 mL ultrapure water. After homogenization with clean glass beads, samples were placed in an ultrasonic bath (30 min) and then centrifuged at 10,000 × −ΔΔCT method was used to calculate relative expression level of target genes.RNA of colon tissues was extracted using a commercial kit , and RNA concentration and quality were evaluated with an ultra-micro spectrophotometer  and 1% agarose gel electrophoresis. One thousand nanograms of RNA was reverse transcribed to cDNAs using a HiFiScript reverse transcriptase mixture with genomic DNA remover . The SYBR green  method was used to quantify gene expression levels with a real-time quantitative PCR (RT-qPCR). The 10-μL mixture including 0.2 μM primer and 2 μL diluted cDNAs was run on the LightCycler 96 RT-qPCR system (Roche), followed by predenaturation at 95°C for 60 s and 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 45 s. GAPDH was used as an internal reference. All the primers for target genes are listed in Table S3. Standard and dissolution curves were obtained to confirm efficiency and specificity of the primers. The 2g for 10 min, protein concentrations of the supernatant were measured using a bicinchoninic acid (BCA) assay kit . Nuclear proteins from colon tissues were extracted using a commercial nuclear and cytoplasmic protein extraction kit . All samples were diluted to 3 μg/μL solution with RIPA lysis buffer and SDS-PAGE loading buffer . After protein denaturation at 100°C for 10 min, samples were subjected to SDS-PAGE electrophoresis, in which about 60 μg proteins was added. The electrophoretic buffer consisted of 14.4 g/L glycine, 3.03 g/L Tris, and 1 g/L SDS in deionized water. Conditions for electrophoresis and transfer to the membrane were constant at 20 mA and 200 mA, respectively. Transfer membrane buffer included 14.4 g/L glycine, 3.03 g/L Tris, and 20% methanol (vol/vol) in deionized water.About 50 mg colonic tissue was placed in radioimmunoprecipitation assay (RIPA) lysis buffer  containing phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitors and was homogenized with a homogenizer at −20°C. After centrifugation at 12,000 × After the proteins were transferred to 0.2 μm PVDF membranes, these membranes were blocked with 5% skim milk powder in PBS for 2 h at room temperature. Then the membranes were incubated overnight in primary antibodies diluted 1:1,000 with 5% skim milk powder solution. Antibody solution contained primary antibodies for S100A9 , TNF-α , NF-κB P50 , claudin-3 , claudin-7 , occludin , Bcl-2 , Bax , GAPDH , β-tubulin , and PCNA . After three washes for 5 min each, fluorescently labeled secondary antibodies, including donkey anti-mouse IgG  or goat anti-rabbit IgG , were incubated with the membranes for 4 h at 4°C. After three washings, the membranes were read by an Odyssey infrared imager . Tubulin and GAPDH were used as internal references to correct loading errors for total proteins, while PCNA was used as an internal reference for nuclear proteins.Colonic tissues fixed in 4% paraformaldehyde were removed and neatly trimmed. Segments (0.5 cm) of tissues were placed in embedding boxes and washed overnight with running water. The tissues in the embedding boxes were dehydrated with an increasing gradient of ethanol from 70% to 100%. After treatment with xylene, colonic tissues were immersed in paraffin at 65°C for 2 h and then embedded by cooling. Subsequently, tissues were cut into 0.2-μm slices, and slices were fixed to antislip slides. These samples were dewaxed with xylene and treated with a reducing gradient of ethanol ranging from 100% to 70%. To complete the rehydration process, samples were rinsed with tap water for 2 min. Next, the samples were stained with hematoxylin solution (5 g/L) for 5 min and immersed in 1% hydrochloric acid for 10 s to separate the staining. After a second staining with eosin aqueous solution for about 15 s, the samples were rapidly dehydrated with a gradient ethanol aqueous solution from 70% to 100% and treated in xylene for 10 min to finish the lucency process. Finally, slices were sealed with cedar oil and observed with the BX 53 microscope system .P values ranging from 0.05 to 0.10 are considered to indicate a tendency.All data are presented as means and standard errors (SE). Contrast analysis was conducted to analyze differences. In the 16S rDNA sequencing analysis, the α diversity was evaluated by Chao, Sobs, Shannon, and Simpson indexes. The β diversity was analyzed by PCoA based on the weighted normalized UniFrac method. Diversity analysis and data visualization were performed using Qiime2 software. Correlation analysis was based on Spearman’s rank correlation coefficient and was carried out by R . Other statistics and plots were constructed using SPSS 24  and GraphPad Prism 8.0.1 software . SRR15092578 to SRR15092596. The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.Sequencing data generated in this study were deposited in Sequence Read Archive (SRA) with the SRA accession numbers