Sublethal Levels of Antibiotics Promote Bacterial Persistence in Epithelial Cells

Abstract Antibiotic therapy and host cells frequently fail to eliminate invasive bacterial pathogens due to the emergence of antibiotic resistance, resulting in the relapse and recurrence of infections. Bacteria evolve various strategies to persist and survive in epithelial cells, a front‐line barrier of host tissues counteracting invasion; however, it remains unclear how bacteria hijack cellular responses to promote cytoplasmic survival under antibiotic therapy. Here, it is demonstrated that extracellular bacteria show invasive behavior and survive in epithelial cells in both in vivo and in vitro models, to increase antibiotic tolerance. In turn, sublethal levels of antibiotics increase bacterial invasion through promoting the production of bacterial virulence factors. Furthermore, antibiotic treatments interrupt lysosomal acidification in autophagy due to the internalized bacteria, using Bacillus cereus and ciprofloxacin as a model. In addition, it is found that sublethal levels of ciprofloxacin cause mitochondrial dysfunction and reactive oxygen species (ROS) accumulation to impair lysosomal vascular tape ATPase (V‐ATPase) to further promote bacterial persistence. Collectively, these results highlight the potential of host cells mediated antibiotic tolerance, which markedly compromises antibiotic efficacy and worsens the outcomes of infection.


Introduction
The escalating crisis of antibiotic resistance calls for new antibiotics and strategies to combat bacterial pathogens associated infections. [1] Discovering new antibiotics is challenging nowadays, [2] it is therefore crucial that alternative solutions are urgently required to address this problem. One approach is to develop ways to revitalize existing antibiotics, [3][4][5] to kill/inhibit multidrug resistant pathogens. To achieve such goal, we need further mechanistic understandings of the diverse ways by which bacteria survive under antibiotic therapy. Indeed, the relapse and recurrence of infections after treatments suggest that many failures of antibiotic therapy are caused by antibiotic tolerance of bacterial pathogens. [6,7] Unlike antibiotic resistant bacteria which inherit or acquire mutations, [8] antibiotic tolerance is the capability of individual bacteria or bacterial populations to survive antibiotic stresses without genetic changes. [9,10] Phenotypic tolerance to antibiotics in bacteria with a transient, dormant, or non-dividing status is usually induced by intermittent antibiotic exposures, [11,12] starvation, [13,14] or host environment. [15] Antibiotic tolerance can facilitate the evolution of antibiotic resistance. [16,17] For instance, Salmonella Typhimurium forms persisters to promote the dissemination of antibiotic resistance plasmids. [18] However, it remains largely unclear what the driving force for the emergence of antibiotic tolerance is, particularly in vivo.
Upon infections particularly persistent infections, [19,20] sophisticated defense responses are sequentially activated in hosts to clear bacterial invaders, [21][22][23] together with other therapeutic strategies including antibiotic therapy. Epithelial cells consist of a front-line barrier counteracting such invasion in hosts. [24,25] Epithelial cells play a crucial role in bridging the interactions between bacteria and host responses, [26,27] which may determine the efficacy of antibiotics and even the outcomes as well. [28] Epithelial cells usually harness multiple defensive mechanisms against bacterial invasion including cell integrity, rapid cell turnover, apoptosis, and autophagy. [29,30] On the other hand, many bacteria evolve adaptive strategies to circumvent the clearance by modulating cellular signals. Compared to obligate and facultative intracellular bacterial pathogens such as Mycobacterium tuberculosis and S. Typhimurium, [31] many extracellular bacterial pathogens such as Staphylococcus aureus, are able to www.advancedsciencenews.com www.advancedscience.com invade, survive, and persist in the cytosol of epithelial cells. [32] Once such bacteria survive in host cells, they act as "Trojan horses" to tolerate various stresses including antibiotic therapy. For example, survival of S. aureus within cells increases its ability against the treatment of hundreds of folds of vancomycin. [33] Meanwhile, other extracellular bacteria such as Bacillus cereus, Escherichia coli, Enterococcus faecalis, and Vibrio parahaemolyticus have also been shown to replicate in diverse cells. [34][35][36][37] Therefore, we hypothesized that survival of extracellular bacteria in epithelial cells served as a reservoir to elude antibiotic treatments. The persistence of bacteria in the cytosol may enable the emergence of antibiotic tolerance to cause the recurrent infections.
In this study, we first observed that eight species of extracellular bacteria could invade epithelial cells in both in vitro and in vivo models. Sublethal levels of antibiotics promoted the production of virulence factors to enhance bacterial invasion. Then, we found the low level of ciprofloxacin caused mitochondrial dysfunction and ROS accumulation by inhibiting lysosomal V-ATPase to further promote B. cereus persistence. Lastly, the internalized bacteria (including E. coli and B. cereus) survived in the cytoplasm by paralyzing the acidification of autophagosomes.

Epithelial Cells Protect Internalized Bacteria from Antibiotic Treatments
To get better understanding of post-antibiotic expansion of bacterial pathogens in clinic, [28,38,39] we employed a mouse model orally infected with B. cereus and E. coli. We observed the presence of B. cereus and E. coli in the epithelial cells, particularly in the ileum ( Figure S1A,B, Supporting Information). Then we prepared primary rat intestinal epithelial cells (RIECs) and infected with each of multiple pathogens, including four Gram-positive bacteria Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus, and Streptococcus suis, and four Gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and V. parahaemolyticus. Consistently, we found that all bacteria could invade RIECs (Figure S1C, Supporting Information) and similar phenomenon was observed in diverse epithelial cell lines including rat small intestine cell (IEC-6) cells (Figure 1A), lung carcinoma cell (A549), human hepatocellular carcinoma (HepG2), and African green monkey kidney cell (Vero cells) ( Figure S1D-F, Supporting Information). Unlike macrophages such as RAW 264.7 cells which actively phagocytose bacteria, non-phagocytic immune cells such as mouse hybridoma cells (S/P20 cells) could not be infected ( Figure S1G, Supporting Information). These findings suggested that extracellular bacteria could invade epithelial cells in both in vivo and in vitro models.
To compare the efficacy of antibiotics against bacteria in the presence and absence of cells, we used IEC-6 cells as a model. Compared to the extracellular minimum bactericidal concentrations (MBCs) of eight bacteria, we observed a significant increase of intracellular MBCs with 107 to 667-fold increases ( Figure 1B). It indicated that antibiotic efficacy dramatically decreased against internalized bacteria, although these bacteria were susceptible to antibiotics in the absence of cells. Subsequently, we tested the intracellular MBCs of multiple antibiotics with different modes of action against B. cereus. It showed that the values of intracellular MBCs significantly increased in the presence of diverse cells ( Figure 1C), suggesting a common mechanism of antibiotic tolerance. Additionally, we extended to examine the antibacterial activity against E. coli, S. aureus, and V. parahaemolyticus in different types of epithelial cells. Consistent with the observation in B. cereus, we found dramatically increased antibiotic tolerance (Table S5, Supporting Information). Altogether, these results suggested that epithelial cells could protect internalized bacteria from high levels of antibiotic treatments.

Sublethal Levels of Antibiotics Facilitate Bacterial Invasion to Epithelial Cells
To dissect epithelial cells mediated antibiotic tolerance, we first focused on the effect of antibiotics on bacterial invasion, which is the restricted step to form the cell-mediated tolerance. It is well known that bacterial toxins can facilitate bacteria entry into host cells. [40][41][42][43][44] Therefore, we measured the production of two major toxins in B. cereus (non-hemolytic enterotoxin, Nhe) and S. aureus ( -toxin, AT) in the presence of sublethal levels of antibiotics. Results showed that such antibiotics promoted toxin productions in a time dependent manner (Figure 2A,B). In turn, Nhe and AT facilitated the invasion of B. cereus and S. aureus, consistent with the notion that bacterial toxins are crucial to accelerate invasion. [40,41,44] Correspondingly, addition of neutralizing antibodies mAb 1E11 or MED14893* could abolish the potentiated invasion ( Figure 2C). Furthermore, we found that these bacterial toxins damaged the integrity of cell membrane, as indicated by the release of choline and lactate dehydrogenase (LDH) ( Figure 2D,E). Lastly, we deciphered the involved signal pathways during invasion. According to our previous findings that both factor associated suicide (Fas) and signal-regulating kinase (ASK1) are important to trigger apoptosis in B. cereus associated infections, [45] we therefore quantified bacterial numbers in mutant cells with the deletion of either fas or ASK1 genes. Interestingly, the invasion of B. cereus dramatically postponed in the mutants, particularly when both signals were simultaneously inhibited ( Figure 2F). It indicated that B. cereus could hijack cellular responses to coordinate invasion. Additionally, we evaluated the role of other bacterial virulence factors such as phospholipase in bacterial invasion. Exogenous addition of phospholipase C (PLC) derived from B. cereus could promote the invasion for either S. aureus or E. coli ( Figure 2G). Although the invasion of B. cereus was not enhanced by additional PLC, its specific inhibitor (D609) reduced the invasion of B. cereus. Taken together, these data suggested that bacteria could harness versatile virulence factors to achieve the invasion of epithelial cells.
Next, we investigated how antibiotics modulated the intracellular lifestyle of invasive bacteria. First, we observed that the proliferation of persistent B. cereus in epithelial cells in a time depend manner ( Figure 3A). To quantify the efficacy of bacteria invading epithelial cells, we found that the increased multiplicity of infection (MOI, the number of bacteria that are added per cell during infection) and long infection time resulting in more internalized bacteria, survival and replication in cells (Figure S2, Supporting Information). Meanwhile, we observed that sublethal levels of antibiotics had no effect on the growth of    Table S2, Supporting Information). Compared to the abundant extracellular antibiotics, accumulated antibiotics in cells consisted only about 0.28-14.34% of the total ( Figure S3C, Supporting Information). Given that sufficient levels of antibiotics are prerequisite to inhibit bacterial growth, [13,14,17] such low levels of antibiotics in the cytosol could not reach sufficient concentrations to kill/inhibit bacteria. Thus, we used sublethal levels of antibiotics to treat infected mice and observed that such antibiotics could promote B. cereus internalization ( Figure 3B,C). Moreover, antibiotic treatments promoted the internalization of B. cereus in a time dependent manner ( Figure S4A, Supporting Information). In addition, long-term exposures to sublethal levels of antibiotics except tetracycline advanced the survival of internalized B. cereus ( Figure S4B-D, Supporting Information). It might be due to either the intrinsic property of tetracycline or that partial bacteria escaped from the epithelial cells treated with tetracycline.
Although B. cereus is a spore forming bacterium, we found that the vegetative cells comprised a large proportion of bacte-rial numbers in IEC-6 cells ( Figure S5A,B, Supporting Information), although the increased numbers of spores were observed as well. It is in agreement with that sub-lethal levels of antibiotics promoted diverse non-spore-forming bacteria invading epithelial cells (Figures 1 and 2). Furthermore, the nutrients in the intracellular environment are limited for the survival of invaded bacteria. [13] Compared to the abounding nutrients in media (Figure S5C, Supporting Information), we found the upregulation of starvation response-related genes in both B. cereus (yjbM and yawC) and E. coli (reclA and spoT), under antibiotic treatments ( Figure S5D, Supporting Information). It revealed that bacteria adapted to the conditions of deprived nutrients in epithelial cytoplasm. It is worth noting that epithelial cells always induce autophagy upon starvation. [46,47]

Sublethal Levels of Antibiotics Promote Bacterial Persistence by Inducing Autophagy Arrest
Host cells always initiate multiple strategies such as autophagy to defense invasive bacteria. [47][48][49] To better understand bacterial Adv. Sci. 2020, 7,1900840  survival in cells, we hypothesized that antibiotics promoted bacteria persistence in the cytosol through hijacking autophagy. We dissected the complicated signaling cascade of autophagy using two markers (light chain 3, LC3; p62/sequestosome-1, p62/SQSTM1). [50] We first found that B. cereus interrupted the process of autophagy in a time dependent manner by regulating the expression of LC3-II and p62 ( Figure S6, Supporting Information). Meanwhile, antibiotic treatments aggravated autophagy arrest with the increase of dual florescence labeled LC3 (Figure 4A), while GFP labeled LC3 was quenched its GFP florescence in the acidic environment due to the fusion of autophagosome and lysosome. [51,52] Furthermore, antibiotics induced the increase of www.advancedsciencenews.com www.advancedscience.com numbers of p62 puncta ( Figure 4B), denoting the interruption of lysosomal degradation. [30,48,51] In addition, antibiotics upregulated the expression of LC3-II and p62 in IEC-6 cells infected with B. cereus based on Western blot analysis ( Figure 4C). These results showed that antibiotics facilitated the survival of B. cereus through suppressing autophagy. Similarly, we observed fluorescent patterns and increased expression of LC3 and p62 in IEC-6 cells infected with E. coli ( Figure S7, Supporting Information).
We next sought to elucidate the autophagy arrest of infected cells, using the inducer rapamycin and inhibitor chloroquine of autophagy. Antibiotics mediated autophagy arrest was rescued by rapamycin while enhanced by chloroquine ( Figure S8A-C, Supporting Information). It suggested that antibiotics promoted bacteria inducing autophagy arrest. Internalized bacteria are usually delivered to the lysosomes for further degradation. The acidic microenvironment plays a critical role in maintaining the activity of lysosome. [53] Thus, we tracked the acidic lysosomes in IEC-6 cells and observed the decreased numbers of co-localized B. cereus with acidic organelles in the presence of antibiotics (Figure 4D,E). To further characterize the reduced acidic organelles, we found that the labeled lysosomal membrane protein (LAMP1) was not colocalized with bacteria under antibiotic treatment (Figure S8D, Supporting Information). These results suggested that the process of acidification was impeded, to interrupt the degradation of engulfed bacteria.

Sublethal Level of Ciprofloxacin Cause Mitochondrial Dysfunction and Inhibition of Lysosomal V-ATPase
To further characterize how antibiotics impair lysosomal acidification, we focused on the lysosomal V-ATPase complex, the workhorse for maintaining the acidic environment in lysosomes. [54] We observed that antibiotic treatment promoted bacterial survival in the presence of specific inhibitor of V-ATPase bafilomycin A1 using ciprofloxacin as an example ( Figure 5A; Figure S9A, Supporting Information). Bafilomycin A1 led to the inhibition of ATP6V0D1 ( Figure 5B; Figure S9B, Supporting Information), a subunit of V0-sector for H + transporting. These findings are consistent with previous reports that Streptococcus pyogenes and S. Typhimurium modulate V-ATPase to inhibit lysosomal acidification. [55,56] Activity of V-ATPase plays a crucial role in the function of mammalian target of rapamycin (mTOR). [57] We found that ciprofloxacin promoted the expression of the activated form of mTOR (phosphorylated mTOR, p-mTOR) ( Figure S8C, Supporting Information), confirming the inhibition of V-ATPase. [58] V-ATPase particularly the V1-sector, is responsible for ATP hydrolysis to maintain the pH gradient. [59] Therefore, we tested the expression of ATP6V1D (a subunit of V1-sector) and found the inhibited expression of ATP6V1D under ciprofloxacin treatment ( Figure 5C; Figure S9C, Supporting Information). Correspondingly, the ATP level was significantly decreased under long-term antibiotic treatments, whereas the compensatory ATP accumulation occurred at the early stage ( Figure 5D).
In addition, we found that inhibition of lysosomal acidification further decreased the activity of acid phosphatase (ACP) in the presence of ciprofloxacin (Figure 6A). Decreased activity of ACP retards the clearance of damaged mitochondria, [60,61] leading to the inhibition of lysosomal acidification. Consistent with previous observations, [62,63] we found that ciprofloxacin caused morphological damage and dysfunction on mitochondria ( Figure 6B), resulting in ROS accumulation ( Figure 6C), with the decrease of membrane potential ( Figure 6D,E). Exogenous addition of ROS scavenger NAC (N-acetyl cysteine) could reverse the inhibition of ATP6V0D1 ( Figure 6F). Taken together, these results demonstrated that sublethal levels of antibiotics induced mitochondrial dysfunction resulting in ROS accumulation, which may contribute to the inhibition of lysosomal acidification. [64,65] To further decipher the cellular responses, transcriptome analysis was employed to evaluate the gene expression of IEC-6 cells infected with bacteria under ciprofloxacin treatment. Compared to the profile of cells infected with E. coli, there were fewer changes of gene expression in the cells infected with B. cereus, particularly in the presence of ciprofloxacin (Figure 7A,B). Interestingly, we found that the upregulation of genes related to inflammatory cytokine interleukin-8 (IL-8) was presented in all treatments ( Figure 7C). Then we quantified IL-8 and confirmed that ciprofloxacin treatment enhanced the production of IL-8 ( Figure 7D). Although the regulation of IL-8 remains unclear in the infected cells, implying that sublethal levels of antibiotics trigger a cascade of inflammation responses. [66] Recently, the toxin Nhe of B. cereus has been demonstrated to activate the NLRP3 inflammasome. [67] Thus, further works are needed to elucidate the inflammatory response pathway in host cells mediated antibiotic tolerance.

Discussion
Extracellular bacteria such as B. cereus, E. coli, and S. aureus usually cause systematic infections due to the translocation from the initially persisted sites. [22] Failure of antibiotic therapy in clinic includes the emergence of antibiotic resistance and/or tolerance, [3,6] resulting in the disruption of normal microbiome particularly for gastrointestinal infections. [15] Pathogenic bacteria subsequently invade the epithelium before the recovery of colonization resistance. [68] Survival of bacteria in the cytosol of epithelial cells therefore obtains many benefits when extracellular bacteria manage to counteract the clearance of host. On one hand, such invasive bacteria are much easier to penetrate the first barrier for sequential dissemination from the infected sites. On the other, these bacteria acting as "Trojan horses" in epithelial cells achieve an economic way to sustain antibiotic stresses without genetic costs. In the present work, we found that many extracellular bacteria could invade and survive in diverse epithelial cells in both in vivo and in vitro models (Figure 1; Figure S1, Supporting Information), which is consistent with previous reports for E. faecalis and V. parahaemolyticus. [29,35] Consequently, epithelial cells provide special niches for these bacteria to tolerate multiple antibiotics, because antibiotics cannot accumulate to enough levels in the cytosol to kill/inhibit bacteria. [69] Figure 5. Sublethal levels antibiotics inhibited lysosomal V-ATPase to facilitate bacteria survival. A) Inhibition of acidified lysosomes enhancing the intracellular survival of B. cereus. Bafilomycin A1 (100 nm) was used to inhibited V-ATPase in IEC-6 cells for 1 h. Rapamycin (100 nm) was employed to inhibit autophagy. The numbers of internalized bacteria were counted by the CFU assay. Results are shown as means ± SEM (n = 6, **p < 0.01, ***p < 0.001). Scale bar: 20 µm. B) Expression of ATP6V1D in lysosomes using Western blot. IEC-6 cells were infected with B. cereus NVH0075/95 (MOI = 40) under ciprofloxacin treatment (0.5 µg mL −1 ). All proteins were normalized to the levels of -actin (compared to sole bacterial infectious group). Results are shown as means ± SEM (*p < 0.05; **p < 0.001). C) Increase of ATP levels under antibiotic exposure. IEC-6 cells were infected with B. cereus under antibiotic treatments (0.5 µg mL −1 ciprofloxacin, 0.25 µg mL −1 erythromycin, 4 µg mL −1 tetracycline, 0.625 µg mL −1 rifampin, and 2 µg mL −1 vancomycin). The release of ATP in the supernatants were measured by normalizing the ATP levels to the amount of proteins. Data are shown as mean ± SEM for at least three replicates (**p < 0.01). D) Expression of ATP6V0D in the lysosome of IEC-6 cells. Cells were infected with B. cereus NVH0075/95 (MOI = 40) with the treatment of 0.5 µg mL −1 ciprofloxacin. Cells were pre-incubated with bafilomycin A1 (100 × 10 −9 m) for 1 h to inhibit V-ATPase. Rapamycin (100 × 10 −9 m) targeting mTOR was used as an inducer of autophagy. All proteins were normalized to the levels of -actin. Data are showed as means ± SEM (*p < 0.05, **p < 0.001, n = 3).
The side effects of antibiotics on disrupting host microbiome have been well studied. For instance, antibiotic treatment depletes commensal butyrate-producing Clostridia in the gut, leading to the increased epithelial oxygenation and expansion of S. Typhimurium. [6] Furthermore, antibiotic-associated diarrhea (AAD) is caused after antibiotic therapy, notoriously known for Clostridium difficile infection (CDI). [70] However, the underlying mechanisms of post antibiotic expansion remain unclear. We here provide an alternative explanation that antibiotics promote colonization of extracellular bacteria in epithelial cells to cause secondary infections when the level of antibiotics decrease or fade away.
Host cells often eliminate the infected cells through apoptosis, autophagy, recruiting immune cells and other strategies. It has been shown that epithelial autophagy is essential for the defense against invasive bacterial pathogens. [29] Surprisingly, we observed that the low levels of antibiotics could facilitate bacterial survival through autophagy arrest in epithelial cells (Figure 3; Figure S7, Supporting Information). Antibiotics blocked the fusion of autophagosome and lysosome by upregulating LC3 and p62 ( Figure 3A,B). Similarly, V. parahaemolyticus and Legionella pneumophila modulate lysosomal acidification for survival. [71,72] We found that the inhibition of V-ATPase impaired lysosomal acidification (Figure 4). In addition, both mitochondrial dysfunctions and damaged cell membrane contribute to ROS accumulation ( Figure 4; Figure S9, Supporting Information), to further paralyze cellular homeostasis facilitating bacterial survival in the cytosol.

Conclusion
We describe a general observation that both Gram-positive and Gram-negative bacteria, known for their extracellular lifestyles, can invade and survive in diverse epithelial cells. Such adap-tion endows bacteria with the tolerance to multiple antibiotics ( Figure 7E). Sublethal levels of antibiotics not only promote the production of bacterial toxins to increase invasion, but also cause mitochondrial dysfunction and ROS accumulation resulting in autophagy arrest. Our findings provide a framework for host www.advancedsciencenews.com www.advancedscience.com cells mediated antibiotic tolerance in both in vivo and in vitro models, which will shed light on the better use of antibiotics and development of alternative strategies to either target the internalized bacteria or to boost the cellular defense of host cells, to reduce the recurrence of infections.

Experimental Section
Bacterial Strains and Mammalian Cells: Eight extracellular bacterial strains were used in this study (Table S1, Supporting Information). Four epithelial cell lines, two immune cell lines and two kinds of primary cell lines, were employed in this work (Table S2, Supporting Information). Δfas or ΔASK1 cells were mutants of Vero cells constructed by CRISPR-Cas9 knockout assay, according to the previously published method. [45] Addition of NQDI1 (500 nm, Sigma-Aldrich) in Δfas cells was to obtain the double knockdown of both Fas and ASK1 proteins on Vero cells, because NQDI1 was used as a specific inhibitor of ASK1. [73] More details of the bacterial strains and mammalian cells used in this study are provided in Supporting Information.
5-week-old female ICR mice (n ≥ 5) were infected intragastrically with 200 µL bacteria (B. cereus NVH0075/95 and E. coli ATCC25922) in 0.9% saline solution at 1 × 10 9 CFUs per mouse for 24 h. Meanwhile, the mice solely treated with saline solution were as the no antibiotic control. While infected mice were treated with 0.5 µg mL −1 ciprofloxacin and 2 µg mL −1 tetracycline were served as antibiotic treatments. More experimental details could be found in the supporting information.
Ethics Statement: All animal protocols were approved by the Genentech Institutional Animal Care and Use Committee at the China Agricultural University (SYXK, 2016-0008). The experimental procedures involving mice and rats were gained an approval (SCXK, 2016-006).
Confocal Laser Scanning Microscopy Analysis: For static images, fixed and stained intestinal or cellular samples were captured by a Leica SP8 confocal microscope. 3D images were taken by capture all the X-, Y-, and Z-axis sections. For analyzing the location of internalized bacteria, the Zaxis section was cut every 1 or 2 µm. Images were analyzed and merged by the LAS AF Lite software (Leica).
Antimicrobial Activity Analysis: Extracellular minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were used to determine the antimicrobial activity of antibiotics in extracellular environment. The intracellular MBCs of antibiotics were used to define the antimicrobial activity of antibiotics for that internalized bacteria in cytoplasm, according to a previous publication [33] with slight modifications. More details of the experimental protocols of antimicrobial activity used in this work were expanded in Supporting Information.
Virulence Factor Assays: Two kinds of bacterial toxins including nonhemolytic enterotoxin of B. cereus (Nhe) and -toxin of S. aureus (AT), were tested. Nhe (23 ng mL −1 ), AT (50 ng mL −1 , Sigma-Aldrich), and their corresponding neutralizing antibodies (anti-NheB mAb 1E11, 2 µg mL −1 and anti--toxin mAb, MEDI4893*, 5 µg mL −1 , Sigma-Aldrich) were involved as well. Ribbit anti-IgG antibody (2 µg mL −1 , Beyotime) was used as a negative control. IEC-6 cells, bacteria (B. cereus, S. aureus, and E. coli), and sole bacterial toxin in the presence or absence of its antagonist were simultaneously added. After incubation for 2 h, the numbers of internalized bacteria were counted as previous description above. Lastly, the damage of phospholipid resulting in increased levels of choline were detected by a Phospholipid Assay Kit (Sigma-Aldrich) and lactate dehydrogenase (LDH) in the media from damaged cells were determined by a LDH Release Kit (Beyotime), according to their instructions.