A novel 12-membered ring non-antibiotic macrolide EM982 attenuates cytokine production by inhibiting IKKß and IκBα phosphorylation.

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.

Degradation of EGFR on lung epithelial cells by neutrophil elastase contributes to the aggravation of pneumococcal pneumonia.

Pneumococcus is the main cause of bacterial pneumonia. Pneumococcal infection has been shown to cause elastase, an intracellular host defense factor, to leak from neutrophils. However, when neutrophil elastase (NE) leaks extracellularly, it can degrade host cell surface proteins such as epidermal growth factor receptor (EGFR) and potentially disrupt the alveolar epithelial barrier. In this study, we hypothesized that NE degrades the extracellular domain (ECD) of EGFR in alveolar epithelial cells and inhibits alveolar epithelial repair. Using SDS-PAGE, we showed that NE degraded the recombinant EGFR ECD and its ligand epidermal growth factor, and that the degradation of these proteins was counteracted by NE inhibitors. Furthermore, we confirmed the degradation by NE of EGFR expressed in alveolar epithelial cells in vitro. We showed that intracellular uptake of epidermal growth factor and EGFR signaling was downregulated in alveolar epithelial cells exposed to NE and found that cell proliferation was inhibited in these cells These negative effects of NE on cell proliferation were abolished by NE inhibitors. Finally, we confirmed the degradation of EGFR by NE in vivo. Fragments of EGFR ECD were detected in bronchoalveolar lavage fluid from pneumococcal pneumonia mice, and the percentage of cells positive for a cell proliferation marker Ki67 in lung tissue was reduced. In contrast, administration of an NE inhibitor decreased EGFR fragments in bronchoalveolar lavage fluid and increased the percentage of Ki67-positive cells. These findings suggest that degradation of EGFR by NE could inhibit the repair of alveolar epithelium and cause severe pneumonia.

Pneumococcus downregulates the molecular weight of the extracellular domain of the epidermal growth factor receptor of alveolar epithelial cells.

Pneumococcus is themajor cause of bacterial and invasive pneumococcal infections. Disrupting the alveolarepithelial barrier is an important step in the pathogenesis of invasivepneumococcal infections. The epidermal growth factor receptor (EGFR) maintainsthe integrity of the alveolar epithelial barrier. In this study, we showed that secretory pneumococcal molecules decrease the molecular weight of EGFR without peptide degradation and inhibit alveolar epithelial cell proliferation via EGFR.

Biofilm-derived membrane vesicles exhibit potent immunomodulatory activity in Pseudomonas aeruginosa PAO1.

Pathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesicles in Gram-negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic-derived MVs (p-MVs) and biofilm-derived MVs (b-MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b-MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p-MVs. The mRNA expression levels of Toll-like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b-MV-mediated cytokine induction, suggesting that lipopolysaccharide of native b-MVs is the origin of the immune response. In addition, heat-treated or homogenized b-MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen-associated molecular patterns in lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.

Lipoprotein signal peptidase-deficient Streptococcus pneumoniae exhibits impaired Toll-like receptor 2-stimulatory activity.

Streptococcus pneumoniae is a causative agent of community-acquired pneumonia. Upon pneumococcal infection, innate immune cells recognize pneumococcal lipoproteins via Toll-like receptor 2 and induce inflammation. Here, we generated a strain of S. pneumoniae deficient in lipoprotein signal peptidase (LspA), a transmembrane type II signal peptidase required for lipoprotein maturation, to investigate the host immune response against this strain. Triton X-114 phase separation revealed that lipoprotein expression was lower in the LspA-deficient strain than in the wild-type strain. Additionally, the LspA-deficient strain decreased nuclear factor-κB activation and cytokine production in THP-1 cells, indicating impaired innate immune response against the strain.

Pneumococcal proteins ClpC and UvrC as novel host plasminogen binding factors.

Two plasminogen binding proteins were identified from a mouse infected with Streptococcus pneumoniae. The pneumococcal proteins were annotated as ATP-dependent Clp protease ATP-binding subunit (ClpC) and excinuclease ABC subunit C (UvrC) using the isobaric tags for relative and absolute quantification (iTRAQ) method. Recombinants of both proteins showed significant binding to plasminogen and were found to promote plasminogen activation by tissue-type plasminogen activator. In addition, ClpC and UvrC were LytA-dependently released into the culture supernatant and bound to the bacterial surface. These results suggest that S. pneumoniae releases ClpC and UvrC by autolysis and recruits them to the bacterial surface, where they bind to plasminogen and promote its activation, contributing to extracellular matrix degradation and tissue invasion.

Roles of membrane vesicles from Streptococcus mutans for the induction of antibodies to glucosyltransferase in mucosal immunity.

Glucosyltransferase (Gtf) B and GtfC from Streptococcus mutans are key enzymes for the development of biofilm-associated diseases such as dental caries. Gtfs are involved in membrane vesicles (MVs) and function in the formation of biofilms by initial colonizers such as Streptococcus mitis and Streptococcus oralis on the tooth surface. Therefore, MVs may be important virulence factors and targets for the prevention of biofilm-associated disease. To clarify how GtfB encoded by gtfB and GtfC encoded by gtfC associate with MVs and whether MVs are effective as a mucosal immunogen to induce the production of antibodies against Gtfs, MVs from S. mutans UA159 wild-type (WT), gtfB-, gtfC- and gtfB-C- were extracted from culture supernatants by ultracentrifugation and observed by scanning electron microscopy. Compared with GtfB, GtfC was mainly contained in MVs and regulated the size and aggregation of MVs, and the biofilm formation of S. mutans. The intranasal immunization of BALB/c mice with MVs plus a TLR3 agonist, poly(I-C), was performed 2 or 3 times for 5 weeks, with an interval of 2 or 3 weeks. MVs from all strains caused anti-MV IgA and IgG antibody production. In quality analysis of these antibodies, the IgA and IgG antibodies produced by immunization with MVs from WT and gtfB- strains reacted with Gtfs in the saliva, nasal wash and serum but those produced by immunization with MVs from gtfC- and gtfB-C- strains did not. S. mutans MVs mainly formed by GtfC are an intriguing immunogen for the production of anti-Gtf antibodies in mucosal immunogenicity.

Steric microstructure of mixed-species biofilm formed by interaction between Lactobacillus plantarum ML11-11 and Saccharomyces cerevisiae.

The mixed-species biofilm of Lactobacillus plantarum ML11-11 (LAB) and yeast had a double-layered structure with the ground layer composed of LAB cells, and the upper layer composed of coaggregates of LAB and yeast cells. The ability of LAB to adhere to both, the solid surface and the yeast cells, enabled the formation and maintenance of the biofilm as an ecosystem for LAB and yeast.

Effects of Complex DNA and MVs with GTF Extracted from Streptococcus mutans on the Oral Biofilm.

Streptococcus mutans is one of the principal pathogens for the development of dental caries. Oral biofilms formed by S. mutans are constructed of insoluble glucan formation induced by the principal enzymes, GTF-I and GTF-SI, in sucrose-containing conditions. However, as another means of biofilm formation, extracellular DNA (eDNA) and membrane vesicles (MVs) are also contributors. To explore the roles of eDNA and MVs for biofilm formation, short and whole size pure DNAs, two types of sub-purified DNAs and MVs were extracted from S. mutans by beads destruction, treatment of proteinase K, and ultracentrifugation of culture supernatant, and applied into the biofilm formation assay using the S. mutans UA159 gtfBC mutant, which lost GTF-I and GTF-SI, on a human saliva-coated 96 well microtiter plate in sucrose-containing conditions. Sub-purified DNAs after cell lysis by beads destruction for total 90 and 180 s showed a complex form of short-size DNA with various proteins and MVs associated with GTF-I and GTF-SI, and induced significantly higher biofilm formation of the S. mutans UA159.gtfBC mutant than no sample (p < 0.05). Short-size pure DNA without proteins induced biofilm formation but whole-size pure DNA did not. Moreover, the complex form of MV associated with GTFs and short-size DNA showed significantly higher biofilm formation of initial colonizers on the human tooth surface such as Streptococcus mitis than no sample (p < 0.05). The short-size DNAs associated with MVs and GTFs are important contributors to the biofilm formation and may be one of additional targets for the prevention of oral biofilm-associated diseases.

Screening of lactic acid bacteria that can form mixed-species biofilm with Saccharomyces cerevisiae.

The abilities of lactic acid bacteria (LAB) to form mixed-species biofilm with Saccharomyces cerevisiae in a static co-culture were investigated out of 168 LAB stock cultures, and two Lactobacillus plantarum strains (D71 and E31) and one Leuconostoc mesenteroides strain K01 were found to form mixed-species biofilm with S. cerevisiae BY4741. SEM observation showed that there was no significant difference in morphological properties among these three mixed-species biofilms and they resembled that formed by S. cerevisiae with L. plantarum ML11-11 previously isolated from a brewing sample of Fukuyama pot vinegar. The co-aggregation assays showed that L. plantarum D71 and L. plantarum E31 could co-aggregate with S. cerevisiae similarly to L. plantarum ML11-11, while L. mesenteroides K01 had no ability to co-aggregate with yeast. The above results indicate that aggregation followed by direct cell-to-cell contact is required for mixed-species biofilm formation between these L. plantarum strains and S. cerevisiae, though some different mechanism may be involved in biofilm formation between L. mesenteroides strain and S. cerevisiae.

Effective degradation of various bacterial toxins using ozone ultrafine bubble water.

Infectious and foodborne diseases pose significant global threats, with devastating consequences in low- and middle-income countries. Ozone, derived from atmospheric oxygen, exerts antimicrobial effects against various microorganisms, and degrades fungal toxins, which were initially recognized in the healthcare and food industries. However, highly concentrated ozone gas can be detrimental to human health. In addition, ozonated water is unstable and has a short half-life. Therefore, ultrafine-bubble technology is expected to overcome these issues. Ultrafine bubbles, which are nanoscale entitles that exist in water for considerable durations, have previously demonstrated bactericidal effects against various bacterial species, including antibiotic-resistant strains. This present study investigated the effects of ozone ultrafine bubble water (OUFBW) on various bacterial toxins. This study revealed that OUFBW treatment abolished the toxicity of pneumolysin, a pneumococcal pore-forming toxin, and leukotoxin, a toxin that causes leukocyte injury. Silver staining confirmed the degradation of pneumolysin, leukotoxin, and staphylococcal enterotoxin A, which are potent gastrointestinal toxins, following OUFB treatment. In addition, OUFBW treatment significantly inhibited NF-κB activation by Pam3CSK4, a synthetic triacylated lipopeptide that activates Toll-like receptor 2. Additionally, OUFBW exerted bactericidal activity against Staphylococcus aureus, including an antibiotic-resistant strain, without displaying significant toxicity toward human neutrophils or erythrocytes. These results suggest that OUFBW not only sterilizes bacteria but also degrades bacterial toxins.

C-Terminal Lysine Residue of Pneumococcal Triosephosphate Isomerase Contributes to Its Binding to Host Plasminogen.

The main causative agent of pneumonia, Streptococcus pneumoniae, is also responsible for invasive diseases. S. pneumoniae recruits human plasminogen for the invasion and colonization of host tissues. We previously discovered that S. pneumoniae triosephosphate isomerase (TpiA), an enzyme involved in intracellular metabolism that is essential for survival, is released extracellularly to bind human plasminogen and facilitate its activation. Epsilon-aminocaproic acid, a lysine analogue, inhibits this binding, suggesting that the lysine residues in TpiA are involved in plasminogen binding. In this study, we generated site-directed mutant recombinants in which the lysine residue in TpiA was replaced with alanine and analyzed their binding activities to human plasminogen. Results from blot analysis, enzyme-linked immunosorbent assay, and surface plasmon resonance assay revealed that the lysine residue at the C-terminus of TpiA is primarily involved in binding to human plasminogen. Furthermore, we found that TpiA binding to plasminogen through its C-terminal lysine residue was required for the promotion of plasmin activation by activating factors.

A bivalent outer membrane vesicle-based intranasal vaccine to prevent infection of periodontopathic bacteria.

Periodontal disease has become a serious public health problem, not only causing tooth loss, but also inducing chronic disorders of extra-oral organs. The present study assessed an intranasal vaccine strategy to prevent periodontal disease using outer membrane vesicles (OMVs) of two major periodontopathic bacteria, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa). We compared the morphology, composition, and immune activity between OMVs of Pg strain ATCC 33277 and Aa strain Y4. Aa OMVs had a smoother surface and stronger lipid A activity compared to Pg OMVs. The in vitro immune activity elicited by Aa OMVs in macrophage-like cells was remarkably stronger than that of Pg OMVs. Intranasal immunization of mice with Aa OMVs alone resulted in robust, humoral immune responses in blood and saliva. Despites the intrinsically low mucosal immunogenicity of Pg OMVs alone, using Aa OMVs as a mucosal adjuvant strongly enhanced Pg-specific immune responses, resulting in both serum IgG and salivary IgA, both of which aggregated Pg and Aa cells. Furthermore, Aa OMVs were found to be a more potent mucosal adjuvant than Poly(I:C) in the context of enhancing the production of Pg-specific IgG (especially IgG2a) and IgA. In addition, in a randomized, blinded study, mice oral challenged with Pg and Aa after intranasal immunization with Pg OMVs and Aa OMVs had significantly decreased numbers of both microorganisms compared to mock-immunized mice. Furthermore, in an intracerebral injection mouse model, there were no serious adverse effects on the brain even after administrating a dose of OMVs as same as that used for intranasal administration. Taken together, the bivalent OMV intranasal vaccine may be effective in preventing colonization of periodontopathic bacteria in the oral cavity and related systemic disorders associated with periodontal diseases.

Macrolides Decrease the Proinflammatory Activity of Macrolide-Resistant Streptococcus pneumoniae.

Over the past 2 decades, the prevalence of macrolide-resistant Streptococcus pneumoniae (MRSP) has increased considerably, due to widespread macrolide use. Although macrolide usage has been proposed to be associated with treatment failure in patients with pneumococcal diseases, macrolides may be clinically effective for treating these diseases, regardless of the susceptibility of the causative pneumococci to macrolides. As we previously demonstrated that macrolides downregulate the transcription of various genes in MRSP, including the gene encoding the pore-forming toxin pneumolysin, we hypothesized that macrolides affect the proinflammatory activity of MRSP. Using HEK-Blue cell lines, we found that the supernatants from macrolide-treated MRSP cultures induced decreased NF-κB activation in cells expressing Toll-like receptor 2 and nucleotide-binding oligomerization domain 2 compared to the supernatants from untreated MRSP cells, suggesting that macrolides inhibit the release of these ligands from MRSP. Real-time PCR analysis revealed that macrolides significantly downregulated the transcription of various genes encoding peptidoglycan synthesis-, lipoteichoic acid synthesis-, and lipoprotein synthesis-related molecules in MRSP cells. The silkworm larva plasma assay demonstrated that the peptidoglycan concentrations in the supernatants from macrolide-treated MRSP cultures were significantly lower than those from untreated MRSP cultures. Triton X-114 phase separation revealed that lipoprotein expression decreased in macrolide-treated MRSP cells compared to the lipoprotein expression in untreated MRSP cells. Consequently, macrolides may decrease the expression of bacterial ligands of innate immune receptors, resulting in the decreased proinflammatory activity of MRSP. IMPORTANCE To date, the clinical efficacy of macrolides in pneumococcal disease is assumed to be linked to their ability to inhibit the release of pneumolysin. However, our previous study demonstrated that oral administration of macrolides to mice intratracheally infected with macrolide-resistant Streptococcus pneumoniae resulted in decreased levels of pneumolysin and proinflammatory cytokines in bronchoalveolar lavage fluid samples compared to the levels in samples from untreated infected control mice, without affecting the bacterial load in the fluid. This finding suggests that additional mechanisms by which macrolides negatively regulate proinflammatory cytokine production may be involved in their efficacy in vivo. Furthermore, in this study, we demonstrated that macrolides downregulated the transcription of various proinflammatory-component-related genes in S. pneumoniae, which provides an additional explanation for the clinical benefits of macrolides.

The Pneumococcal Protein SufC Binds to Host Plasminogen and Promotes Its Conversion into Plasmin.

Streptococcus pneumoniae causes otitis media, sinusitis, and serious diseases such as pneumonia and bacteremia. However, the in vivo dynamics of S. pneumoniae infections and disease severity are not fully understood. In this study, we investigated pneumococcal proteins detected in the bronchoalveolar lavage fluid of an S. pneumoniae-infected mouse, which were assumed to be expressed during infection. Analysis of three proteins with unknown infection-related functions revealed that recombinant Fe-S cluster assembly ATP-binding protein (SufC) binds to the host plasminogen and promotes its conversion into plasmin. SufC was detected in the bacterial cell-surface protein fraction, but it had no extracellular secretory signal. This study suggests that S. pneumoniae releases SufC extracellularly through LytA-dependent autolysis, binding to the bacterial cell surface and host plasminogen and promoting its conversion into plasmin. The recruitment of plasmin by S. pneumoniae is considered useful for bacterial survival and spread, and SufC is suggested to facilitate this process.

Erythromycin Restores Osteoblast Differentiation and Osteogenesis Suppressed by Porphyromonas gingivalis Lipopolysaccharide.

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1, indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1.

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11.

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell-cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.

Mutants of Lactobacillus plantarum ML11-11 deficient in co-aggregation with yeast exhibited reduced activities of mixed-species biofilm formation.

Lactic acid bacteria (LAB) mutants deficient in inter-species co-aggregation with yeast were spontaneously derived from Lactobacillus plantarum ML11-11, a significant mixed-species biofilm former in static co-cultures with budding yeasts. These non-co-aggregative mutants also showed significant decreases in mixed-species biofilm formation. These results suggest the important role of co-aggregation between LAB and yeast in mixed-species biofilm formation. Cell surface proteins obtained by 5 M LiCl extraction from the wild-type cells and non-co-aggregative mutant cells were analyzed by SDS-PAGE. There was an obvious difference in protein profiles. The protein band at 30 kDa was present abundantly in the wild-type cell surface fraction but was significantly decreased in the mutant cells. This band assuredly corresponded to the LAB surface factors that contribute to co-aggregation with yeasts.

Glycine Induction Method: Effective Production of Immunoactive Bacterial Membrane Vesicles with Low Endotoxin Content.

Bacteria are known to release nanometer scale proteoliposomes termed bacterial membrane vesicles (MVs), and it is considered that native and bioengineered MVs would be applicable for development of acellular vaccines and novel drug delivery systems in medical settings. However, important considerations for manufacturing purposes include the varied productivity of MV among bacterial species and strains, as well as endotoxicity levels due to the lipopolysaccharide component. The method for MV induction using glycine described here is simple and provides a solution to these problems. Glycine weakens bacterial peptidoglycans and significantly increases bacterial MV formation, while the relative endotoxin activity of glycine-induced MVs is extremely reduced as compared to that of noninduced MVs. Nevertheless, glycine-induced MVs elicit strong immune responses at levels nearly equivalent to those of noninduced MVs. Taken together, the present method for induction by glycine is convenient for research studies of bacterial MVs and has potential for use in medical applications including vaccine development.

Scanning electron microscopy of Escherichia coli encapsulated in a spacerized graphene sandwich.

Electron microscopy of biological materials such as bacteria allows multifaceted analysis to understand their structure and function with high resolution, which is difficult to achieve with optical microscopy. However, the samples are damaged or broken by electron beam irradiation and by the vacuum environment. Here, we observed bacteria in a suspension encapsulated in a graphene sandwich that prevents electron beam damage without the need for fixation. Specifically, we demonstrated in situ scanning electron microscopy observation of Escherichia coli in a graphene sandwich containing a perforated membrane as a spacer, encapsulating non-immobilized E. coli between the graphene layers. However, E. coli activity, such as division, was not observed, although the irradiated cells grew slightly when resuspended under optimal culture conditions. Our findings suggest that the graphene sandwich methodology enables the observation of wet E. coli cells by electron microscopy but requires refinement to allow the live imaging of biological materials.