Natural photosensitizer-loaded in micellar copolymer to prevent bovine mastitis: A new post-dipping protocol on milking.

Good management practices such as post-dipping applications (post-milking immersion bath) contribute to the dairy cattle health during lactation and minimize the appearance of mastitis (an infection in the mammary gland). The post-dipping procedure is performed conventionally using iodine-based solutions. The search for therapeutic modalities that are not invasive and do not cause resistance to the microorganisms that cause bovine mastitis instigates the interest of the scientific community. In this regard, antimicrobial Photodynamic Therapy (aPDT) is highlighted. The aPDT is based on combining a photosensitizer (PS) compound, light of adequate wavelength, and molecular oxygen (3O2), which triggers a series of photophysical processes and photochemical reactions that generate reactive oxygen species (ROS) responsible for the inactivation of microorganisms. The present investigation explored the photodynamic efficiency of two natural PS: Chlorophyll-rich spinach extract (CHL) and Curcumin (CUR), both incorporated into the Pluronic® F127 micellar copolymer. They were applied in post-dipping procedures in two different experiments. The photoactivity of formulations mediated through aPDT was conducted against Staphylococcus aureus, and obtained a minimum inhibitory concentration (MIC) of 6.8 mg mL-1 for CHL-F127 and 0.25 mg mL-1 for CUR-F127. Only CUR-F127 inhibited Escherichia coli growth with MIC 0.50 mg mL-1. Concerning the count of microorganisms during the days of the application, a significant difference was observed between the treatments and control (Iodine) when the teat surface of cows was evaluated. For CHL-F127 there was a difference for Coliform and Staphylococcus (p < 0.05). For CUR-F127 there was a difference for aerobic mesophilic and Staphylococcus (p < 0.05). Such application decreased bacterial load and maintained the milk quality, being evaluated via total microorganism count, physical-chemical composition, and somatic cell count (SCC).

Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures.

To dissect the antibiotic role of nanostructures from chemical moieties belligerent to both bacterial and mammalian cells, here we show the antimicrobial activity and cytotoxicity of nanoparticle-pinched polymer brushes (NPPBs) consisting of chemically inert silica nanospheres of systematically varied diameters covalently grafted with hydrophilic polymer brushes that are non-toxic and non-bactericidal. Assembly of the hydrophilic polymers into nanostructured NPPBs doesn't alter their amicability with mammalian cells, but it incurs a transformation of their antimicrobial potential against bacteria, including clinical multidrug-resistant strains, that depends critically on the nanoparticle sizes. The acquired antimicrobial potency intensifies with small nanoparticles but subsides quickly with large ones. We identify a threshold size (dsilica ~ 50 nm) only beneath which NPPBs remodel bacteria-mimicking membrane into 2D columnar phase, the epitome of membrane pore formation. This study illuminates nanoengineering as a viable approach to develop nanoantibiotics that kill bacteria upon contact yet remain nontoxic when engulfed by mammalian cells.

Aptamer-Functionalized DNA-Silver Nanocluster Nanofilm for Visual Detection and Elimination of Bacteria.

As a new type of nanomaterial, DNA-templated silver nanoclusters (DNA-AgNCs) have been widely studied because of their fluorescence and antibacterial properties. In this study, we combined the DNA-AgNCs with aptamers of bacteria to achieve a novel approach for the visual detection and effective elimination of bacteria. The aptamers of Staphylococcus aureus (S. aureus) were linked to G-rich sequences to achieve fluorescence enhancement when approaching the DNA-AgNCs. The capture of aptamers not only realized the visual monitoring of bacteria but also promoted the antibacterial effects. Additionally, a fluorescent nanofilm with excellent selectivity and antibacterial activity in the detection and elimination of S. aureus was developed based on the DNA-AgNCs. These aptamer-functionalized DNA-AgNCs show significant potential for many applications in food packaging and biomedical engineering.

Temperature-sensitive poly(N-isopropylacrylamide)-chitosan hydrogel for fluorescence sensors in living cells and its antibacterial application.

It is meaningful and challenging to design and develop a fluorescent probe for living cell temperature sensors since it should have good cell compatibility and high-resolution features. In this work, the temperature-sensitive polymer of PA-loaded cysteine (Cys) modified chitosan (Cs) grafted PNIPAM (Cs-Cys-PN/PA) with aggregation-induced emission enhancement (AIEE) properties that reversible hydrogel in an aqueous solution is synthesized. Here, we interpret the temperature stimulus as a monochromatic signal through the AIEE active reversible hydrogel of Cs-Cys-PN. In addition, the cytotoxicity test shown that Cs-Cys-PN has good biocompatibility. Cs-Cys-PN can be used to build antibacterial drugs carrier, thereby providing a new platform of self-released drugs for the treatment of bacterial infections.

Comparison of the Phytochemical Composition and Antibacterial Activities of the Various Extracts from Leaves and Twigs of Illicium verum.

Previous studies have revealed the numerous biological activities of the fruits of Illicium verum; however, the activities of its leaves and twigs have remained undiscovered. The study aimed to investigate the phytochemical components and antibacterial activity of the various extracts from the leaves and twigs of Illicium verum. The herbal extracts were prepared by supercritical CO2 extraction (SFE) and 95% ethanol extraction, followed by partition extraction based on solvent polarity. Analysis of antimicrobial activity was conducted through the usage of nine clinical antibiotic- resistant isolates, including Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. Among the tested samples, the SFE extracts exhibited broader and stronger antibacterial activities against the test strains, with a range of MIC between 0.1-4.0 mg/mL and MBC between 0.2-4.5 mg/mL. Observations made through scanning electron microscopy revealed potential mechanism of the antimicrobial activities involved disruption of membrane integrity of the test pathogens. Evaluation of the chemical composition by gas chromatography-mass spectrometry indicated the presence of anethole, anisyl aldehyde, anisyl acetone and anisyl alcohol within the SFE extracts, demonstrating significant correlations with the antibacterial activities observed. Therefore, the leaves and twigs of Illicium verum hold great potential in being developed as new natural antibacterial agents.

Antimicrobial Activities of LL-37 Fragment Mutant-Poly (Lactic-Co-Glycolic) Acid Conjugate against Staphylococcus aureus, Escherichia coli, and Candida albicans.

Various peptides and their derivatives have been reported to exhibit antimicrobial activities. Although these activities have been examined against microorganisms, novel methods have recently emerged for conjugation of the biomaterials to improve their activities. Here, we prepared CKR12-PLGA, in which CKR12 (a mutated fragment of human cathelicidin peptide, LL-37) was conjugated with poly (lactic-co-glycolic) acid (PLGA), and compared the antimicrobial and antifungal activities of the conjugated peptide with those of FK13 (a small fragment of LL-37) and CKR12 alone. The prepared CKR12-PLGA was characterized by dynamic light scattering and measurement of the zeta potential, critical micellar concentration, and antimicrobial activities of the fragments and conjugate. Although CKR12 showed higher antibacterial activities than FK13 against Staphylococcus aureus and Escherichia coli, the antifungal activity of CKR12 was lower than that of FK13. CKR12-PLGA showed higher antibacterial activities against S. aureus and E. coli and higher antifungal activity against Candida albicans compared to those of FK13. Additionally, CKR12-PLGA showed no hemolytic activity in erythrocytes, and scanning and transmission electron microscopy suggested that CKR12-PLGA killed and disrupted the surface structure of microbial cells. Conjugation of antimicrobial peptide fragment analogues was a successful approach for obtaining increased microbial activity with minimized cytotoxicity.

The Co-Culture of Staphylococcal Biofilm and Fibroblast Cell Line: The Correlation of Biological Phenomena with Metabolic NMR1 Footprint.

Staphylococcus aureus is one of the most prevalent pathogens associated with several types of biofilm-based infections, including infections of chronic wounds. Mature staphylococcal biofilm is extremely hard to eradicate from a wound and displays a high tendency to induce recurring infections. Therefore, in the present study, we aimed to investigate in vitro the interaction between S. aureus biofilm and fibroblast cells searching for metabolites that could be considered as potential biomarkers of critical colonization and infection. Utilizing advanced microscopy and microbiological methods to examine biofilm formation and the staphylococcal infection process, we were able to distinguish 4 phases of biofilm development. The analysis of staphylococcal biofilm influence on the viability of fibroblasts allowed us to pinpoint the moment of critical colonization-12 h post contamination. Based on the obtained model we performed a metabolomics analysis by 1H NMR spectroscopy to provide new insights into the pathophysiology of infection. We identified a set of metabolites related to the switch to anaerobic metabolism that was characteristic for staphylococcal biofilm co-cultured with fibroblast cells. The data presented in this study may be thus considered a noteworthy but preliminary step in the direction of developing a new, NMR-based tool for rapid diagnosing of infection in a chronic wound.

Accelerated antimicrobial discovery via deep generative models and molecular dynamics simulations.

The de novo design of antimicrobial therapeutics involves the exploration of a vast chemical repertoire to find compounds with broad-spectrum potency and low toxicity. Here, we report an efficient computational method for the generation of antimicrobials with desired attributes. The method leverages guidance from classifiers trained on an informative latent space of molecules modelled using a deep generative autoencoder, and screens the generated molecules using deep-learning classifiers as well as physicochemical features derived from high-throughput molecular dynamics simulations. Within 48 days, we identified, synthesized and experimentally tested 20 candidate antimicrobial peptides, of which two displayed high potency against diverse Gram-positive and Gram-negative pathogens (including multidrug-resistant Klebsiella pneumoniae) and a low propensity to induce drug resistance in Escherichia coli. Both peptides have low toxicity, as validated in vitro and in mice. We also show using live-cell confocal imaging that the bactericidal mode of action of the peptides involves the formation of membrane pores. The combination of deep learning and molecular dynamics may accelerate the discovery of potent and selective broad-spectrum antimicrobials.

The Effects of Luminescent CdSe Quantum Dot-Functionalized Antimicrobial Peptides Nanoparticles on Antibacterial Activity and Molecular Mechanism.

BACKGROUND: With the development of bacterial resistance, the range of effective antibiotics is increasingly becoming more limited. The effective use of nanoscale antimicrobial peptides (AP) in therapeutic and diagnostic methods is a strategy for new antibiotics. METHODS: Combining both AP and cadmium selenide (CdSe) into a composite material may result in a reagent with novel properties, such as enhanced antibacterial activity, fluorescence and favorable stability in aqueous solution. RESULTS: AP-loaded CdSe NPs (AP-CdSe NPs) showed strong antibacterial activity against multidrug-resistant (MDR) Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) in vitro and in vivo. Colony-forming unit (CFU) and minimum inhibitory concentration (MIC) assays showed that AP-CdSe NPs have highly effective antibacterial activity. The quantitative analysis of apoptosis by flow cytometry analysis further confirmed that MDR E. coli and S. aureus treated with AP-CdSe NPs had death rates of 98.76% and 99.13%, respectively. Also, AP-CdSe NPs was found to inhibit bacterial activity in an in vivo bacteremia model in mice infected with S. aureus. In addition, the antibacterial mechanism of AP-CdSe NPs was determined by RNA sequencing analysis. Gene ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis revealed the molecular mechanism of the antibacterial effect of AP-CdSe NPs. Importantly, histopathology analysis, and hematological toxicity analysis indicated that AP-CdSe NPs had few side effects. CONCLUSION: These results demonstrate that AP loaded on CdSe NPs had a higher water solubility, bioavailability and antibacterial effect compared with raw AP. This study reports findings that are helpful for the design and development of antibacterial treatment strategies based on AP.

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA).

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Manipulation of Autophagy and Apoptosis Facilitates Intracellular Survival of Staphylococcus aureus in Human Neutrophils.

Polymorphonuclear neutrophils (PMN) are critical for first line innate immune defence against Staphylococcus aureus. Mature circulating PMN maintain a short half-life ending in constitutive apoptotic cell death. This makes them unlikely candidates as a bacterial intracellular niche. However, there is significant evidence to suggest that S. aureus can survive intracellularly within PMN and this contributes to persistence and dissemination during infection. The precise mechanism by which S. aureus parasitizes these cells remains to be established. Herein we propose a novel mechanism by which S. aureus subverts both autophagy and apoptosis in PMN in order to maintain an intracellular survival niche during infection. Intracellular survival of S. aureus within primary human PMN was associated with an accumulation of the autophagic flux markers LC3-II and p62, while inhibition of the autophagy pathway led to a significant reduction in intracellular survival of bacteria. This intracellular survival of S. aureus was coupled with a delay in neutrophil apoptosis as well as increased expression of several anti-apoptotic factors. Importantly, blocking autophagy in infected PMN partially restored levels of apoptosis to that of uninfected PMN, suggesting a connection between the autophagic and apoptotic pathways during intracellular survival. These results provide a novel mechanism for S. aureus intracellular survival and suggest that S. aureus may be subverting crosstalk between the autophagic and apoptosis pathways in order to maintain an intracellular niche within human PMN.

Functional Identification of Serine Hydroxymethyltransferase as a Key Gene Involved in Lysostaphin Resistance and Virulence Potential of Staphylococcus aureus Strains.

Gaining an insight into the mechanism underlying antimicrobial-resistance development in Staphylococcus aureus is crucial for identifying effective antimicrobials. We isolated S. aureus sequence type 72 from a patient in whom the S. aureus infection was highly resistant to various antibiotics and lysostaphin, but no known resistance mechanisms could explain the mechanism of lysostaphin resistance. Genome-sequencing followed by subtractive and functional genomics revealed that serine hydroxymethyltransferase (glyA or shmT gene) plays a key role in lysostaphin resistance. Serine hydroxymethyltransferase (SHMT) is indispensable for the one-carbon metabolism of serine/glycine interconversion and is linked to folate metabolism. Functional studies revealed the involvement of SHMT in lysostaphin resistance, as ΔshmT was susceptible to the lysostaphin, while complementation of the knockout expressing shmT restored resistance against lysostaphin. In addition, the ΔshmT showed reduced virulence under in vitro (mammalian cell lines infection) and in vivo (wax-worm infection) models. The SHMT inhibitor, serine hydroxymethyltransferase inhibitor 1 (SHIN1), protected the 50% of the wax-worm infected with wild type S. aureus. These results suggest SHMT is relevant to the extreme susceptibility to lysostaphin and the host immune system. Thus, the current study established that SHMT plays a key role in lysostaphin resistance development and in determining the virulence potential of multiple drug-resistant S. aureus.

Preparation of Celery Essential Oil-Based Nanoemulsion by Ultrasonication and Evaluation of Its Potential Anticancer and Antibacterial Activity.

INTRODUCTION: Plants have always been a significant source of natural active components with biological properties. Celery seed oil (extracted from Apium graveolens) has several potential applications, but its therapeutic uses in the form of nanoemulsion formulation need to be investigated further in order to meet the demand in cancer treatment, and to alleviate the prevailing crisis arising from increased antimicrobial resistance. METHODS: The therapeutic potential of celery seed oil was investigated through the formulation and testing of a nanoemulsion developed with Tween 80 (a non-ionic surfactant) and the utilization of an ultrasonication technique. Anticancer and apoptotic properties of the formulation were evaluated through MTT and Annexin V-FITC assays. The clonogenic assay aided in the identification of the antiproliferative properties of the formulation on oral squamous cell carcinoma. The antimicrobial study was supported by agar well diffusion assay, membrane integrity test and scanning electron microscopy. RESULTS: Experiments identified relevant parameters, including optimal surfactant concentration and emulsification time. GC-MS analysis identified various components in the celery oil, but not their biological activities. A sonication time of 20 min resulted in a droplet diameter of 23.4 ± 1.80 nm. The IC50 concentration of the optimal nanoemulsion formulation against SAS cells was 1.4 µL/mL. At this concentration, cell proliferation was significantly reduced through inhibition of the anchorage-independent cell growth by disrupting colony formation and inducing cell death (apoptosis) of cancer cells. The nanoemulsion was also treated with a microbial suspension of S. aureus, and displayed antibacterial properties through lipid membrane fusion, causing cytoplasmic leakage as verified through agar well diffusion and membrane permeability assays. Scanning electron microscopy revealed complete distortion of the bacterial pathogen. CONCLUSION: The results in this study present celery as a possible constituent for cancer therapeutics and as a candidate for aggressive, yet safe cancer treatment. The celery-based nanoemulsion has the potential to act as a key alternative to standard antibiotic therapy.

Porous poly(lactic acid) based fibres as drug carriers in active dressings.

PURPOSE: The polymeric porous surface of fibres (PLA) may influence the kinetics of release of biologically active compounds (gentamicin, G and ethacridine lactate, R) affecting development of a bacterial biofilm. METHODS: The porous fibres with different morphology were manufactured by the electrospinning method from ternary systems composed of PLA and selected solvents. Fibres morphology was examined using a scanning electron microscopy (SEM), their structure was analyzed by FT-IR ATR spectroscopy and differential scanning calorimetry (DSC). Changes in the drug release profile were measured using ICP/UV-Vis methods and the resulting bactericidal or bacteriostatic properties were tested by two-layer disk diffusion test in relation to various drug incorporation methods. RESULTS: The porous fibres can be applied to produce drug-bearing membranes. The spectroscopic studies confirmed incorporation of gentamicin into the fibres and the presence of ethacridine lactate on their surface. Bimodal fibres distribution (P3) promoted faster release of gentamicin and ethacridine lactate from P3G and P3R materials. The electrospinning process coupled with the vapor induced phase separation influenced the glass transition temperature of the porous polymer fibres. The pre/post-electrospinning modification influenced the glass transition, maximum temperature of cold crystallization and melting point of the porous membrane, compared to the neat polymer. The polylactide fibres with gentamicin showed strong bactericidal effect on Gram-positive bacteria, while fibres with ethacridine lactate were bacteriostatic. CONCLUSIONS: The obtained fibres with complex surface morphology can be used as a membrane in active dressings as they make it possible to control the release profile of the active compounds.

Machine learning-driven electronic identifications of single pathogenic bacteria.

A rapid method for screening pathogens can revolutionize health care by enabling infection control through medication before symptom. Here we report on label-free single-cell identifications of clinically-important pathogenic bacteria by using a polymer-integrated low thickness-to-diameter aspect ratio pore and machine learning-driven resistive pulse analyses. A high-spatiotemporal resolution of this electrical sensor enabled to observe galvanotactic response intrinsic to the microbes during their translocation. We demonstrated discrimination of the cellular motility via signal pattern classifications in a high-dimensional feature space. As the detection-to-decision can be completed within milliseconds, the present technique may be used for real-time screening of pathogenic bacteria for environmental and medical applications.

Bacterial aggregate size determines phagocytosis efficiency of polymorphonuclear leukocytes.

The ability of bacteria to aggregate and form biofilms impairs phagocytosis by polymorphonuclear leukocytes (PMNs). The aim of this study was to examine if the size of aggregates is critical for successful phagocytosis and how bacterial biofilms evade phagocytosis. We investigated the live interaction between PMNs and Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Staphylococcus epidermidis using confocal scanning laser microscopy. Aggregate size significantly affected phagocytosis outcome and larger aggregates were less likely to be phagocytized. Aggregates of S. epidermidis were also less likely to be phagocytized than equally-sized aggregates of the other three species. We found that only aggregates of approx. 5 µm diameter or smaller were consistently phagocytosed. We demonstrate that planktonic and aggregated cells of all four species significantly reduced the viability of PMNs after 4 h of incubation. Our results indicate that larger bacterial aggregates are less likely to be phagocytosed by PMNs and we propose that, if the aggregates become too large, circulating PMNs may not be able to phagocytose them quickly enough, which may lead to chronic infection.

Cryo-EM structure of the ribosome functional complex of the human pathogen Staphylococcus aureus at 3.2 Å resolution.

Staphylococcus aureus is a bacterial pathogen and one of the leading causes of healthcare-acquired infections in the world. The growing antibiotic resistance of S. aureus obliges us to search for new drugs and treatments. As the majority of antibiotics target the ribosome, knowledge of its detailed structure is crucial for drug development. Here, we report the cryo-EM reconstruction at 3.2 Å resolution of the S. aureus ribosome with P-site tRNA, messenger RNA, and 10 RNA modification sites previously not assigned or visualized. The resulting model is the most precise and complete high-resolution structure to date of the S. aureus 70S ribosome with functional ligands.

Distinct oligomeric structures of the YoeB-YefM complex provide insights into the conditional cooperativity of type II toxin-antitoxin system.

YoeB-YefM, the widespread type II toxin-antitoxin (TA) module, binds to its own promoter to autoregulate its transcription: repress or induce transcription under normal or stress conditions, respectively. It remains unclear how YoeB-YefM regulates its transcription depending on the YoeB to YefM TA ratio. We find that YoeB-YefM complex from S.aureus exists as two distinct oligomeric assemblies: heterotetramer (YoeB-YefM2-YoeB) and heterohexamer (YoeB-YefM2-YefM2-YoeB) with low and high DNA-binding affinities, respectively. Structures of the heterotetramer alone and heterohexamer bound to promoter DNA reveals that YefM C-terminal domain undergoes disorder to order transition upon YoeB binding, which allosterically affects the conformation of N-terminal DNA-binding domain. At TA ratio of 1:2, unsaturated binding of YoeB to the C-terminal regions of YefM dimer forms an optimal heterohexamer for DNA binding, and two YefM dimers with N-terminal domains dock into the adjacent major grooves of DNA to specifically recognize the 5'-TTGTACAN6AGTACAA-3' palindromic sequence, resulting in transcriptional repression. In contrast, at TA ratio of 1:1, binding of two additional YoeB molecules onto the heterohexamer induces the completely ordered conformation of YefM and disassembles the heterohexamer into two heterotetramers, which are unable to bind the promoter DNA optimally due to steric clashes, hence derepresses TA operon transcription.

The effect of Staphylococcus aureus on the electrochemical behavior of porous Ti-6Al-4V alloy.

Ti-6Al-4V alloy has been widely investigated for biomedical applications due to its low density, high specific strength, and favorable corrosion resistance. However, some reported failures have imposed a challenge to improve bone regeneration and fixation, as well as antibacterial properties. A further opportunity for solving this problem is the introduction of porosity. However, this can induce metallic release and corrosion product formation. In this work, a Ti-6Al-4V alloy was exposed to Hank's solution, sterilized and inoculated with Staphylococcus aureus at 37 °C. Surface analysis was carried out by SEM-EDS and XPS. Electrochemical measurements were also performed using chronopotentiometry at open circuit potential, polarization curves, and electrochemical impedance spectroscopy. After exposure, FE-SEM showed some colonies of S. aureus on the sample with 22% porosity. However, XPS analysis revealed that the presence of bacterium influenced the composition of the oxide layer, even more drastically with the increase in added porosity. Moreover, the impedance analysis showed De Levie's behavior, revealing a reduction of pore resistance and modulus of the impedance in the low frequency range in inoculated medium, and polarization curves showed that the passivity potential range was decreased, whereas the passivity current increased in the presence of the S. aureus.

The architecture of the Gram-positive bacterial cell wall.

The primary structural component of the bacterial cell wall is peptidoglycan, which is essential for viability and the synthesis of which is the target for crucial antibiotics1,2. Peptidoglycan is a single macromolecule made of glycan chains crosslinked by peptide side branches that surrounds the cell, acting as a constraint to internal turgor1,3. In Gram-positive bacteria, peptidoglycan is tens of nanometres thick, generally portrayed as a homogeneous structure that provides mechanical strength4-6. Here we applied atomic force microscopy7-12 to interrogate the morphologically distinct Staphylococcus aureus and Bacillus subtilis species, using live cells and purified peptidoglycan. The mature surface of live cells is characterized by a landscape of large (up to 60 nm in diameter), deep (up to 23 nm) pores constituting a disordered gel of peptidoglycan. The inner peptidoglycan surface, consisting of more nascent material, is much denser, with glycan strand spacing typically less than 7 nm. The inner surface architecture is location dependent; the cylinder of B. subtilis has dense circumferential orientation, while in S. aureus and division septa for both species, peptidoglycan is dense but randomly oriented. Revealing the molecular architecture of the cell envelope frames our understanding of its mechanical properties and role as the environmental interface13,14, providing information complementary to traditional structural biology approaches.