Laboratory Media Research Articles

Cystic fibrosis sputum media induces an overall loss of antibiotic susceptibility in Mycobacterium abscessus

AbstractMycobacterium abscessus complex (MABSC) comprises a group of environmental microorganisms, which are a concerning cause of opportunistic respiratory infections in patients with cystic fibrosis or bronchiectasis. Only 45.6% of MABSC treatments are successful, and therefore this is a need to discover new antimicrobials that can treat these pathogens. However, the transferability of outcomes to the clinic is flawed by an inability to accurately represent the lung environment within the laboratory. Herein, we apply two preestablished formulations of sputum media (ACFS and SCFM1) to MABSC antibiotic susceptibility testing. Using conventional broth microdilution, we have observed strain and antibiotic dependent alterations in antimicrobial sensitivity in each sputum media compared standard laboratory media (7H9), with an overall reduction in susceptibility within the physiologically relevant conditions. We provide a timely contribution to the field of M. abscessus antibiotic discovery by emphasising the need for improved physiological relevance.

Physical characterization of agar-based biodegradable films derived from nonhazardous laboratory waste

Globally, approximately 2.12 billion tons of waste are annually disposed of, with laboratories significantly contributing across diverse waste streams. Effective waste management strategies are crucial to mitigate environmental impact and promote sustainability within scientific communities. This study addresses the challenges by introducing a novel method that transforms laboratory media waste into a valuable biopolymer named “Agastic.” The process involves repurposing agar extracted from bulk laboratory waste, blending it with bio-based plasticizers to produce Agastic sheets exhibiting mechanical properties comparable to traditional bioplastics. Using response surface methodology (RSM) and central composite design (CCD), optimal concentrations of agar (1.5–2.5% w/v), glycerol (0.25–1% v/v), and ethanolamine (0.5–1.5% v/v) were determined. Predictions from Design Expert software indicated impressive tensile strength up to 14.31 MPa for AGA-1 and elongation at break up to 52% for AGA-2. Fourier Transform Infrared Spectroscopy (FTIR) analysis confirmed agarose structural features in AGA-1 and AGA-2. Thermogravimetric analysis (TGA) showed polysaccharide-related breakdown between 38°C and 280°C in AGA-1, peaking at 299.36°C; AGA-2 exhibited diverse thermal decomposition up to 765°C, suggesting their biodegradable potential in packaging applications. Scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) analysis confirmed nontoxic nature of Agastic and preserved morphological integrity in both samples. Soil degradation studies revealed AGA-1 and AGA-2 losing 71.31% and 70.88% of weight, respectively, over 15 days. This innovation provides a sustainable pathway to repurpose laboratory waste into eco-friendly bioplastics, particularly suitable for moisture-sensitive packaging such as nursery applications. These findings underscore Agastic films’ promise as environmentally friendly alternatives to traditional plastics, supporting circular bioeconomy principles and significantly reducing ecological impacts associated with plastic waste.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Transposon mutagenesis screen in Klebsiella pneumoniae identifies genetic determinants required for growth in human urine and serum.

Klebsiella pneumoniae is a global public health concern due to the rising myriad of hypervirulent and multidrug-resistant clones both alarmingly associated with high mortality. The molecular mechanisms underpinning these recalcitrant K. pneumoniae infection, and how virulence is coupled with the emergence of lineages resistant to nearly all present-day clinically important antimicrobials, are unclear. In this study, we performed a genome-wide screen in K. pneumoniae ECL8, a member of the endemic K2-ST375 pathotype most often reported in Asia, to define genes essential for growth in a nutrient-rich laboratory medium (Luria-Bertani [LB] medium), human urine, and serum. Through transposon directed insertion-site sequencing (TraDIS), a total of 427 genes were identified as essential for growth on LB agar, whereas transposon insertions in 11 and 144 genes decreased fitness for growth in either urine or serum, respectively. These studies not only provide further knowledge on the genetics of this pathogen but also provide a strong impetus for discovering new antimicrobial targets to improve current therapeutic options for K. pneumoniae infections.

Effectiveness of organic acids for inactivating pathogenic bacteria inoculated in laboratory media and foods: an updated minireview.

Food processing industries commonly employ organic acids (OAAs) to determine bacterial contamination in acidified and fermented foods. OAAs are believed to possess potent antimicrobial properties by permeating cell membranes, altering proton and anion concentrations in the cytoplasm due to their lipophilic undissociated forms. The bacteriostatic or bactericidal effects of OAAs are influenced by various factors including microbial physiology, environmental pH, and acid dissociation ratios. Despite their utility, the precise mechanisms underlying OAA-mediated inhibition of pathogenic bacteria remain incompletely understood. Therefore, the objectives of this review are to compile a selected area of researches that focus on the current propensity of different OAAs for inactivating food-borne pathogens, and then to present a theoretical insight on the use of OAAs to prevent and control pathogenic bacteria present in acidic/acidified foods and their mode of mechanisms.

Growth fitness, virulence, and heat tolerance of Salmonella Typhimurium variants resistant to food preservation methods

To study potential ramifications of antimicrobial resistance, we carried out adaptive laboratory evolution assays (ALE) to isolate three resistant variants (RVs) of Salmonella enterica Typhimurium, employing three different types of food preservation methods: 1) an emergent technology, plasma-activated water (PAW), leading to variant RV-PAW; a traditional method, heat, leading to variant RV-HT, and a natural antimicrobial compound, carvacrol, leading to variant RV-CAR. The variant resistant to plasma-activated water, RV-PAW, had mutations in rpoA and rpoD; it showed increased tolerance to heat in orange juice but ultimately did not pose a significant threat, as it exhibited a fitness cost at refrigeration temperature (8 °C), whereas its virulence against Caenorhabditis elegans decreased. The variant resistant to heat, RV-HT, had mutations in flhC, dnaJ: it exhibited a fitness cost at high growth temperatures (43 °C) and induced morphofunctional alterations in C. elegans. The variant resistant to carvacrol, RV-CAR, had mutations in sseG, flhA, wbaV, lon; this variant not only exhibited significantly higher thermotolerance in both laboratory media and food models but also effectively increased its growth fitness at refrigeration temperatures while retaining its virulence, evidenced by the highest percentage of Smurf phenotype in C. elegans.To address these challenges, we applied a process combining thermal treatment with citral, with the aim of leveraging the sublethal damage caused in RVs by heat treatments in orange juice. This approach achieves enhanced microbial inactivation without having to escalate the intensity of the thermal treatment. The result was particularly encouraging in the case of RV-CAR, the most challenging strain, for which we improved lethality by up to 3 log10 inactivation cycles.

Evaluation of phenotypic and genotypic methods for the identification and characterization of bacterial isolates recovered from catheter-associated urinary tract infections.

Urinary tract infections are the most common hospital-acquired infection, 80% of which are associated with catheterization. Diagnostic methods may influence the reported identities of these pathogens, and phenotypic testing under laboratory conditions may not reflect infection phenotypes. This study aimed to evaluate the efficacy of diagnostic methods and whether medium composition alters phenotypes by characterizing catheter-associated urinary tract infection isolates from a UK hospital. We compared five bacterial identification methods, including biochemical testing, matrix-assisted laser desorption/ionization biotyping, and genome sequencing, finding differences in genus- or species-level identifications. Antibiotic susceptibility comparisons between phenotypic assays and genomic predictions showed high agreement only in multidrug-resistant strains. To determine whether growth rate and biofilm formation were affected by medium composition, strains were grown in both planktonic and biofilm states. Low planktonic growth and significant biofilm formation were observed in artificial urine compared to rich laboratory media, underscoring the importance of assay design. This study highlights the risks of relying on a single diagnostic method for species identification, advocating for whole-genome sequencing for accuracy. It emphasizes the continued importance of phenotypic methods in understanding antibiotic resistance in clinical settings and the need for characterization conditions that mirror those encountered by pathogens in the body.

Competitive fitness of asymptomatic bacteriuria E. coli strain 83972 against uropathogens in human urine.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. The main causative agent of UTI is uropathogenic Escherichia coli (UPEC). There is an immediate need for novel prophylactic and treatment strategies against UTI because of the increasing incidence of antimicrobial resistance among uropathogens. ABU 83972, an asymptomatic bacteriuria-causing E. coli strain, prevents UTI by suppressing the colonization of UPEC. However, the nature of competition and growth repression of UPEC by ABU 83972 is unclear and is the subject of our investigation. Here, we characterized the growth kinetics of ABU 83972 and uropathogens in human urine and laboratory media. Next, we performed a series of competitive co-culture experiments where ABU 83972 and uropathogens were inoculated at a 1:1 ratio in human urine and in various media, and their relative abundance was determined. In human urine, ABU 83972 outcompeted UPEC and additional uropathogens, reaching up to 90% of the total population after 24 hours of incubation. In contrast, UPEC outcompeted ABU 83972 in LB and M9 minimal media and exhibited superior colonization than ABU 83972 in the mouse urinary bladder. Since engineered living materials (ELMs) can be used to retain an organism of interest in a particular location, we developed ABU 83972-containing ELMs that effectively outcompeted UPEC in human urine. In summary, our work establishes that ABU 83972 outcompetes UPEC in a milieu- and cell-density-dependent manner, highlighting the importance of the metabolites and nutrients found in the human urine as determinants of the competitive fitness of ABU 83972.

Up-regulation of Retrograde Response in yeast increases glycerol and reduces ethanol during wine fermentation

Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are key factors for the biotechnological success of the yeast Saccharomyces cerevisiae during food-producing fermentations. One such pathway is Retrograde Response, which controls the alpha-ketoglutarate supply required for the synthesis of amino acids like glutamate and lysine. Repressor MKS1 is linked with the TORC1 complex and negatively regulates this pathway. Deleting MKS1 from a variety of industrial strains causes glycerol to increase during winemaking, brewing and baking. This increase is accompanied by a reduction in ethanol production during grape juice fermentation in four commercial wine strains. Interestingly, this does not lead volatile acidity to increase because acetic acid levels actually lower. Aeration during winemaking usually increases acetic acid levels, but this effect reduces in the MKS1 mutant. Despite the improvement in the metabolites of oenological interest, it comes at a cost given that the mutant shows slower fermentation kinetics when grown in grape juice, malt and laboratory media and using glucose, sucrose and maltose as carbon sources. The deletion of RTG2, an activator of Retrograde Response that acts as an antagonist of MKS1, also results in a defect in wine fermentation speed. These findings suggest that the deregulation of this pathway causes a fitness defect. Therefore, manipulating repressor MKS1 is a promising approach to modulate yeast metabolism and to produce low-ethanol drinks.

Dormancy-like Phenotype of Aggregatibacter actinomycetemcomitans: Survival during Famine.

Microbes frequently experience nutrient deprivations in the natural environment and may enter dormancy. Aggregatibacter actinomycetemcomitans is known to establish long-term infections in humans. This study examined the dormancy-like phenotype of an A. actinomycetemcomitans strain D7S-1 and its isogenic smooth-colony mutant D7SS. A tissue culture medium RPMI-1640 was nutrient-deficient (ND) and unable to support A. actinomycetemcomitans growth. RPMI-1640 amended with bases was nutrient-limited (NL) and supported limited growth of A. actinomycetemcomitans less than the nutrient-enriched (NE) laboratory medium did. Strain D7S-1, after an initial 2-log reduction in viability, maintained viability from day 4 to day 15 in the NL medium. Strain D7SS, after 1-log reduction in viability, maintained viability from day 3 to day 5. In contrast, bacteria in the NE medium were either non-recoverable (D7S-1; >6-log reduction) or continued to lose viability (D7SS; 3-log reduction) on day 5 and beyond. Scanning and transmission electron microscopy showed that A. actinomycetemcomitans in the NL medium formed robust biofilms similar to those in the NE medium but with evidence of stress. A. actinomycetemcomitans in the ND medium revealed scant biofilms and extensive cellular damage. We concluded that A. actinomycetemcomitans grown in the NL medium exhibited a dormancy-like phenotype characterized by minimum growth, prolonged viability, and distinct cellular morphology.

Inactivation of Listeria monocytogenes on a laboratory medium and enoki mushrooms using organic acid and essential oil vapors

The aim of this study was to determine the antimicrobial activity of organic acid (OA) and essential oil (EO) vapors against Listeria monocytogenes on a laboratory medium and enoki mushrooms. Among 14 OA and 8 EO vapors tested, 3 OA vapors (acetic, formic, and propionic acid) and 3 EO vapors (cinnamon bark, citronella, and lemongrass) showed relatively strong antilisterial activity. On tryptic soy agar (TSA), all three OA vapors had a minimum inhibitory concentration (MIC) of 12.5 mg/L, while the minimum lethal concentration (MLC) of formic acid vapor was the lowest, at 50 mg/L, followed by acetic acid vapor (200 mg/L) and propionic acid vapor (400 mg/L). The MIC values of lemongrass, cinnamon bark, and citronella EO vapors were 25, 100, and 100 mg/L, respectively. The MLC of lemongrass EO vapor was 100 mg/L and MLCs of other EO vapors were >400 mg/L. Formic acid and lemongrass EO vapors showed a partial synergism on TSA (fractional inhibitory concentration index of 0.7500). For L. monocytogenes inoculated on enoki mushrooms, formic acid vapor at a concentration of ≥50 mg/L, as well as formic acid and lemongrass vapors in combination (1:2 vol ratio) at ≥ 75 mg/L, inactivated L. monocytogenes. These results indicate that formic acid vapor is effective at controlling the growth of L. monocytogenes on enoki mushrooms.

Bacteriological Profile of Common Lab Contaminants Responsible for Spoilage in Biological Laboratory

In biological laboratories, contamination can arise from various sources, including airborne particles, equipment, reagents, and human error. Common contaminants responsible for spoilage in biological laboratories include bacteria, fungi, virus etc. Bacteria are ubiquitous in the environment and can contaminate laboratory samples, cultures, and media. There is a possibility of transferring microorganisms from one laboratory to the other during the course of activities that involves moving of materials. In this present work we had been identify three bacterial species like Staphylococcus aureus, Klebsiella pneumonia, Enterococcus Sp. which responsible for lab spoilage. Therefore measures like cleaning of lab, using protective gears, autoclaving of glassware needed to minimize lab contamination.

Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.

Bacterial proteome adaptation during fermentation in dairy environments

The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.

Phenotypic and genotypic characterization of Campylobacter coli isolates from the Vietnamese poultry production network; a pilot study

IntroductionChanging farming practices and the associated increase in the use of antibiotics are amongst the main drivers shaping the global increase of Campylobacter infections. The effects farming practices have on Campylobacter species, need to be studied at the global scale, particularly in emerging middle-income countries, where the demand for low-cost poultry meat is rising. While Campylobacter jejuni causes the majority of poultry associated diarrhoea, Campylobacter coli causes a significant amount of disease but are relatively understudied. In this study we characterised seven C. coli strains isolated from chicken faeces and chicken meat in Thai Nguyen province, Vietnam.MethodsWhole Genome Sequencing and phenotypic assays (growth, motility, antimicrobial resistance testing, virulence assay) were performed to reveal the genetic relatedness and pathophysiological characteristics of the isolates. ResultsAll isolates were resistant to ciprofloxacin and nalidixic acid but susceptible to phenicols. Three were resistant to macrolides azithromycin and erythromycin. Six isolates were classified as multi-drug resistant. All isolates had similar growth rates in laboratory culture media, while five were hyper-motile. Lethality towards a tractable host-model system, larvae of the greater wax moth Galleria mellonella, often used to determine Campylobacter virulence, was demonstrated for the first time for C. coli. DiscussionMultilocus sequence typing data identified five ST’s all within the C. coli ST-828 clonal complex and were previously reported in North American (ST-829), European (ST-1586), and Asia (ST-5511) from patients suffering from gastroenteritis, emphasising the global spread of these strains. This work highlights the importance of further research into this understudied global threat.

Evalution of Using Virtual Laboratory Media to Learning Geometry in Mathematic Programe Open University

The current condition of the virtual laboratory is developing so rapidly, one indicator is the number of Mathematics students in open university who are looking for references related to practical geometry courses via online between lectures, this raises concern as a lecturer in adding value to lectures, which are adapted to the 21st century learning era today by creating virtual laboratories based on Virtual Reality, research methods using the ADDIE R & D model (Analysis, Design, Develop, Implementation and Evaluation), the science virtual lab product has not been developed much in Indonesia, another thing that makes the appeal of this virtual lab product is that it is able to display virtual reality which can increase motivation and learning outcomes. Student, based on the validation of media experts and material experts on the virtual geometry lab product, it scores 91 and 94 meaning that the virtual geometry lab product is very suitable for use, then more than 90% of lecturers and students at the Open University are very happy to use it. This research has shown that using virtual geometry labs in geometry learning at the Open University shows that all students show enthusiasm in using virtual geometry lab products as a supplement to geometry learning inside and outside the classroom. This was shown that after limited testing of virtual geometry lab products in the open university mathematics education study program, it showed that the majority had completed B+ or a score above 85 in geometry learning.

Active pH regulation facilitates Bacillus subtilis biofilm development in a minimally buffered environment.

Biofilms provide individual bacteria with many advantages, yet dense cellular proliferation can also create intrinsic metabolic challenges including excessive acidification. Because such pH stress can be masked in buffered laboratory media-such as MSgg commonly used to study Bacillus subtilis biofilms-it is not always clear how such biofilms cope with minimally buffered natural environments. Here, we report how B. subtilis biofilms overcome this intrinsic metabolic challenge through an active pH regulation mechanism. Specifically, we find that these biofilms can modulate their extracellular pH to the preferred neutrophile range, even when starting from acidic and alkaline initial conditions, while planktonic cells cannot. We associate this behavior with dynamic interplay between acetate and acetoin biosynthesis and show that this mechanism is required to buffer against biofilm acidification. Furthermore, we find that buffering-deficient biofilms exhibit dysregulated biofilm development when grown in minimally buffered conditions. Our findings reveal an active pH regulation mechanism in B. subtilis biofilms that could lead to new targets to control unwanted biofilm growth.IMPORTANCEpH is known to influence microbial growth and community dynamics in multiple bacterial species and environmental contexts. Furthermore, in many bacterial species, rapid cellular proliferation demands the use of overflow metabolism, which can often result in excessive acidification. However, in the case of bacterial communities known as biofilms, these acidification challenges can be masked when buffered laboratory media are employed to stabilize the pH environment for optimal growth. Our study reveals that B. subtilis biofilms use an active pH regulation mechanism to mitigate both growth-associated acidification and external pH challenges. This discovery provides new opportunities for understanding microbial communities and could lead to new methods for controlling biofilm growth outside of buffered laboratory conditions.

Epithelial Cell Infection Analyses with Shigella.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Development of Android-based virtual laboratory media at vocational school: effects on students’ cognitive skills

<span lang="EN-US">Vocational school students are not only required to master competency skills but also cognitive skills. Engineering mechanics learning is a basic calculation lesson that students should master. This research aimed to develop and identify the effectiveness of Android-based virtual laboratory (V-lab) media to improve students’ cognitive skills in learning engineering mechanics at vocational school in Surakarta. The subjects comprised 70 students of first grade vocational school majoring in civil engineering in Surakarta. This research was a research and development (R&D) study to develop an Android-based virtual laboratory media. Testing the N-Gain analysis in the experimental and control classes was to determine the effectiveness of virtual laboratory media in improving students’ cognitive thinking skills. Data were obtained from questionnaires and pretest and post-test test sheets. The developed virtual laboratory media was considered ‘very appropriate’ by media, material, and practitioner experts. The average value of N-Gain for the experimental class was 78.19% which was included in the effective category. Meanwhile, in the control class, the average N-Gain value reached 54.07%, which was included in the less effective category. This indicates that the use of Android-based virtual laboratory media for learning is more effective than conventional learning media.</span>

Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.