Antibiotic Screening Research Articles

Streptomyces yaizuensis sp. nov., a berninamycin C-producing actinomycete isolated from sponge.

While screening for antibiotics in a marine sample, we discovered a berninamycin C-producing actinomycete, designated YSPA8T, isolated from a sponge. A polyphasic approach was used to determine the taxonomic position of the strain. Strain YSPA8T formed sympodially branched aerial mycelia that ultimately segment into chains of spores. Comparative and phylogenetic analyses of the 16S rRNA gene sequence showed that strain YSPA8T were closely related to Streptomyces clavuligerus ATCC 27064T (99.66%), Streptomyces amakusaensis NRRL B-3351T (98.69%), Streptomyces inusitatus NBRC 13601T (98.48%), and 'Streptomyces jumonjinensis' JCM 4947 (98.41%). The phylogenetic tree using the 16S rRNA gene sequences, and both phylogenomic trees suggested that the closest relative of strain YSPA8T was S. clavuligerus ATCC 27064T. The average nucleotide identity, digital DNA-DNA hybridization, and average amino acid identity values between strain YSPA8T and S. clavuligerus ATCC 27064T were 84.1%, 28.9%, and 82.5%, respectively, which were below the thresholds of 95%, 70%, and 95% for a prokaryotic conspecific assignment. The G + C of the strain YSPA8T was 72.6%. Whole-cell hydrolysates of strain YSPA8T contained LL-diaminopimelic acid. The predominant menaquinones were MK-9(H6) (49%) and MK-9(H8) (48%), and the major fatty acids were C16:0 (26.8%), C16:1 ω7c/ω6c (17.2%), iso-C16:0 (16.0%), and iso-C15:0 (12.5%). The major phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, and other unidentified phospholipids. Based on the phenotypic, phylogenetic, genomic, and chemotaxonomic data, strain YSPA8T represents a novel species of the genus Streptomyces, and the proposed name for this species is Streptomyces yaizuensis sp. nov. The type strain is YSPA8T (=NBRC 115866T = TBRC 17196T).

Repurposing MALDI-TOF MS for effective antibiotic resistance screening in Staphylococcus epidermidis using machine learning

The emergence of Staphylococcus epidermidis as a significant nosocomial pathogen necessitates advancements in more efficient antimicrobial resistance profiling. However, existing culture-based and PCR-based antimicrobial susceptibility testing methods are far too slow or costly. This study combines machine learning with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to develop predictive models for various antibiotics using a comprehensive dataset containing thousands of S. epidermidis isolates. Optimized machine learning models utilized feature selection and achieved high AUROC scores ranging from 0.80 to 0.95 while maintaining AUPRC scores up to 0.97. Shapley Additive exPlanations were employed to analyze relevant features and assess the significance of corresponding protein biomarkers while also verifying that predictive power was derived from the detection of proteins rather than noise. Antimicrobial resistance models were validated externally to evaluate model performance outside the original data collection site. The approaches and findings in this study demonstrate a significant advancement in rapid, cost-effective antimicrobial resistance profiling, offering a promising solution for improving treatments for nosocomial infections and being potentially applicable to other microbial pathogens in the future.

Beta-Lactamase production and antibiotic susceptibility screening of Staphylococus aureus isolated from ready to eat fruits sold in some parts of Offa Metropolis, Nigeria

The global menace of community-acquired antibiotic resistance of Beta Lactamase-producing Staphylococcus aureus has been traced to the increased consumption of Ready-to-eat Foods/Fruits. Samples each of ready-to-eat whole and sliced fruits (sliced pawpaw, apple, sliced watermelon, garden egg, cucumber, pear, guava, sliced coconut, berry and date fruit) were collected randomly from vendors in Offa, Kwara State, in Nigeria. Isolation and characterization of Staphylococcus aureus from the samples were done. The isolates were screened for Beta-Lactamase production and susceptibility to some antibiotics using standard microbiological techniques. A total of twenty-two (Twenty coagulase-positive and two coagulase-negative) Staphylococcus aureus was isolated. The total Staphylococal count was highest in sliced pawpaw (23.30 ± 2.75 × 10⁵ cfu/g) while the least was recorded in apple (3.0 ± 0.01 × 10⁵ cfu /g). Twenty (20) of the isolates were recorded to be Beta Lactamase producers. All the Beta Lactamase producers were 100 % resistant to Aztreoname, 80 % to Amoxicillin Clavulate, 45 and 35 % to Ceftazidine and Ceftriaxone. Thirty percent of the isolates were found to be susceptible to Ceftazidine only while 25 % were susceptible to Ceftriaxone only. The study concluded that increased incidence of Community-Acquired Multidrug-Resistant Staphylococcus aureus could be traceable to the consumption of unhygienic processed Ready-to-eat Fruits. The ripple effects of which could be dangerous to human health.

Construction and optimization of a genetic transformation system for efficient expression of human insulin-GFP fusion gene in flax

The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon, and a fusion gene expression vector of insulin combined with green fluorescent protein (GFP) was constructed. The optimization of the flax callus culturing was undertaken, and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved. The critical concentration values of hygromycin on the flax hypocotyl development, as well as on its differentiated callus, were explored by the method of antibiotic gradient addition, and the application of antibiotic screening for the verification of positive calluses was assessed. The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed, as confirmed through polymerase chain reaction and Western blotting. In conclusion, we have established a flax callus culture system suitable for insulin expression. By optimizing the conditions of the flax callus induction, transformation, screening, and verification of a transgenic callus, we have provided an effective way to obtain insulin. Moreover, the herein-employed flax callus culture system could provide a feasible, cheap, and environmentally friendly platform for producing bioactive proteins.Graphical

Screening and Selection of Antibiotics for Enhanced Production of Astaxanthin by Haematococcus lacustris.

Haematococcus lacustris (Girod-Chantrans) Rostafinski (Chlorophyta) is the richest microalgal source of astaxanthin. Natural astaxanthin from H. lacustris has been widely studied and used for commercial production worldwide. In this study, we examined the effects of 11 antibiotics (dihydrostreptomycin sulphate, neomycin, chloramphenicol, penicillin, streptomycin, ampicillin, kanamycin, gentamycin, hygromycin B, tetracycline, and paromomycin) on the biomass dry weight, growth, and astaxanthin yield of H. lacustris using Jaworski's medium without a nitrogen source. Astaxanthin content in H. lacustris was improved in the presence of ampicillin (0.25 g/L, 0.5 g/L, 1 g/L), chloramphenicol (0.25 g/L), and penicillin (0.25 g/L, 0.5 g/L, 1 g/L) in comparison to the control on day 15. The greatest increase in astaxanthin content on day 15 (6.69-fold) was obtained with the addition of penicillin (0.5 g/L) in comparison to the control. Similarly, on day 15, the cell numbers were also the highest for the H. lacustris culture grown with the addition of penicillin (0.5 g/L).

Dual-lanthanide cyclodextrin-polyoxometalate fluorescent assembly for rapid and sensitive detection of ciprofloxacin

Quick screening and in-situ detection of trace antibiotics in environmental samples is needed for better control and monitoring of antibiotics pollution. Herein, a novel dual-lanthanide (Tb3+ and Eu3+) cyclodextrin-polyoxometalate fluorescent assembly (CD-LnPOM) has been developed for ciprofloxacin (CIP) detection. The "ratiometric" fluorescence response of CD-LnPOM enables specific detection of CIP within 5 s over a wide concentration range (0–100 μM) with a detection limit of 0.266 nM. When CIP presents, cyclodextrin acts as a host and encapsulates CIP in the cavity through host-guest interaction, which enhances the antenna sensitization between Tb3+ and CIP. Meanwhile, due to the more robust coordination between CIP and Tb3+, CIP blocks the energy transfer from Tb3+ to Eu3+ and recovers the sensitization of Tb3+ by POM. Based on the developed CD-LnPOM probe, a portable sensing device based on digital image color analysis is successfully demonstrated for real river water and antibiotic sludge samples. The developed fluorescent probe enables rapid and in-situ detection of trace antibiotics, which offers a new method for antibiotics screening and monitoring.

A high-throughput assay identifies molecules with antimicrobial activity against persister cells.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Synthesis of some 2-azitidinones (β-lactams) as antibiotic mimics and screening of their antimicrobial activity

Synthesis of some 2-azitidinones (β-lactams) as antibiotic mimics and screening of their antimicrobial activity

Antibiotic adherence in dermatologic surgery: a Multicenter prospective cohort study.

Understanding patient non-adherence to prescribed antibiotics can inform clinical practices, patient counseling, and antibiotic efficacy study design in dermatology. The primary objective was to determine the rate of and reasons for antibiotic non-adherence in the dermatologic surgery setting. The secondary objective was to test the applicability of previously studied survey questions for antibiotic non-adherence screening in the dermatologic surgery setting. Five academic outpatient dermatologic surgery centers across the United States conducted one multicenter prospective cohort study. Dermatologic surgery patients ≥ 18 years of age who were prescribed an antibiotic were included as part of this study. 15.2% (42/276) of patients did not adhere to their antibiotic regimen after dermatologic surgery. Most common reasons for incomplete antibiotic courses included forgotten antibiotics (42.9%,18/42) and side effects (28.6%, 12/42). Previously evaluated questions to identify and predict non-adherence had modest performance in the dermatologic surgery setting (Area under the curve of 0.669 [95% CI (0.583-0.754)]). Antibiotic non-adherence after skin surgery is prevalent and commonly due to reasons that physicians can address with patients.

Actinobacteria: Smart Micro-Factories for The Health Sector.

Antibiotics are considered "wonder drugs" due to the fact that they are the most extensively utilised medication in the world. They are used to cure a broad spectrum of diseases and lethal infections. A variety of bacteria and fungi produce antibiotics as a result of secondary metabolism; however, their production is dominated by a special class of bacteria, namely Actinobacteria. Actinobacteria are gram-positive bacteria with high G+C content and unparalleled antibiotic-producing ability. They produce numerous polyenes, tetracyclines, β-lactams, macrolides, and peptides. Actinobacteria are ubiquitous in nature and are isolated from various sources, such as marine and terrestrial endophytes of plants and air. They are studied for their relative antibiotic-producing ability along with the mechanism that the antibiotics follow to annihilate the pathogenic agents that include bacteria, fungi, protozoans, helminths, etc. Actinobacteria isolated from endophytes of medicinal plants have amassed significant attention as they interfere with the metabolism of medicinal plants and acquire enormous benefits from it in the form of conspicuous novel antibiotic-producing ability. Actinobacteria is not only an antibiotic but also a rich source of anticancer compounds that are widely used owing to its remarkable tumorigenic potential. Today, amongst Actinobacteria, class Streptomyces subjugates the area of antibiotic production, producing 70% of all known antibiotics. The uniqueness of bioactive Actinobacteria has turned the attention of scientists worldwide in order to explore its potentiality as effective "micronanofactories". This study provides a brief overview of the production of antibiotics from Actinobacteria inhabiting diverse environments and the methods involved in the screening of antibiotics.

Rapid testing of antibiotic residues to increase food safety awareness of animal origin.

Antibiotics are used to improve growth, reduce disease, and decrease mortality in animals grown for food. The government regulates and prohibits the use of antibiotics, in particular, the use of antibiotic growth promoter (AGP) in livestock; however, it is not yet known whether the use of antibiotics is in accordance with regulations so that there are no antibiotic residues in food of animal origin. To ensure food safety of animal origin and to raise awareness of food safety, it is necessary to detect antibiotic residues in fish, eggs, and chicken meat from Yogyakarta Special Province through monitoring and monitoring. To ensure food safety and regulatory compliance in food samples, antibiotic residue screening techniques are essential. A number of methods, such as time-consuming and costly chromatographic and spectroscopic methods, have been developed for the detection of antibiotic residues in food samples; however, not all laboratories have these facilities. Therefore, a rapid diagnosis of food of animal origin is required. The purpose of this study was to rapidly test antibiotic residues by using Premi®test kits (R-Biopharm AG, Germany) to increase awareness of food safety of animal origin. We tested 345 animal-based food samples from traditional markets, supermarkets, and central markets in five districts of Yogyakarta Special Province for antibiotic residues using rapid test kits and observation questionnaires to identify risk factors. The presence of antibiotic residues in food-animal origin samples from the Yogyakarta region had an antibiotic residue level of 9.28% (32/345), consisting of fish samples 11.3% (18/97), eggs 15.65% (1/114), and chicken meat samples 0.87% (13/102). The highest percentage of samples positive for residual antibiotics was 21.9% (7/32) from supermarket meat samples. The highest amounts of antibiotic residues were found in fish samples collected from Sleman Regency, up to 25% (8/32), whereas in supermarket fish samples, there were as high as 18.8% (6/32). Antibiotic residues in animal-based food can be attributed to various factors, including product source, transportation conditions, and environmental conditions. The widespread distribution of antibiotic residues in fish comes from environmental conditions during maintenance, distribution, and retailing. Monitoring antibiotic residue prevalence in food-animal origins, particularly chicken meat, eggs, and fish, is crucial for improving animal food quality and safety.

Development of a universal antibiotic resistance screening system for efficient enrichment of C-to-G and A-to-G base editing

To expand the scope of genomic editing, a C-to-G transversion-based editor called CGBE has been developed for precise single-nucleotide genomic editing. However, limited editing efficiency and product purity have hindered the development and application of CGBE. In this study, we introduced the Puromycin-Resistance Screening System, referred to as CGBE/ABE-PRSS, to select genetically modified cells via the CGBE or ABE editors. The CGBE/ABE-PRSS system significantly improves the enrichment efficiency of CGBE- or ABE-modified cells, showing enhancements of up to 59.6 % compared with the controls. Our findings indicate that the CGBE/ABE-PRSS, when driven by the CMV promoter, results in a higher enrichment of edited cells compared to the CAG and EF1α promoters. Furthermore, we demonstrate that this system is compatible with different versions of both CGBE and ABE, enabling various cell species and simultaneous multiplexed genome editing without any detectable random off-targets. In conclusion, our developed CGBE/ABE-PRSS system facilitates the selection of edited cells and holds promise in both basic engineering and gene therapy applications.

Analysis of endophytic bacterial diversity in seeds of different genotypes of cotton and the suppression of Verticillium wilt pathogen infection by a synthetic microbial community

BackgroundIn agricultural production, fungal diseases significantly impact the yield and quality of cotton (Gossypium spp.) with Verticillium wilt posing a particularly severe threat.ResultsThis study is focused on investigating the effectiveness of endophytic microbial communities present in the seeds of disease-resistant cotton genotypes in the control of cotton Verticillium wilt. The technique of 16S ribosomal RNA (16S rRNA) amplicon sequencing identified a significant enrichment of the Bacillus genus in the resistant genotype Xinluzao 78, which differed from the endophytic bacterial community structure in the susceptible genotype Xinluzao 63. Specific enriched strains were isolated and screened from the seeds of Xinluzao 78 to further explore the biological functions of seed endophytes. A synthetic microbial community (SynCom) was constructed using the broken-rod model, and seeds of the susceptible genotype Xinluzao 63 in this community that had been soaked with the SynCom were found to significantly control the occurrence of Verticillium wilt and regulate the growth of cotton plants. Antibiotic screening techniques were used to preliminarily identify the colonization of strains in the community. These techniques revealed that the strains can colonize plant tissues and occupy ecological niches in cotton tissues through a priority effect, which prevents infection by pathogens.ConclusionThis study highlights the key role of seed endophytes in driving plant disease defense and provides a theoretical basis for the future application of SynComs in agriculture.

Detection of Methicillin Resistance and Methicillin Sensitive Staphylococcus Aureus from Various Clinical Specimens in Tertiary Care Hospital

INTRODUCTION : Staphylococcus aureus is one of the most common Gram-positive pyogenic bacteria responsible for variety of disease. Methicillin resistance Staphylococcus aureus (MRSA) has become a major cause of hospital associated infection. MRSA can also acquire resistance to multiple alternative antimicrobials, further complicating treatment of infection. For the severe infection of MRSA, vancomycin considered one of the last treatments relatively new agents such as Linezolid and Daptomycin is used. MATERIAL AND METHODS: 141 Staphylococcus aureus were isolated from samples such as pus, sputum, urine, blood, body fluid, wound swab received in microbiology department of Krishna institute of medical sciences, karad. They were conformed by microscopy, culture, and biochemical reaction. Then MRSA were detected by using disc diffusion test using cefoxitin (30ug) disc. RESULTS: Majority of Staphylococcus aureus were isolated from pus samples 71 (50.35%). Out of 141 isolates, 81 (57.44%) were MRSA and 60 (42.55%) were MSSA. In MRSA and MSSA isolates, higher percentage of resistance was seen in Penicillin 78 (96.29%) and 55 (90.16%) respectively. CONCLUSION :The present study emphasized the burden of MRSA in patients at our hospital. The determination of antibiotic sensitivity patterns and screening for MRSA among Staphylococcus aureus isolates are important for controlling the clinical Staphylococcal infection. The antibiotic sensitivity pattern helps in judicious use of antimicrobial agents.

Evaluation of culture conditions for sewage-based surveillance of antibiotic resistance in Klebsiella pneumoniae

BackgroundRecent studies have shown promise in predicting clinical antibiotic resistance rates from sewage data. Few have focused on Klebsiella pneumoniae, despite its virulence and importance as carrier of antibiotic resistance. Several media have been suggested for the isolation of K. pneumoniae from complex samples. However, comprehensive evaluations of culture protocols for isolation of K. pneumoniae from sewage are lacking. MethodsHere, influent samples from a major Swedish sewage treatment plant were used to evaluate ten culture conditions in parallel: cultivation on Brilliant green containing Inositol-Nitrate-Deoxycholate agar (BIND), Bruce agar, Klebsiella ChromoSelect Selective agar®, MacConkey-Inositol-Carbenicillin, or Simmons Citrate Agar with Inositol (SCAI) incubated at either 37°C or 42°C for 44 h. The culture conditions were compared based on colony counts of presumed K. pneumoniae and identification precision assessed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. ResultsThe sensitivity was lowest for BIND, whereas it was similar for the other media irrespective of incubation temperature. For four media, a better precision was observed after incubation at 42°C compared to 37°C, to a large extent explained by a lower frequency of captured Klebsiella oxytoca. SCAI incubated at 42°C showed the highest precision (84.4%). By combining this protocol with subsequent antibiotic resistance screening of collected isolates, low resistance rates in sewage K. pneumoniae were revealed, potentially reflecting the local resistance landscape. ConclusionWhen combined with downstream analyses, SCAI incubated at 42°C could be a valuable culture protocol for sewage-based studies on various aspects of K. pneumoniae epidemiology including antibiotic resistance prevalence.

Re-investigation of in vitro activity of acetohydroxyacid synthase I holoenzyme from Escherichia coli

Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N-hydroxy-N-phenylacetamide and N-hydroxy-N-phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C–N bonds displays promising potential.

Investigation of Uropathological Bacterial Profile and Antibiotic Susceptibility Patterns Among Pregnant Women in Rawalpindi and Islamabad: A Comprehensive Analysis

Background: Urinary tract infections (UTIs) represent a significant health concern for pregnant women, potentially leading to adverse outcomes for both mother and child. The anatomical and physiological changes during pregnancy increase the risk of UTIs, making the study of their bacteriological profile and antibiotic susceptibility patterns especially pertinent. Objective: This study aimed to assess the bacteriological profile and antibiotic susceptibility patterns of UTIs among pregnant women in Rawalpindi and Islamabad, to identify the most prevalent pathogens and their resistance to commonly used antibiotics. Methods: A cross-sectional study was conducted on 165 early morning mid-stream urine samples collected from pregnant women attending the Railway General Hospital, Rawalpindi, over four months. Samples were analyzed for UTI-causing organisms using Cysteine Lactose Electrolyte Deficient (CLED) medium, MacConkey agar, and blood agar plates. Gram staining and biochemical tests were utilized for bacterial identification, while the Kirby-Bauer disk diffusion method determined antibiotic susceptibility. Statistical analysis was performed using SPSS version 25. Results: The study population predominantly consisted of residents from Rawalpindi (94.5%). The highest incidence of UTIs was observed in the age group of 31-36 years (34.5%). Staphylococcus aureus (28%) and Pseudomonas aeruginosa (30%) were the most prevalent pathogens. High resistance rates were noted against ampicillin and azithromycin among gram-positive isolates, whereas gram-negative bacteria showed considerable resistance to ciprofloxacin. However, gentamicin displayed relative effectiveness across various pathogens. Conclusion: The study highlights a significant prevalence of antibiotic-resistant UTIs among pregnant women in the studied regions, with Staphylococcus aureus and Pseudomonas aeruginosa being the dominant pathogens. The findings call for an urgent need to implement effective antibiotic stewardship and screening programs during pregnancy to mitigate the risk of UTIs and their associated complications.

Multifactorial genetic control and magnesium levels govern the production of a Streptomyces antibiotic with unusual cell density dependence.

Streptomyces bacteria are renowned both for their antibiotic production capabilities and for their cryptic metabolic potential. Their metabolic repertoire is subject to stringent genetic control, with many of the associated biosynthetic gene clusters being repressed by the conserved nucleoid-associated protein Lsr2. In an effort to stimulate new antibiotic production in wild Streptomyces isolates, we leveraged the activity of an Lsr2 knockdown construct and successfully enhanced antibiotic production in the wild Streptomyces isolate WAC07094. We determined that this new activity stemmed from increased levels of the angucycline-like family member saquayamycin. Saquayamycin has both antibiotic and anti-cancer activities, and intriguingly, beyond Lsr2-mediated repression, we found saquayamycin production was also suppressed at high density on solid or in liquid growth media; its levels were greatest in low-density cultures. This density-dependent control was exerted at the level of the cluster-situated regulatory gene sqnR and was mediated in part through the activity of the PhoRP two-component regulatory system, where deleting phoRP led to both constitutive antibiotic production and sqnR expression. This suggests that PhoP functions to repress the expression of sqnR at high cell density. We further discovered that magnesium supplementation could alleviate this density dependence, although its action was independent of PhoP. Finally, we revealed that the nitrogen-responsive regulators GlnR and AfsQ1 could relieve the repression exerted by Lsr2 and PhoP. Intriguingly, we found that this low density-dependent production of saquayamycin was not unique to WAC07094; saquayamycin production by another wild isolate also exhibited low-density activation, suggesting that this spatial control may serve an important ecological function in their native environments.IMPORTANCEStreptomyces specialized metabolic gene clusters are subject to complex regulation, and their products are frequently not observed under standard laboratory growth conditions. For the wild Streptomyces isolate WAC07094, production of the angucycline-family compound saquayamycin is subject to a unique constellation of control factors. Notably, it is produced primarily at low cell density, in contrast to the high cell density production typical of most antibiotics. This unusual density dependence is conserved in other saquayamycin producers and is driven by the pathway-specific regulator SqnR, whose expression is influenced by both nutritional and genetic elements. Collectively, this work provides new insights into an intricate regulatory system governing antibiotic production and indicates there may be benefits to including low-density cultures in antibiotic screening platforms.

Photodynamic patterning of living bacterial biofilms with high resolutions for information encryption and antibiotic screening

AbstractControlling the growth of bacterial biofilms in a specific pattern greatly enhances the study of cell‐to‐cell interactions and paves the way for expanding their biological applications. However, the development of simple, cost‐effective, and highly resolved biopatterning approaches remains a persistent challenge. Herein, a pioneering photodynamic biopatterning technique for the creation of living bacterial biofilms with customized geometries at high resolutions is presented. First of all, an outstanding aggregation‐induced emission photosensitizer is synthesized to enable efficient photodynamic bacterial killing at a low concentration. By combining with custom‐designed photomasks featuring both opaque and transparent patterns, the viability of photosensitizer‐coated bacteria is successfully manipulated by controlling the degree of light transmittance. This process leads to the formation of living bacterial biofilms with specific patterns replicated from the photomask. Such an innovative strategy can be employed to generate living bacterial biofilms composed of either mono‐ or multispecies, with a spatial resolution of approximately 24 µm. Furthermore, its potential applications in information storage/encryption and antibiotic screening are explored. This study provides an alternative way to understand and investigate the intricate interactions among bacteria within 3D biofilms, holding great promise in the controlled fabrication of dynamic biological systems for advanced applications.

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.