Inhibition Of Cell Wall Synthesis Research Articles

Networking and co-occurrence of virulent and multidrug resistant environmental bacteria in different aquatic systems: A gap in MDR-virulence transfer?

Co-occurrence of resistance and virulence is often overlooked in aquatic bacteria as environmental reservoirs, while transmission of these characteristics to clinically significant strains present unforeseen problems in future. In this investigation, environmental bacteria identified concurrently from multiple aquatic habitats viz., groundwater, canal, river and coastal waters were profiled for antibiotic resistance, metal tolerance, virulence factors and genes coding for these determinants. Strains from polluted river and canal exhibited higher resistance and virulence, especially Pseudomonas gessardii and P. fluorescens displayed high antibiotic resistance index (ARI > 0.6-0.8) with Alkaline Protease and Phospholipase production. Opportunistic pathogens including Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, Corynebacterium and Comamonas testosteroni expressed all three virulence factors with relatively low resistance. However, V. vulnificus and V. alginolyticus exhibited multiclass antibiotic resistance (5/6 classes). Metagenomic analysis revealed that genes corresponding to beta-lactam resistance were significantly higher (p < 0.05) in freshwater than seawater, while multidrug resistance gene were higher (p < 0.05) in seawater. In all aquatic bodies, abundant virulence genes belonged to secretion system proteins followed by motility related genes. Culturable bacteria revealed differential distribution of positive and negative correlation between 31 targeted genes with expressed resistance and virulence. Among Acinetobacter, significant positive correlation was found between Phospholipase production, other virulence genes (OVGs) and resistance to DNA Synthesis Inhibitors (DSI). In Pseudomonas, positive correlation was detected between toxin genes (toxA, eta, hlyA and stx) and resistance to cell wall synthesis inhibitors (CSI) as well as with OVGs and adhesion genes (eae, afa, papC and papA). Network analysis displayed unique clustering of genes ncc, arsB, strA, merA and intI dominated by non-pathogens and distinct clustering of genes pho, erm, nfsA, trh, lasB, tdh and invA by Vibrio. This investigation extends insight on co-occurring resistance and virulence in aquatic reservoir bacteria that could pose serious threats to public health in future.

Aktivitas Daya Hambat Isolat Actinomycetes dengan Lama Fermentasi yang Berbeda terhadap Pertumbuhan Klebsiella pneumoniae

Actinomycetes are filamentous, Gram-positive bacteria widely distributed in soil and can produce secondary metabolites useful as antibiotics. The synthesis of secondary metabolites of Actinomycetes is influenced by the growth conditions of each isolate, including the fermentation period. Klebsiella pneumoniae is an opportunistic bacterial pathogen causing lower respiratory tract infection. Objectives: To determined the inhibition activity of Actinomycetes with different fermentation periods against the growth of K.pneumoniae. Methods: Actinomycetes were isolated from soil in Kebun Raya Bogor and grown on Starch Casein Agar (SCA) medium before being fermented in liquid media consisting of 2% of mannitol, 2% of peptone, and 1% of glucose with an incubation time of 6,7, and 8 days. An antibacterial activity test was conducted on Mueller Hinton Agar (MHA) medium using the well diffusion method. Results: The activity of Actinomycetes isolates with fermentation duration of 6,7 and 8 were able to inhibit the growth of K. pneumoniae with an average inhibition zone of 4,46 mm; 4,94 mm; and 5,04 mm, respectively. Actinomycetes' inhibitory mechanism ranges from inhibition of cell wall synthesis to nucleic acid and protein synthesis. One Way ANOVA test showed significant differences between the inhibition zone produced by each fermentation group. Conclusion: Actinomycetes isolates have antibacterial activity against the growth of K. pneumoniae with the highest activity achieved by Actinomycetes with a fermentation time of 8 days.Keywords: Actinomycetes, antibacterial, fermentation, Klebsiella pneumoniae

Identification and Biochemical Characterization of Pyrrolidinediones as Novel Inhibitors of the Bacterial Enzyme MurA.

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC50 = 4.5 μM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

Computational and data mining studies to understand the distribution and dynamics of Temoneria (TEM) β-lactamase and their interaction with β-lactam and β-lactamase inhibitors

β-lactams are large group of antibiotics widely used to suppress the bacterial growth by inhibiting cell wall synthesis. Bacterial resistance against β-lactam antibiotics is primarily mediated through the production of Temoneria (TEM) β-lactamase (BLs), with almost 474 variants identified in Lactamase Engineering Database (LacED). The present study aims to develop a model to track the evolution of TEM BLs and their interactions with β-lactam and BLs inhibitors through data mining and computational approaches. Further, the model will be used to predict the effective combinations of β-lactam and BLs inhibitors to treat the bacterial infection harbouring emerging variants of β-lactamase. The molecular docking study results demonstrated that most TEM mutants recorded the least binding energy to penicillin and cephalosporin (I/II/III/IV/V generations) class of antibiotics. On the contrary, the same mutants recorded higher binding energy to carbapenem and Monobactam class of antibiotics. Among the BLs inhibitors, tazobactam recorded the least binding energy against most of the TEM mutants, indicating that it can lower the catalytic activity of TEM BLs, thereby potentiating antibiotic action. Similarly, data mining work has assisted us in creating a database of TEM mutants that has comprehensive data on mutations, bacterial diversity, Km, MIC, and IRT types. It has been noted that earlier released antibiotics like amoxicillin and ampicillin had lower Km and higher MIC values, which indicates the prevalence of bacterial resistance. By analysing the differential binding energy (ΔBE) of the selected TEM mutants against β-lactam and BLs inhibitors, the most effective combination of β-lactam (carbapenem and monobactam class of antibiotics) and BLs inhibitors (tazobactam) was identified, to cure bacterial diseases/infections and to prevent similar antibiotic resistance outbreaks. Therefore, our study opens a new avenue in developing strategies to manage antibiotic resistance in bacteria.

Chemical Constitution, In-silico Molecular Docking Studies and Antibacterial Activity of Flower Essential Oil of Artabotrys hexapetalus

The isolation of the volatile constituents from the flowers of Artabotrys hexapetalus was carried out using a simple headspace solvent-trapping technique and identified by GC-MS analysis. The major compounds are ethyl acetate 53.6%, isobutyl acetate (29.4%) and ethyl benzoate (14.2%). The odour of the solution obtained from this method was found to be similar to that of the fresh flowers. Further the essential oil from A. hexapetalus was obtained for the first time from India by hydro distillation using a Clevenger type apparatus and analysed by GC-MS. The plant yielded 1.26%, of the essential oils from the flower. The analysis lead to the identification of 28 compounds representing 96.17% of the total oil. The essential oil consists of predominantly oxygenated sesquiterpenes (51.91%) followed by sesquiterpenes (43.31%) and small quantities of monoterpenes (1.24%) and other compounds (1.34%). The main constituents of the essential oil obtained from the flowers of A.hexapetalus are β-caryophyllene (18.69%), caryophyllene oxide (14.54%), cubenol (12.53%) and ledol (11.5%). The essential oil showed antibacterial activity against bacterial strains Streptococcus pneumonia, Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa exhibiting a zone of inhibition of 16.4, 15.7, 17.5 and 14.5 mm and MIC value of 2.5, 5.0, 2.5, 5.0 mg/ml respectively. Molecular docking analysis indicated that the essential oil constituents are nucleic acid and cell wall synthesis inhibitors. So it is worth to include this in cosmetics and fragrances.

A New Perspective on the Antimicrobial Mechanism of Berberine Hydrochloride Against Staphylococcus aureus Revealed by Untargeted Metabolomic Studies.

Berberine hydrochloride (BBR) is a natural product widely used in clinical medicine and animal production. It has a variety of antimicrobial effects, but its complex antimicrobial mechanism has not been clarified. This study aimed to discover the metabolic markers and gain a new perspective on the antibacterial mechanism of BBR. The effects of different inhibitory concentrations of BBR on the survival and growth of standard strain Staphylococcus aureus ATCC 25923 were analyzed by the bacteriostatic activity test. Differences in intracellular metabolites of S. aureus following 19 μg/ml BBR exposure for 1 h were investigated by combining non-targeted metabolomics techniques of gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The results showed that the minimum inhibitory concentration of BBR against S. aureus was 51 μg/ml. A total of 368 and 3,454 putative metabolites were identified by GC-MS and LC-MS analyses, respectively. Principal component analysis showed the separation of intracellular metabolite profiles between BBR-exposed samples and non-exposed controls. Pathway activity profiling analysis indicated a global inhibition of metabolisms by BBR exposure, while enhancement was also found in nucleic acid metabolism, amino sugar, and nucleotide sugar metabolism. Several metabolic markers were screened out mainly based on their variable importance of projection values. Two pyridine dicarboxylic acids were significantly downregulated, suggesting the reduction of stress resistance. The oxidized phospholipid (PHOOA-PE) was accumulated, while lipid antioxidant gamma-tocopherol was decreased, and farnesyl PP, the synthetic precursor of another antioxidant (staphyloxanthin), was decreased below the detection threshold. This evidence indicates that BBR reduced the antioxidant capacity of S. aureus. Accumulation of the precursors (UDP-GlcNAc, CDP-ribitol, and CDP-glycerol) and downregulation of the key metabolite D-Ala-D-Ala suggest the inhibition of cell wall synthesis, especially the peptidoglycan synthesis. Metabolites involved in the shikimate pathway (such as 3-dehydroshikimate) and downstream aromatic amino acid synthesis were disturbed. This study provides the first metabolomics information on the antibacterial mechanism of BBR against S. aureus. The key metabolic markers screened in this study suggest that the shikimate pathway, staphyloxanthin synthesis, and peptidoglycan biosynthesis are new directions for further study of BBR antibacterial mechanism in the future.

SsAGM1-Mediated Uridine Diphosphate-N-Acetylglucosamine Synthesis Is Essential for Development, Stress Response, and Pathogenicity of Sclerotinia sclerotiorum.

The necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen. S. sclerotiorum can cause Sclerotinia stem rot in more than 600 species of plants, which results in serious economic losses every year. Chitin is one of the most important polysaccharides in fungal cell walls. Chitin and β-Glucan form a scaffold that wraps around the cell and determines the vegetative growth and pathogenicity of pathogens. UDP-GlcNAc is a direct precursor of chitin synthesis. During the synthesis of UDP-GlcNAc, the conversion of GlcNAc-6P to GlcNAc-1P that is catalyzed by AGM1 (N-acetylglucosamine-phosphate mutase) is a key step. However, the significance and role of AGM1 in phytopathogenic fungus are unclear. We identified a cytoplasm-localized SsAGM1 in S. sclerotiorum, which is homologous to AGM1 of Saccharomyces cerevisiae. We utilized RNA interference (RNAi) and overexpression to characterize the function of SsAGM1 in S. sclerotiorum. After reducing the expression of SsAGM1, the contents of chitin and UDP-GlcNAc decreased significantly. Concomitantly, the gene-silenced transformants of SsAGM1 slowed vegetative growth and, importantly, lost the ability to produce sclerotia and infection cushion; it also lost virulence, even on wounded leaves. In addition, SsAGM1 was also involved in the response to osmotic stress and inhibitors of cell wall synthesis. Our results revealed the function of SsAGM1 in the growth, development, stress response, and pathogenicity in S. sclerotiorum.

CTX-M-producing Escherichia coli strains: resistance to temocillin, fosfomycin, nitrofurantoin and biofilm formation.

Aim: ESBL-producing and bacterial biofilms-forming Escherichia coli are associated with antimicrobial treatment failure. This study aimed to investigate the phenotypic resistance mechanisms of CTX-M E. coli against old antibiotics - cell wall synthesis inhibitors temocillin, nitrofurantoin and fosfomycin. Materials & Methods: Susceptibility to old antibiotics testing was performed using disk diffusion method, biofilm formation was evaluated spectrophotometrically, and PCR was used for the determination of CTX-M type. Results & conclusion: Temocillin was active against nearly 93%, nitrofurantoin and fosfomycin, respectively, 91.7% and 98.6% of tested E. coli. Thus, it demonstrated to be a good alternative therapeutic option against ESBL infections. Bacteria resistant to old antibiotics had CTX-M-15 or CTX-M-15, TEM-1 and OXA-1 combinations. No significant association was found between CTX-M E. coli resistance to temocillin, nitrofurantoin and fosfomycin; however, the level of biofilm formation was found as not affected by the type of CTX-M β-lactamases.

Aerosolization triggers immediate antibiotic resistance in bacteria

Antibiotic resistance poses a threat in many institutions including hospitals, meat processing facilities and other agricultural and occupational entities. The goal of this research is to uncover how bacteria react to environmental conditions including aerosolization and delineate factors that trigger expression of antibiotic resistance genes (ARGs) using the laboratory strain Escherichia coli (E. coli) MG 1655 as surrogate bacterium. Fresh mid-log phase bacterial suspensions were aerosolized at different environmental conditions for 5 different time durations into a sterile chamber and collected using a bioaerosol collector. Each sample was analyzed based on its response to each environmental factor by antibiotic susceptibility testing and polymerase chain reaction (PCR) using specific primers to target ARGs. Strong resistance was detected to cell wall and protein synthesis inhibitors Cephalothin, and Gentamicin after 5 min compared to 30 min and 45 min of aerosolizations in reference to viable cell concentrations. Resistance to the highest number of antibiotics was detected in the shortest aerosolization samples while exposure for longer aerosolization durations caused an increased amount of stress which bacteria could not alleviate, indicating the involvement of different mechanisms to respond to short-term and long-term exposures. A small amount of resistance was detected after intermediate durations of aerosolization, maybe due to a transient response of the cells to compensate for mechanical, osmotic and other stresses during aerosolization. This study is the first to demonstrate that aerosolization triggers antibiotic resistance within minutes in E. coli.

Identification of a Pseudosubstrate Stimulatory Motif Uncovers Novel Roles of Cdc14 Phosphatase in Fungal Cell Wall Integrity

Fungal pathogens are a growing threat to human health and global food security as they are becoming increasingly resistant to the few existing antifungal treatments. Cdc14 is a protein phosphatase primarily known for its essential role in controlling mitotic exit in the model yeast Saccharomyces cerevisiae. However, in other fungal species Cdc14 is non‐essential and the reasons for its strict conservation across Dikarya remain unclear. Recently, Cdc14 was shown to be required for host infection by several plant pathogenic fungi. Here, we show that Cdc14 is also required for virulence in the human pathogen, Candida albicans.These observations, coupled with our structural and biochemical knowledge of Cdc14 phosphatases, suggest it could be a useful target for antifungal development. In our search for differences between fungal and animal Cdc14 enzymes that could be exploited for antifungal design, we discovered an invariant motif in the otherwise disordered C‐terminal tail of fungal Cdc14 orthologs. The motif resembles the recognition sequence of optimal Cdc14 substrates. We provide evidence from molecular modeling and enzyme kinetic studies that this motif is a “pseudosubstrate” that binds the Cdc14 active site and functions to accelerate the rate‐limiting catalytic step, providing a plausible mechanism for dynamic control of intracellular Cdc14 activity. The turnover rate for Cdc14 lacking a functional pseudosubstrate motif is reduced by an order of magnitude compared to the wild‐type enzyme. Both S. cerevisiaeand C. albicanscells expressing Cdc14 variants with point mutations in this motif exhibit a specific and pronounced sensitivity to cell wall stresses, including treatment with echinocandin antifungal drugs that inhibit cell wall synthesis. Moreover, signaling through the cell wall integrity pathway is chronically elevated in these cells, indicative of a cell wall structural defect. Preliminary evidence in synchronized S. cerevisiaecultures with reduced Cdc14 activity suggests that cell wall integrity is compromised around the time of cytokinesis and septation, consistent with its known functions in these processes. We are currently testing if the pseudosubstrate motif is required for virulence of C. albicans.Our findings reveal 1) a novel contribution to catalysis of a structural element outside the conserved catalytic core of Cdc14 enzymes and 2) novel roles of Cdc14 in promoting cell wall integrity and pathogenesis. More detailed characterization of the mechanisms by which Cdc14 contributes to cell wall integrity and host infection in human pathogenic fungi should help assess its future value for antifungal development.

Linearized teixobactin is inactive and after sequence enhancement, kills methicillin-resistant Staphylococcus aureus via a different mechanism.

Staphylococcus aureus is a highly adaptable pathogen that can rapidly develop resistance to conventional antibiotics such as penicillin. Recently, teixobactin was discovered from uncultivated soil bacteria by using the i-chip technology. This depsipeptide forms an ester bond between the backbone C-terminal isoleucine carboxylic acid and the hydroxyl group of threonine at position 8. Also, it contains multiple nonstandard amino acids, making it costly to synthesize. This study reports new peptides designed by linearizing teixobactin. After linearization and conversion to normal amino acids, teixobactin lost its antibacterial activity. Using this inactive template, a series of peptides were designed via hydrophobic patching and residue replacements. Three out of the five peptides were active. YZ105, only active against Gram-positive bacteria, however, showed the highest cell selectivity index. Different from teixobactin, which inhibits cell wall synthesis, YZ105 targeted the membranes of methicillin-resistant S. aureus (MRSA) based on kinetic killing, membrane permeation, depolarization, and scanning electron microscopy studies. Moreover, YZ105 could kill nafcillin-resistant MRSA, Staphylococcal clinical strains, and disrupted preformed biofilms. Taken together, YZ105, with a simpler sequence, is a promising lead for developing novel anti-MRSA agents.

Role of Faropenem in Treatment of Pediatric Infections: The Current State of Knowledge.

Carbapenems play an important role in the management of bacterial infections. Meropenem, imipenem, ertapenem, and faropenem are carbapenems with the broadest antibacterial spectrum and strong antibacterial activity. Faropenem is a novel oral carbapenem with an advantage over other parenteral carbapenems in the series. Like other β-lactam antibiotics, faropenem inhibits cell wall synthesis by inhibiting penicillin-binding proteins (PBPs). Faropenem is stable against β-lactamase and has a low propensity for bacterial resistance. Faropenem has demonstrated excellent in-vitro and clinical activity in adult infections with a broad spectrum of activity. Faropenem also has a favorable safety profile. These activities of the faropenem created the interest of researchers in exploring its use in the treatment of pediatric infections. After promising outcomes in-vitro and clinical evaluation in children, faropenem is now approved in some parts of the world for the treatment of pediatric infections. Faropenem oral dry syrup is available for the treatment of a wide range of pediatric infections, including upper respiratory tract infections, urinary tract infections, dermatological infections, and bacterial periodontal infections in children. The current recommended clinical dose in pediatric patients is 15 mg/kg/ day, divided into three doses. The availability of faropenem dry syrup has expanded the current therapeutic options for treating pediatric infections.In this review, we have put light on the in-vitro and clinical studies of faropenem dry syrup in pediatric patients, along with its molecular and pharmacological basics.

Molecular Docking andin silicoEvaluation of Phytochemicals of Bioactive Methanolic Extract ofIpomoea mauritianaJacq. as Anti-Bacterial Agents

Antibacterial treatment has grown difficult due to the increasing growth in bacterial infections, as well as their tolerance to most first-line antibiotics. This is a severe danger to the world’s human health in the 21st century, necessitating further research to identify drugs with improved antibacterial effects and broad-spectrum functions. This study aimed to discover anti-bacterial agents through the molecular docking and in silico approach. Most responsive thirty (32) compounds on UPLC-Q-TOF/MS analysis were selected from our previous report to get the hit compound(s) against inhibition of cell wall synthesis, inhibition of protein synthesis, interference with nucleic acid synthesis, inhibition of a metabolic pathway, inhibition of membrane function and inhibition of adenosine triphosphate (ATP) synthase. From the molecular docking results, we afforded six compounds for cell wall synthesis protein, four compounds for protein synthesis protein, five for nucleic acid synthesis protein, three for metabolic pathway protein, four for membrane function protein and three for ATP synthase protein which eventually undergoes the pharmacokinetic and drug-likeness properties to obtain lead compound(s). Finally, we discovered that compounds Turpinionosides B, Polydatin, Ledebouriellol, and Pterodontoside A have the strongest binding interactions with cell wall synthesis, inhibition of protein synthesis and inhibition of metabolic pathway synthesis, interference with nucleic acid synthesis and inhibition of ATP synthase, inhibition of membrane function proteins, respectively. These compounds have the potential to become an anti-bacterial therapeutic candidate due to their promising pharmacological properties.

External Stresses Affect Gonococcal Type 4 Pilus Dynamics.

Bacterial type 4 pili (T4P) are extracellular polymers that serve both as adhesins and molecular motors. Functionally, they are involved in adhesion, colony formation, twitching motility, and horizontal gene transfer. T4P of the human pathogen Neisseria gonorrhoeae have been shown to enhance survivability under treatment with antibiotics or hydrogen peroxide. However, little is known about the effect of external stresses on T4P production and motor properties. Here, we address this question by directly visualizing gonococcal T4P dynamics. We show that in the absence of stress gonococci produce T4P at a remarkably high rate of ∼200 T4P min–1. T4P retraction succeeds elongation without detectable time delay. Treatment with azithromycin or ceftriaxone reduces the T4P production rate. RNA sequencing results suggest that reduced piliation is caused by combined downregulation of the complexes required for T4P extrusion from the cell envelope and cellular energy depletion. Various other stresses including inhibitors of cell wall synthesis and DNA replication, as well as hydrogen peroxide and lactic acid, inhibit T4P production. Moreover, hydrogen peroxide and acidic pH strongly affect pilus length and motor function. In summary, we show that gonococcal T4P are highly dynamic and diverse external stresses reduce piliation despite the protective effect of T4P against some of these stresses.

Clinical Pharmacokinetics and Pharmacodynamics of Dalbavancin.

Dalbavancin is a synthetic lipoglycopeptide that exerts its antimicrobial activity through two distinct modes of action, inhibition of cell wall synthesis and an anchoring mechanism. Compared with previous glycopeptide antibiotics, dalbavancin demonstrates improved antibacterial potency against Gram-positive organisms and a long half-life of approximately 1week, which is longer in tissues (e.g., skin, bone) than plasma. These factors facilitated the development of single-dose or once-weekly dosing regimens to treat acute bacterial skin and skin structure infections (ABSSSI). Dalbavancin exhibits dose-proportional pharmacokinetics and is highly protein bound (93%). Despite being highly protein bound, it has a steady-state volume of distribution >10L and distributes widely into the skin, bone, peritoneal space, and epithelial lining fluid, but not cerebrospinal fluid. Dalbavancin elimination occurs via a combination of renal (approximately 45%) and non-renal clearance, with dose adjustments recommended only in patients with a creatinine clearance <30mL/min not receiving any form of dialysis. The established pharmacokinetic/pharmacodynamic index associated with bacterial kill is free area under the concentration-time curve over the minimum inhibitory concentration (fAUC/MIC), with a goal 24-h fAUC/MIC of at least 27.1 for Staphylococcus aureus infections. Recent data suggest usefulness in the treatment of infections beyond ABSSSI, with convenient dosing and redosing strategies for complicated infections requiring extended treatment durations. Additional studies are needed to confirm these preliminary findings.

THCz: Small molecules with antimicrobial activity that block cell wall lipid intermediates

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop invitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials.

Exogenous proanthocyanidins improve tolerance of Cu-toxicity by amelioration of oxidative damage and re-programming of gene expression in Medicago sativa.

Excess copper (Cu) in soil due to industrial and agricultural practices can result in reduced plant growth. Excess Cu resulted in severely retarded root growth with severe discoloration of Alfalfa (Medicago sativa) and Medicago truncatula. Growth in the presence of hydrogen peroxide resulted in similar symptoms that could be partially recovered by the addition of the reductant ascorbic acid revealing damage was likely due to oxidative stress. The addition of proanthocyanidins (PAs) in the presence of Cu prevented much of the damage, including plant growth and restoration of lignin synthesis which was inhibited in the presence of excess Cu. Transcriptome analyses of the impact of excess Cu and the amelioration after PAs treatment revealed that changes were enriched in functions associated with the cell wall and extracellular processes, indicating that inhibition of cell wall synthesis was likely the reason for retarded growth. Excess Cu appeared to induce a strong defense response, along with alterations in the expression of a number of genes encoding transcription factors, notably related to ethylene signaling. The addition of PAs greatly reduced this response, and also induced novel genes that likely help ameliorate the effects of excess Cu. These included induction of genes involved in the last step of ascorbic acid biosynthesis and of enzymes involved in cell wall synthesis. Combined, these results show that excess Cu causes severe oxidative stress damage and inhibition of cell wall synthesis, which can be relieved by the addition of PAs.

Reduced Susceptibility and Resistance to Vancomycin of Staphylococcus aureus: A Review of Global Incidence Patterns and Related Genetic Mechanisms

Staphylococcus aureus is a Gram-positive bacterium causing a wide range of infections ranging from cutaneous infections to endocarditis and bacteremia. Beta-lactamases such as penicillin and, subsequently, methicillin have been used in the treatment of S. aureus infections. With the emergence of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin, a bacterial cell wall synthesis inhibitor, has been used as the treatment of choice for MRSA infections.However, over the past few decades, there have been reports of reduced susceptibility and resistance of S. aureus to vancomycin globally, most recently from Michigan, United States, in July 2021. Based on the minimum inhibitory concentration (MIC) of the antibiotic against S. aureus, there are three strains of resistance, vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-resistant Staphylococcus aureus (VRSA), and heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA).The increasing prevalence of VISA and VRSA infections is a cause of global concern. This qualitative review of peer-reviewed research publications aims to describe the cases of VISA and VRSA reported in the literature globally and summarizes the genetic mechanisms implicated in their resistance. The most common mechanism implicated in VRSA infections is the vanA operon, while cell wall thickening is responsible for VISA infections. This review aims to perform a global comparison between the MIC corresponding to the strength of resistance to vancomycin and the presence of the vanA operon. In this review, VISA and VRSA are noted to be most susceptible to quinupristin-dalfopristin and linezolid, respectively. Maintaining active systemic surveillance for such infections, employing strict infection control measures, and continuing to mitigate indiscriminate and irrational use of antibiotics are some of the actions that can be undertaken to reduce the incidence and transmission of VISA, VRSA, and hVISA infections worldwide.

Electroporation as an Efficacy Potentiator for Antibiotics With Different Target Sites.

Antibiotic resistance is a global health threat, and there is ample motivation for development of novel antibacterial approaches combining multiple strategies. Electroporation is among the promising complementary techniques – highly optimizable, effective against a broad range of bacteria, and largely impervious to development of resistance. To date, most studies investigating electroporation as an efficacy potentiator for antibacterials used substances permissible in food industry, and only few used clinical antibiotics, as acceptable applications are largely limited to treatment of wastewaters inherently contaminated with such antibiotics. Moreover, most studies have focused mainly on maximal achievable effect, and less on underlying mechanisms. Here, we compare Escherichia coli inactivation potentiation rates for three antibiotics with different modes of action: ampicillin (inhibits cell wall synthesis), ciprofloxacin (inhibits DNA replication), and tetracycline (inhibits protein synthesis). We used concentrations for each antibiotic from 0 to 30× its minimum inhibitory concentration, a single 1-ms electric pulse with amplitude from 0 to 20 kV/cm, and post-pulse pre-dilution incubation either absent (≲1 min) or lasting 60 min, 160 min, or 24 h. Our data show that with incubation, potentiation is significant for all three antibiotics, increases consistently with pulse amplitude, and generally also with antibiotic concentration and incubation time. With incubation, potentiation for ampicillin was rather consistently (although with weak statistical significance) superior to both ciprofloxacin and tetracycline: ampicillin was superior to both in 42 of 48 data points, including 7 with significance with respect to both, while at 60- and 160-min incubation, it was superior in 31 of 32 data points, including 6 with significance with respect to both. This suggests that electroporation potentiates wall-targeting antibiotics more than those with intracellular targets, providing motivation for in-depth studies of the relationship between the mode of action of an antibiotic and its potentiation by electroporation. Identification of substances permissible in foods and targeting the cell wall of both Gram-negative and Gram-positive bacteria might provide candidate antibacterials for broad and strong potentiation by electroporation applicable also for food preservation.

Engineering Escherichia coli to produce and secrete colicins for rapid and selective biofilm cell killing.

Bacterial biofilms are associated with chronic infectious diseases and are highly resistant to conventional antibiotics. Antimicrobial bacteriocins are alternatives to conventional antibiotics and are characterized by unique cell-killing mechanisms, including pore formation on cell membranes, nuclease activity, and cell wall synthesis inhibition. Here, we used cell-free protein synthesis to rapidly evaluate the anti-biofilm activities of colicins E1, E2, and E3. We found that E2 (with DNase activity) most effectively killed target biofilm cells (i.e., the K361 strain) while leaving non-targeted biofilms intact. We then engineered probiotic Escherichia coli microorganisms with genetic circuits to controllably synthesize and secrete colicin E2, which successfully inhibited biofilms and killed pre-formed indicator biofilms. Our findings suggest that colicins rapidly and selectively kill target biofilm cells in multispecies biofilms and demonstrate the potential of using microorganisms engineered to produce antimicrobial colicin proteins as live therapeutic strategies to treat biofilm-associated infections.