Broad-Spectrum Antibacterial Activity of Antioxidant Octyl Gallate and Its Impact on Gut Microbiome.

In this study, we investigated the antibacterial activity of octyl gallate (OG), an antioxidant food additive, against both Gram-positive and Gram-negative bacterial pathogens. OG demonstrated robust bactericidal activity against Gram-positive bacterial pathogens with minimum inhibitory concentrations (MIC) of 4 to 8 µg/mL and minimum bactericidal concentrations (MBC) of 8 to 16 µg/mL in vitro. However, OG exhibited limited antibacterial activity against Gram-negative bacteria, including E. coli, although it could inhibit bacterial growth in vitro. Importantly, OG administration in mice altered the fecal microbiome, significantly reducing microbial diversity, modifying community structure, and increasing the abundance of beneficial bacteria. Additionally, OG displayed low cytotoxicity and hemolytic activity. These findings suggest that OG could be developed as a novel antibacterial agent, particularly against multi-drug-resistant MRSA. Our results provide new insights into the therapeutic potential of OG in modulating the gut microbiome, especially in conditions associated with microbial imbalance, while ensuring food safety.

New Insights into the Biological Functions of Essential TsaB/YeaZ Protein in Staphylococcus aureus.

TsaB/YeaZ represents a promising target for novel antibacterial agents due to its indispensable role in bacterial survival, high conservation within bacterial species, and absence of eukaryotic homologs. Previous studies have elucidated the role of the essential staphylococcal protein, TsaB/YeaZ, in binding DNA to mediate the transcription of the ilv-leu operon, responsible for encoding key enzymes involved in the biosynthesis of branched-chain amino acids-namely isoleucine, leucine, and valine (ILV). However, the regulation of ILV biosynthesis does not account for the essentiality of TsaB/YeaZ for bacterial growth. In this study, we investigated the impact of TsaB/YeaZ depletion on bacterial morphology and gene expression profiles using electron microscopy and deep transcriptomic analysis, respectively. Our results revealed significant alterations in bacterial size and surface smoothness upon TsaB/YeaZ depletion. Furthermore, we pinpointed specific genes and enriched biological pathways significantly affected by TsaB/YeaZ during the early and middle exponential phases and early stationary phases of growth. Crucially, our research uncovered a regulatory role for TsaB/YeaZ in bacterial autolysis. These discoveries offer fresh insights into the multifaceted biological functions of TsaB/YeaZ within S. aureus.

Targeting multidrug resistant Staphylococcus infections with bacterial histidine kinase inhibitors.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), which are not susceptible to current antibiotics has necessitated the development of novel approaches and targets to tackle this growing challenge. Bacterial two-component systems (TCSs) play a central role in the adaptative response of bacteria to their ever-changing environment. They are linked to antibiotic resistance and bacterial virulence making the proteins of the TCSs, histidine kinases and response regulators, attractive for the development of novel antibacterial drugs. Here, we developed a suite of maleimide-based compounds that we evaluated against a model histidine kinase, HK853, in vitro and in silico. The most potent leads were then assessed for their ability to decrease the pathogenicity and virulence of MRSA, resulting in the identification of a molecule that decreased the lesion size caused by a methicillin-resistant S. aureus skin infection by 65% in a murine model.

Antibacterial effect of rose bengal against colistin-resistant gram-negative bacteria.

Increasing drug resistance in Gram-negative bacteria presents significant health problems worldwide. Despite notable advances in the development of a new generation of ß-lactams, aminoglycosides, and fluoroquinolones, it remains challenging to treat multi-drug resistant Gram-negative bacterial infections. Colistin (polymyxin E) is one of the most efficacious antibiotics for the treatment of multiple drug-resistant Gram-negative bacteria and has been used clinically as a last-resort option. However, the rapid spread of the transferable gene, mcr-1 which confers colistin resistance by encoding a phosphoethanolamine transferase that modifies lipid A of the bacterial membrane, threatens the efficacy of colistin for the treatment of drug-resistant bacterial infections. Colistin-resistant strains of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae often reduce their susceptibility to other anti-Gram-negative bacterial agents. Thus, drugs effective against colistin-resistant strains or methods to prevent the acquisition of colistin-resistance during treatment are urgently needed. To perform cell-based screenings of the collected small molecules, we have generated colistin-resistant strains of E. coli, A. baumannii, K. pneumoniae, P. aeruginosa, and S. enterica Typhimurium. In-house MIC assay screenings, we have identified that rose bengal (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein) is the only molecule that displays unique bactericidal activity against these strains at low concentrations under illumination conditions. This article reports the antibacterial activity of a pharmaceutical-grade rose bengal against colistin-resistant Gram-negative bacteria.

Synergistic Potentiation of Antimicrobial and Antibiofilm Activities of Penicillin and Bacitracin by Octyl Gallate, a Food-Grade Antioxidant, in Staphylococcus epidermidis.

Staphylococcus epidermidis is a major nosocomial pathogen that frequently forms biofilms on indwelling medical devices. This study aimed to investigate the synergistic antimicrobial and antibiofilm activities of octyl gallate (OG) in combination with penicillin and bacitracin against S. epidermidis. Antimicrobial synergy was assessed by conducting checkerboard titration assays, and antibiofilm activity was determined with biofilm assays and fluorescence microscopy analysis. The presence of 8 µg/mL of OG increased both the bacteriostatic and bactericidal activities of penicillin and bacitracin against S. epidermidis. It lowered the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of penicillin by eight-fold and those of bacitracin by four-fold. Moreover, when used with penicillin or bacitracin, OG significantly decreased the level of biofilm production by preventing microcolony formation. Furthermore, OG significantly permeabilized the bacterial cell wall, which may explain its antimicrobial synergy with penicillin and bacitracin. Together, these results demonstrate that OG, a food-grade antioxidant, can be potentially used as a drug potentiator to enhance the antimicrobial and antibiofilm activities of penicillin and bacitracin against S. epidermidis.

Identification and Validation of a Novel Antibacterial Compound MZ-01 against Methicillin-Resistant Staphylococcus aureus.

The discovery of new classes of antibiotics is slow, and it is being greatly outpaced by the development of bacterial resistance. This disparity places us in an increasingly vulnerable position because we are running out of safe and effective therapeutic options to treat antibiotic-resistant infections. This is exemplified by the emergence and persistence of hospital-acquired and community-associated methicillin-resistant S. aureus (MRSA), which has markedly narrowed our options for treating life-threatening staph infections. Thus, there is an urgent need to develop novel, potent, preventive, and therapeutic agents. In our current study, we performed a whole-cell screening assay of synthetic libraries for antibacterial activity and identified a novel molecule, MZ-01. MZ-01 exhibited potent bactericidal activity against Gram-positive bacterial pathogens, including MRSA, Streptococcus pyogenes, and Streptococcus pneumoniae, at low concentrations. MZ-01 killed and lysed both the late exponential phase of an S. aureus population and bacteria inside mammalian cells. Furthermore, MZ-01 exhibited low cytotoxicity. These results indicate that MZ-01 is a promising scaffold to guide the development of novel, potent antibacterial agents against multidrug-resistant Gram-positive bacterial pathogens such as MRSA.

Antibacterial Activity of Pharmaceutical-Grade Rose Bengal: An Application of a Synthetic Dye in Antibacterial Therapies.

Rose bengal has been used in the diagnosis of ophthalmic disorders and liver function, and has been studied for the treatment of solid tumor cancers. To date, the antibacterial activity of rose bengal has been sporadically reported; however, these data have been generated with a commercial grade of rose bengal, which contains major uncontrolled impurities generated by the manufacturing process (80-95% dye content). A high-purity form of rose bengal formulation (HP-RBf, >99.5% dye content) kills a battery of Gram-positive bacteria, including drug-resistant strains at low concentrations (0.01-3.13 µg/mL) under fluorescent, LED, and natural light in a few minutes. Significantly, HP-RBf effectively eradicates Gram-positive bacterial biofilms. The frequency that Gram-positive bacteria spontaneously developed resistance to HP-RB is extremely low (less than 1 × 10-13). Toxicity data obtained through our research programs indicate that HP-RB is feasible as an anti-infective drug for the treatment of skin and soft tissue infections (SSTIs) involving multidrug-resistant (MDR) microbial invasion of the skin, and for eradicating biofilms. This article summarizes the antibacterial activity of pharmaceutical-grade rose bengal, HP-RB, against Gram-positive bacteria, its cytotoxicity against skin cells under illumination conditions, and mechanistic insights into rose bengal's bactericidal activity under dark conditions.

Identification of cbiO Gene Critical for Biofilm Formation by MRSA CFSa36 Strain Isolated from Pediatric Patient with Cystic Fibrosis.

The colonization of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has a detrimental effect on the respiratory care of pediatric patients with cystic fibrosis (CF). In addition to being resistant to multiple antibiotics, S. aureus also has the ability to form biofilms, which makes the infection more difficult to treat and eradicate. In this study, we examined the ability of S. aureus strains isolated from pediatric patients with CF to form biofilms. We screened a transposon mutant library of MRSA and identified a putative cobalt transporter ATP binding domain (cbiO) that is required for biofilm formation. We discovered that deleting cbiO creating a cbiO null mutant in CFSa36 (an MRSA strain isolated from a patient with cystic fibrosis) significantly hinders the ability of CFSa36 to form biofilm. The complementation of cbiO restored the ability of the cbiO deletion mutant to generate biofilm. Interestingly, we revealed that incorporating extra copper ions to the chemically defined medium (CDM) complemented the function of cbiO for biofilm formation in a dose-dependent manner, while the addition of extra iron ions in CDM enhanced the effect of cbiO null mutation on biofilm formation. In addition, neither the addition of certain extra amounts of copper ions nor iron ions in CDM had an impact on bacterial growth. Taken together, our findings suggest that cbiO mediates biofilm formation by affecting the transportation of copper ions in the MRSA CFSa36 strain. This study provides new insights into the molecular basis of biofilm formation by S. aureus.

Contribution of Coagulase and Its Regulator SaeRS to Lethality of CA-MRSA 923 Bacteremia.

Coagulase is a critical factor for distinguishing Staphylococcus aureus and coagulase-negative Staphylococcus. Our previous studies demonstrated that the null mutation of coagulase (coa) or its direct regulator, SaeRS, significantly enhanced the ability of S. aureus (CA-MRSA 923) to survive in human blood in vitro. This led us to further investigate the role of coagulase and its direct regulator, SaeRS, in the pathogenicity of CA-MRSA 923 in bacteremia during infection. In this study, we found that the null mutation of coa significantly decreased the mortality of CA-MRSA 923; moreover, the single null mutation of saeRS and the double deletion of coa/saeRS abolished the virulence of CA-MRSA 923. Moreover, the mice infected with either the saeRS knockout or the coa/saeRS double knockout mutant exhibited fewer histological lesions and less neutrophils infiltration in the infected kidneys compared to those infected with the coa knockout mutant or their parental control. Furthermore, we examined the impact of coa and saeRS on bacterial survival in vitro. The null mutation of coa had no impact on bacterial survival in mice blood, whereas the deletion mutation of saeRS or coa/saeRS significantly enhanced bacterial survival in mice blood. These data indicate that SaeRS plays a key role in the lethality of CA-MRSA 923 bacteremia, and that coagulase is one of the important virulence factors that is regulated by SaeRS and contributes to the pathogenicity of CA-MRSA 923.

Identification and Characterization of Pleiotropic High-Persistence Mutations in the Beta Subunit of the Bacterial RNA Polymerase.

Mutations conferring resistance to bactericidal antibiotics reduce the average susceptibility of mutant populations. It is unknown, however, how those mutations affect the survival of individual bacteria. Since surviving bacteria can be a reservoir for recurring infections, it is important to know how survival rates may be affected by resistance mutations and by the choice of antibiotics. Here, we present evidence that (i) Escherichia coli mutants with 100 to 1,000 times increased frequency of survival in ciprofloxacin, an archetypal fluoroquinolone antibiotic, can be readily obtained in a stepwise selection; (ii) the high survival frequency is conferred by mutations in the switch region of the beta subunit of the RNA polymerase; (iii) the switch-region mutations are (p)ppGpp mimics, partially analogous to rpoB stringent mutations; (iv) the stringent and switch region rpoB mutations frequently occur in clinical isolates of E. coli, Acinetobacter baumannii, Mycobacterium tuberculosis, and Staphylococcus aureus, and at least one of them, RpoB S488L, which is a common rifampicin resistance mutations, dramatically increases the survival of a clinical methicillin-resistant S. aureus (MRSA) strain in ampicillin; and (v) the RpoB-associated high-survival phenotype can be reversed by subinhibitory concentrations of chloramphenicol.

Application of Mycobacterium smegmatis as a surrogate to evaluate drug leads against Mycobacterium tuberculosis.

Discovery of new anti-tuberculosis (TB) drugs is a time-consuming process due to the slow-growing nature of Mycobacterium tuberculosis (Mtb). A requirement of biosafety level 3 (BSL-3) facility for performing research associated with Mtb is another limitation for the development of TB drug discovery. In our screening of BSL-1 Mycobacterium spp. against a battery of TB drugs, M. smegmatis (ATCC607) exhibits good agreement with its drug susceptibility against the TB drugs under a low-nutrient culture medium (0.5% Tween 80 in Middlebrook 7H9 broth). M. smegmatis (ATCC607) enters its dormant form in 14 days under a nutrient-deficient condition (a PBS buffer), and shows resistance to a majority of TB drugs, but shows susceptibility to amikacin, capreomycin, ethambutol, and rifampicin (with high concentrations) whose activities against non-replicating (or dormant) Mtb were previously validated.

Environmental factors modulate biofilm formation by Staphylococcus aureus.

Biofilm formation on indwelling medical devices represents an exclusive evasion mechanism for many pathogenic bacteria to establish chronic infections. Staphylococcus aureus is one of the major bacterial pathogens that are able to induce both animal and human infections. The continued emergence of multiple drug-resistant S. aureus, especially methicillin-resistant S. aureus, is problematic due to limited treatment options. Biofilm formation by S. aureus complicates the treatment of methicillin-resistant S. aureus infections. Therefore, elucidating the mechanisms of biofilm formation in this pathogen is important for the development of alternative therapeutic strategies. Various environmental and genetic factors contribute to biofilm formation. In this review, we address the environmental factors and discuss how they affect biofilm formation by S. aureus.

Multilocus Sequence Typing of Staphylococcus aureus.

Multilocus sequence typing (MLST) has been successfully used to differentiate and trace the bacterial species and pathogens that cause outbreaks or epidemics of infectious diseases. MLST provides a powerful solution for molecular epidemiological characterization of bacterial strains, including Staphylococcus aureus, by using the sequences of the internal region of seven housekeeping genes. In previous studies, we utilized MLST to analyze the genotypes of S. aureus isolates from pediatric patients with cystic fibrosis and revealed three prevalent ST types, including ST5, ST30, and ST8 in these isolates. In this chapter, we describe a detailed procedure of MLST for genotyping S. aureus.

Identification of Target Genes Mediated by Two-Component Regulators of Staphylococcus aureus Using RNA-seq Technology.

Transcriptomics enables us to elucidate comprehensive gene expression profiles in given experimental conditions. Global regulators, which include transcriptional regulators and two-component regulatory systems, have evolved in a variety of bacterial systems. They play important roles in bacterial fitness and pathogenesis by regulating target gene expression. Advanced next-generation RNA sequencing technology (RNA-seq) provides a powerful and effective tool to analyze the transcriptome of bacterial cells. In this chapter, we provide a detailed procedure for the investigation of gene expression profiles and identification of target genes, regulons, and/or pathways that are mediated by a regulator. This procedure is done using RNA-seq analysis, which involves RNA purification, mRNA enrichment, decontamination, RNA-seq data analysis, and quantitative real-time reverse transcription PCR.

Metabolomic Profiling of Staphylococcus aureus.

Metabolomics is becoming increasingly important in bioscience research as it provides a comprehensive analytical platform for a better understanding of the metabolic functions of cells and organisms. Recently, microbial metabolomics has been utilized in diverse research areas, including detection and diagnosis of pathogens, metabolic engineering, and drug discovery. An efficient and reproducible method to measure the intracellular metabolites of a specific microbial organism is a key prerequisite for utilizing metabolome analysis in microbiological research. In this chapter, we describe a workflow focusing on the extraction and quantification of intracellular metabolites of Staphylococcus aureus. Fast quenching with chilled methanol is applied to minimize metabolite leakage, while solvent extraction is used to obtain both polar and nonpolar fractions, which are then analyzed by respective liquid chromatography-mass spectrometry (LC-MS) methods for characterizing and quantifying the intracellular metabolites of S. aureus. This protocol is demonstrated to be an efficient method for analyzing polar and nonpolar intracellular metabolites of S. aureus.

Determining Impact of Growth Phases on Capacity of Staphylococcus aureus to Adhere to and Invade Host Cells.

One of the largest concerns in public health is the continual emergence of multidrug-resistant bacterial pathogens. The resistance of bacterial pathogens to specific drugs presents a significant problem because it severely limits treatment options. Staphylococcus aureus is a particularly problematic pathogen that is prevalent in human and animal populations. Data on this bacterium have shown that S. aureus is capable of invading different types of host cells, suggesting that multiple mechanisms are behind its ability to thwart a host immune system and evade the toxicity of some antibiotics. S. aureus produces a myriad of cell wall-associated molecules, such as fibronectin-binding proteins, which assist in the adhesion and invasion of the bacterial cell to a host cell. Understanding the expression of these cell wall-associated molecules at different growth phases will improve general knowledge on how this bacterium can adhere to and invade a host. In our previous work, we found that different types of human MRSA isolates possess different abilities to adhere to and invade epithelial cells. In a recent study we conducted, it was found that S. aureus taken from the exponential phase of growth, when compared to S. aureus taken from the stationary phase, had a noticeable higher ability to invade host cells.

Interaction between two essential, conserved bacterial proteins YeaZ and glycoprotease as a potential antibacterial target in multi-drug-resistant Staphylococcus aureus.

Protein-protein interactions among highly conserved and essential proteins can serve as new targets for antibacterial therapies. One protein-protein interaction between two widely conserved and essential bacterial proteins, YeaZ and its paralog, a putative glycoprotease, is being looked into for its antimicrobial drug potential. These two proteins possess tandem functions, including repression of the branched-chain amino acids biosynthesis and induction of a tRNA modification important in enhancing translation fidelity through anticodon-codon base pairing. Heterodimer formation between these two proteins is essential for Staphylococcus aureus, and other bacterial species including Escherichia coli and Salmonella typhimurium. Such YeaZ-glycoprotease interaction could thus be a target for antimicrobial drugs designed for multi-drug-resistant S. aureus. In this review, we discuss the function, structure, and interaction between these two proteins and their orthologs in other bacteria.

Complete Genome Sequence of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Strain WCUH29.

The hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) strain WCUH29 has been intensively and widely used as a model system for identification and evaluation of novel antibacterial targets and pathogenicity. In this announcement, we report the complete genome sequence of HA-MRSA WCUH29 (NCIMB 40771).

Down-regulation of guanylate binding protein 1 causes mitochondrial dysfunction and cellular senescence in macrophages.

Macrophage polarization is tightly associated with its metabolic reprograming and immune dysfunction. However, the intracellular molecules/pathways that connect these alterations in inflammatory macrophages remain largely unidentified. Herein, we explored the role of guanylate binding protein 1 (Gbp1), an intracellular anti-microbial protein, in regulating polarization, metabolic reprogramming, and cellular aging of macrophages. We showed that Gbp1 expression in inguinal white adipose tissue is significantly decreased in high-fat diet -fed and aged mice. Gbp1 expression is significantly induced by IFNγ and LPS in macrophages but not adipocytes. Downregulation of Gbp1 expression causes macrophage polarization towards a pro-inflammatory phenotype. Gbp1 knockdown (Kd) macrophages have impaired mitochondrial respiratory function, which is further supported by down-regulation of genes encoding electron transport chain components and genes involved in fatty acid oxidation and mitochondrial function. Moreover, we observed Gbp1 is localized in both cytosol and mitochondrial fraction, and Gbp1 Kd macrophages display decreased mitophagy activity. More interestingly, Gbp1 Kd macrophages undergo senescence as evidenced by increased activation of AMPK-p53 pathway and positive staining of ß-galactosidase. These observations suggest that Gbp1 may play an important role in protecting against mitochondrial dysfunction and preserving immune function of macrophages during inflammatory stress and aging.