The acylphloroglucinol rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clinical applications, very little is known about the mechanism of action of rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target. Using live cell, high-resolution, and electron microscopy we demonstrate that rhodomyrtone causes large membrane invaginations with a dramatic increase in fluidity, which attract a broad range of membrane proteins. Invaginations then form intracellular vesicles, thereby trapping these proteins. Aberrant protein localization impairs several cellular functions, including the respiratory chain and the ATP synthase complex. Being uncharged and devoid of a particular amphipathic structure, rhodomyrtone did not seem to be a typical membrane-inserting molecule. In fact, molecular dynamics simulations showed that instead of inserting into the bilayer, rhodomyrtone transiently binds to phospholipid head groups and causes distortion of lipid packing, providing explanations for membrane fluidization and induction of membrane curvature. Both its transient binding mode and its ability to form protein-trapping membrane vesicles are unique, making it an attractive new antibiotic candidate with a novel mechanism of action.

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Staphylococcus pseudintermedius is a zoonotic pathogen that can cause life-threatening infections in animals and humans. The study of methicillin-resistant S. pseudintermedius (MRSP) and its ability to produce biofilms is important to select the most suitable treatment. The prevalence and characteristics of S. pseudintermedius isolated from dogs admitted at the Veterinary Teaching Hospital, Prince of Songkla University, Thailand were assessed. Results showed that 28.30% (15/53) of the isolates were MRSP. Amplification of the mecA gene was observed in 93.33% (14/15) MRSP. Methicillin-resistant strains revealed co-resistant patterns against other antibiotics, including chloramphenicol, clindamycin, tetracycline, clarithromycin, ciprofloxacin, and trimethoprim. In this study, all bacterial isolates produced biofilms, while 90.55% of S. pseudintermedius isolates were strong or moderate biofilm producers. Most (45–60%) of the resistant strains were strong biofilm producers, while the correlation between biofilm production and antibiotic resistance was not statistically significant. This is the first study in southern Thailand to investigate the drug-resistant profile of S. pseudintermedius and its ability to form biofilm. The results will contribute to a better understanding of the emergence and prevalence of antimicrobial resistance in S. pseudintermedius.

Rhodomyrtone, a natural antimicrobial compound, displays potent activity against many Gram-positive pathogenic bacteria, comparable to last-defence antibiotics including vancomycin and daptomycin. Our previous studies pointed towards effects of rhodomyrtone on the bacterial membrane and cell wall. In addition, a recent molecular docking study suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ. In this study, we applied a computational approach (in silico), in vitro, and in vivo experiments to investigate molecular interactions of rhodomyrtone with FtsZ. Using molecular simulation, FtsZ conformational changes were observed in both (S)- and (R)-rhodomyrtone binding states, compared with the three natural states of FtsZ (ligand-free, GDP-, and GTP-binding states). Calculations of free binding energy showed a higher affinity of FtsZ to (S)-rhodomyrtone (−35.92 ± 0.36 kcal mol−1) than the GDP substrate (−23.47 ± 0.25 kcal mol−1) while less affinity was observed in the case of (R)-rhodomyrtone (−18.11 ± 0.11 kcal mol−1). In vitro experiments further revealed that rhodomyrtone reduced FtsZ polymerization by 36% and inhibited GTPase activity by up to 45%. However, the compound had no effect on FtsZ localization in Bacillus subtilis at inhibitory concentrations and cells also did not elongate after treatment. Higher concentrations of rhodomyrtone did affect localization of FtsZ and also affected localization of its membrane anchor proteins FtsA and SepF, showing that the compound did not specifically inhibit FtsZ but rather impaired multiple divisome proteins. Furthermore, a number of cells adopted a bean-like shape suggesting that rhodomyrtone possibly possesses further targets involved in cell envelope synthesis and/or maintenance.

A large-scale surveillance is an important measure to monitor the regional spread of antimicrobial resistance. We prospectively studied the prevalence and molecular characteristics of clinically important Gram-negative bacilli, including Escherichia coli (EC), Klebsiella pneumoniae (KP), Acinetobacter baumannii complex (ABC), and Pseudomonas aeruginosa (PA) from blood, respiratory tract, urine, and sterile sites at 47 hospitals across Thailand. Among 187,619 isolates, 93,810 isolates (50.0%) were critically drug-resistant. Of which, 12,915 isolates (13.8%) were randomly selected for molecular characterization. EC was most commonly isolated from all specimens, except the respiratory tract in which ABC was predominant. Prevalence of extended-spectrum cephalosporin resistance (ESCR) was higher in EC (42.5%) than KP (32.0%), but carbapenem-resistant (CR)-KP (17.2%) was 4.5-fold higher than CR-EC (3.8%). A majority of ESCR-/CR-EC and KP carried bla CTX-M (64.6%-82.1%). bla NDM and bla OXA-48-like were the most prevalent carbapenemase genes in CR-EC/CR-KP (74.9%/52.9% and 22.4%/54.1%, respectively). Besides, 12.9%/23.0% of CR-EC/CR-KP co-carried bla NDM and bla OXA-48-like. Among ABC, 41.9% were extensively drug-resistant (XDR), and 35.7% were multidrug-resistant (MDR), while PA showed XDR/MDR at 6.3%/16.5%. A. baumannii (AB) was the most common species among ABC isolates. The major carbapenemase gene in MDR-AB/XDR-AB was bla OXA-23-like (85.8%/93.0%), which had much higher rates than other ABC species. bla IMP , bla VIM , bla OXA-40-like, and bla OXA-58-like were also detected in ABC at lower rates. The most common carbapenemase gene in MDR-/XDR-PA was bla IMP (29.0%/30.6%), followed by bla VIM (9.5%/25.3%). The findings reiterate an alarming situation of drug resistance that requires serious control measures.

Strong evidence of high potency of rhodomyrtone as a promising antibacterial agent against pathogenic gram-positive bacteria has been clearly demonstrated in our previous work. The aim of this study was to provide insight into early action of rhodomyrtone, an acylphloroglucinol, on membrane damage in multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA). Early effects of rhodomyrtone on the bacterial membrane integrity were detected in a time-course study. Flow cytometry revealed a reduction in green fluorescent emission and increase in uptake of propidium iodide in rhodomyrtone-treated bacterial cells in a concentration- and time-dependent manner. Disruption of cytoplasmic membrane was further monitored by measuring cellular adenosine triphosphate (ATP) and potassium ion (K). Leakage of both ATP and K and significant decrease in intracellular ATP in MRSA were observed following treatment. Pronounced changes in the bacterial ultrastructure and morphology were confirmed by transmission electron microscopy and scanning electron microscopy. Bacterial cell disruption, holes in cell surface, and bulge formations were noted in rhodomyrtone-treated cells. In this study, we provided relevant data to clarify that rhodomyrtone is a bacterial cell membrane-damaging agent. A possible early effect of this novel compound involves bacterial membrane disruption.

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The emergence of drug-resistant bacteria has impacted the outcome of current therapeutics as a threat to global healthcare; novel medicines are urgently needed. Thirteen medicinal plants were collected in Northeastern Thailand, and their crude ethanolic extracts were evaluated for antibacterial activities against Staphylococcus aureus ATCC25923 and Escherichia coli ATCC25922 using the broth micro-dilution method. Piper betle leaf ethanolic extract showed optimal activity against both representative bacterial strains. Activity was also observed against clinical isolates of methicillin-resistant S. aureus (MRSA) and E. coli, with minimal inhibitory concentration (MIC) ranging from 0.31 mg/mL to 2.5 mg/mL and minimal bactericidal concentration (MBC) ranging from 0.62 mg/mL to 2.5 mg/mL. A time-kill study revealed that the extract activity was time- and dose-dependent, and also bactericidal on the tested bacteria. P. betle extract inhibited biofilm formation and promoted biofilm eradication in both S. aureus and E. coli. 4-Allyl-1,2-diacetoxybenzene and eugenol were identified as the most abundant compounds in the extract and may play major roles in the anti-bacterial and anti-biofilm activity. Results suggest that ethanolic P. betle leaf extract shows promise as an alternative method for the prevention of bacterial diseases.