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Microbial persisters are the featured tiny sub-population of microorganisms that are highly tolerant to multiple antimicrobials. Currently, studies on persisters remain a considerable challenge owing to technical limitations. Here, we explored the application of single-cell Raman spectroscopy (SCRS) in the investigation of persisters. Escherichia coli (ATCC 25922) cells were treated with a lethal dosage of ampicillin (100 μg/mL, 32 × MIC, 4 h) for the formation of persisters. The biochemical characters of E. coli and its persisters were assessed by SCRS, and their metabolic activities were labeled and measured with D2O-based single-cell Raman spectroscopy (D2O-Ramanometry). Notable differences in the intensity of Raman bands related to major cellular components and metabolites were observed between E. coli and its ampicillin-treated persisters. Based on their distinct Raman spectra, E. coli and its persister cells were classified into different projective zones through the principal component analysis and t-distributed stochastic neighbor embedding. According to the D2O absorption rate, E. coli persisters exhibited higher metabolic activities than those of untreated E. coli. Importantly, after the termination of ampicillin exposure, these persister cells showed a temporal pattern of D2O intake that was distinct from non-persister cells. To our knowledge, this is the first report on identifying E. coli persisters and assessing their metabolic activities through the integrated SCRS and D2O-Ramanometry approach. These novel findings enhance our understanding of the phenotypes and functionalities of microbial persister cells. Further investigations could be extended to other pathogens by disclosing microbial pathogenicity mechanisms for developing novel therapeutic strategies and approaches.

Microbial persisters enable the development of certain intrinsic strategies for survival with extreme tolerance to multiple antimicrobials. Porphyromonas gingivalis is considered to be the “keystone” periodontopathogen. Indeed, periodontitis, as a highly common inflammatory disease, is the major cause of severe tooth loss and edentulism in adults globally, and yet it is crucially involved in various systemic comorbidities like diabetes. We have recently revealed P. gingivalis persisters-induced perturbation of immuno-inflammatory responses and effective suppression of this key pathogen by bismuth drugs. This study further explored novel approaches to eradicating P. gingivalis persisters through synergistic combination of colloidal bismuth subcitrate (CBS) with traditional antibiotics. P. gingivalis (ATCC 33277) cells in planktonic and biofilm states were cultured to stationary phase, and then treated with metronidazole (100 mg/L), amoxicillin (100 mg/L), CBS, (100 μM) and combinations of these medications, respectively. Persister survival rate was calculated by colony-forming unit. Cell viability and cytotoxicity of CBS were assessed in human gingival epithelial cells (HGECs). Notably, CBS combined with metronidazole enabled the effective eradication of P. gingivalis persisters in planktonic mode, and nearly eliminated their existence in biofilm mode. Importantly, CBS exhibited no effects on the viability of HGECs, along with minimal cytotoxicity (

Candida albicans biofilms display markedly increased antifungal resistance, and the underlying mechanisms remain unclear. This study investigated the signature profiles of C. albicans planktonic cells and biofilms in response to caspofungin (CAS) by mass spectrometry-based shotgun proteomics. We found that C. albicans biofilms were twofold more resistant to CAS with reference to planktonic cells. Notably, 9.6% of C. albicans biofilm cells survived the lethal treatment of CAS (128 μg/ml), confirmed by LIVE/DEAD staining, confocal laser scanning microscopy (CLSM) and scanning electron microscopy analyses. The responses of C. albicans planktonic cells and biofilms to CAS treatment at respective minimum inhibitory concentrations (MICs) were assessed by high-throughput proteomics and bioinformatics approaches. There were 148 and 224 proteins with >twofold difference identified from the planktonic cells and biofilms, respectively. CAS treatment downregulated several cell wall- and oxidative stress-related proteins. Whereas, CAS-induced action was compensated by markedly increased expression of many other proteins involved in cell wall integrity and stress response (e.g., heat shock proteins). Moreover, considerable expression changes were identified in metabolism-associated proteins like glycolysis, tricarboxylic acid (TCA) cycle and ATP biosynthesis. Importantly, various key proteins for cell wall integrity, stress response and metabolic regulation (e.g., PIL1, LSP1, HSP90, ICL1, and MLS1) were exclusively enriched and implicated in C. albicans biofilms. This study demonstrates that C. albicans biofilms undergo highly complicated yet complex regulation of multiple cellular pathways in response to CAS. Signature proteins essential for modulating cell wall integrity, stress response and metabolic activities may account for the antifungal resistance of C. albicans biofilms.

Candida bloodstream infections (CBI) are one of the most common nosocomial infections globally, and they account for a high mortality rate. The increasing global prevalence of drug-resistant Candida strains has also been posing a challenge to clinicians. In this study, we comprehensively evaluated the biofilm formation and production of hemolysin and proteinase of 63 CBI isolates derived from a hospital setting in Hong Kong as well as their antifungal susceptibility both in the presence and in the absence of human serum, using standard methodology. Candida albicans was the predominant species among the 63 CBI isolates collected, and non-albicans Candida species accounted for approximately one third of the isolates (36.5%). Of them, Candida tropicalis was the most common non-albicans Candida species. A high proportion (31.7%) of the CBI isolates (40% of C. albicans isolates, 10% of C. tropicalis isolates, 11% of C. parapsilosis isolates, and 100% of C. glabrata isolates) were found to be resistant to fluconazole. One of the isolates (C. tropicalis) was resistant to amphotericin B. A rising prevalence of drug-resistance CBI isolates in Hong Kong was observed with reference to a previous study. Notably, all non-albicans Candida species, showed increased hemolytic activity relative to C. albicans, whilst C. albicans, C. tropicalis, and C. parapsilosis exhibited proteinase activities. Majority of the isolates were capable of forming mature biofilms. Interestingly, the presence of serum distorted the yeast sensitivity to fluconazole, but not amphotericin B. Taken together, our findings demonstrate that CBI isolates of Candida have the potential to express to varying extent their virulence attributes (e.g., biofilm formation, hemolysin production, and proteinase activity) and these, together with perturbations in their antifungal sensitivity in the presence of serum, may contribute to treatment complication in candidemia. The effect of serum on antifungal activity warrants further investigations, as it has direct clinical relevance to the treatment outcome in subjects with candidemia. [ABSTRACT FROM AUTHOR]