Microbiology spectrum最新文献

Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) offers high sensitivity for the rapid identification of microbial isolates. While predominantly used in bacterial and yeast identification within the clinical laboratory, recent advancements have prompted interest in expanding MALDI-TOF MS applications for the rapid identification of filamentous fungi, particularly clinically relevant molds. However, the implementation of mold identification protocols introduces biosafety concerns due to the potential for spore aerosolization during sample handling and inoculation, posing occupational hazards to laboratory personnel. To evaluate the feasibility and safety of MALDI-TOF MS-based mold identification, we conducted a prospective study comparing an extraction-free approach to two different rapid extraction methods across routine media and specialized fungal plates. Each method was assessed for spectral quality, reproducibility, and identification accuracy using a validated reference library. Importantly, we performed a biosafety validation study to assess the efficacy of the extraction-free approach in inactivating mold spores and hyphal elements. The extraction-free approach generally outperformed the rapid extraction methods, demonstrating more accurate fungal identifications (90.5% vs. 61.9% and 66.7%) and a higher average spectra score; accuracy and spectra scores were further enhanced using specialized fungal media (92.9% vs. 81.0% and 52.4%). Our findings support the use of this extraction-free approach as both a safe and effective method to rapidly identify filamentous fungi using MALDI-TOF MS within the clinical laboratory.IMPORTANCEThis study overviews the implementation of identifying clinically relevant filamentous fungi using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in a clinical laboratory. The methodology applied in this study produced both highly sensitive and specific results with significantly reduced turnaround time compared to traditional standard of care culture-based workup. Faster time to detection has the potential to impact the healthcare treatment of millions of patients each year without requiring significant changes to clinical workflow. This study used multiple approaches to improve the clinical utility and performance of MALDI-TOF MS for fungal identification. Importantly, it also highlights the effectiveness of a developed inactivation method to ensure laboratory personnel safety. This method and workflows are of interest to support clinical microbiology diagnostics and to help aid in infection identification to enhance timely treatment.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) offers fast identification to improve time to appropriate antibiotics in Gram-negative bloodstream infections (GN-BSIs). This retrospective study evaluated patients with GN-BSI PRE- and POST-implementation of rapid identification with and without direct antimicrobial susceptibility testing (AST). Patients aged 0-18 years with GN-BSI before and after implementation of direct blood culture broth MALDI-TOF MS and AST were evaluated. Cohorts were defined by methods of organism identification and AST: traditional culture and AST methods (PRE), direct from broth identification by MALDI-TOF MS with culture-dependent AST (MALDI + cAST) and direct from broth identification MALDI-TOF MS with direct AST (MALDI + dAST). A total of 246 pediatric patients were included, 78 PRE, 92 MALDI + cAST, and 76 MALDI + dAST. Both POST-implementation groups had decreased median time from blood culture collection to identification by over 24 h (16.1 h MALDI + cAST and 15.5 h MALDI + dAST vs 42.9 h PRE; P < 0.0001). MALDI + dAST had decreased median time to susceptibilities (34.3 h MALDI + dAST vs 44.4 h PRE and 51.4 h MALDI + cAST; P < 0.0001) and decreased time to optimal therapy (TTOT) compared with PRE and MALDI + cAST (51.4 h MALDI + dAST vs 57.1 h PRE and 58.2 h MALDI + cAST; P = 0.03). There was no significant difference in time to effective therapy (TTET). Time to discontinuation of Gram-positive coverage decreased in MALDI + cAST and MALDI + dAST cohorts (39.3 h MALDI + cAST and 37.1 h MALDI + dAST vs 49.1 PRE; P = 0.03). The implementation of a rapid ID and AST approach significantly reduced the time to identification and time to susceptibility. However, a 17-h gap in antibiotic optimization highlights the need for additional interventions.IMPORTANCERapid pathogen identification and susceptibility testing is essential for optimizing antimicrobial therapy in pediatric Gram-negative bloodstream infections. This study demonstrates that direct from blood culture broth identification with MALDI-TOF MS along with direct AST led to faster time to susceptibilities and time to optimal therapy in patients with GN-BSI. Despite this, there exists a 17-h gap between results and optimization of antibiotics. This suggests rapid diagnostic testing has not been effectively integrated into clinical practice and the need for active antimicrobial stewardship to improve the impact of these faster laboratory diagnostics.

Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus are major causes of hospital-acquired infections worldwide. However, their genomic features remain underexplored in many regions, particularly in low- and middle-income countries (LMICs). Here, we investigated the genomic diversity, antibiotic resistance, and virulence traits of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus isolates from three tertiary care hospitals in Mexico City. A total of 101 isolates collected between 2006 and 2019 from newborns and adults with diverse infections were analyzed using whole-genome sequencing. MRSA isolates were restricted to clonal complexes CC5 and CC8, whereas MSSA strains displayed a broader diversity across multiple lineages. A distinct clade of CC8-MRSA isolates identified in 2013 among newborns was closely related to the USA300 lineage but carried SCCmec IVc without the Panton-Valentine leukocidin genes and the COMER island. Similarly, a localized cluster of CC5-MSSA isolates from neonates in 2012 showed genomic variations largely driven by prophage-associated elements. MRSA strains carried a higher burden of resistance genes, including fluoroquinolone-associated mutations, whereas MSSA isolates exhibited greater heterogeneity in virulence genes and spa types. Both groups shared a conserved virulence gene repertoire; however, variations in the virulence genes highlighted lineage-specific pathogenic features.

Importance: MRSA and MSSA S. aureus genomes are still largely unstudied in Mexico although they are frequently found in hospitals. This study provides a long-term genomic analysis of MRSA and MSSA isolates from three hospitals in Mexico. Our findings revealed the presence of S. aureus international clones of clonal complexes CC5 and CC8 over a decade, broader diversity in MSSA, and localized clonal transmission within hospitals. The contrasting resistance and virulence profiles of MRSA and MSSA clones underscore the need for genomic surveillance frameworks to inform infection control and antibiotic stewardship in healthcare settings.

Hypervirulent (hvKP) and classical (cKP) strains of Klebsiella pneumoniae are major contributors to serious hospital-acquired infections, and the growing issue of antibiotic resistance highlights the urgent need for new antimicrobial approaches. In this study, we investigated the inhibitory effects of Sarcotoxin II 50, an insect-derived antimicrobial peptide, on K. pneumoniae. Our findings show that Sarcotoxin II 50 exerts potent antibacterial activity by impairing capsular polysaccharide (CPS) production and fimbrial formation, which compromises bacterial adhesion and colonization capacity. In vitro experiments demonstrated that Sarcotoxin II 50 effectively suppressed the growth of cKP. Mechanistic analyses revealed that the peptide interferes with CPS biosynthesis and alters the structure and expression of type 1 fimbriae (fimA) and type 3 fimbriae (mrkA, mrkD, mrkH), which are associated with reduced bacterial tissue invasion and attenuated virulence in a murine abscess model. Overall, this study provides a detailed mechanistic understanding of how Sarcotoxin II 50 inhibits K. pneumoniae both in vitro and in vivo under the tested experimental conditions. These results offer a mechanistic basis for the development of novel antimicrobial therapies targeting K. pneumoniae infections and support the further exploration of this peptide as a promising antimicrobial candidate. Characterization of a new insect peptide, Sarcotoxin II 50, with anti-K. pneumoniae activity from the housefly for the first time.IMPORTANCESarcotoxin II 50 inhibited K. pneumoniae invasion into A549 and BEAS-2B lung epithelial cells. Sarcotoxin II 50 inhibits K. pneumoniae invasion into epithelial cells by affecting capsular polysaccharide synthesis, fimbriae formation, and cell division. This manuscript investigates, for the first time, the function and mechanism of the Sacrotoxin II family member in combating K. pneumoniae, 40 years after its initial identification.

Sustainable food sanitation and preservation technologies are highly needed to meet the increasing demand for healthy and safe food. In this work, we investigated the effects of a recently developed antimicrobial triple formulation (TF) comprised of a natural polyphenol gallic acid amended with the generally recognized as safe materials hydrogen peroxide and DL-lactic acid, on a gram-positive bacterium Listeria innocua during its exponential and stationary growth phases. Our findings revealed the physiological changes associated with the application of this material, as observed using single-cell flow cytometry, photometric assays, as well as confocal and scanning electron microscopy. Both exponential- and stationary-phase L. innocua cells lost culturability after incubation with TF, but their cellular responses were different. After 30 min of contact with the TF, the nonculturable, stationary-phase cells maintained membrane integrity, membrane potential, stable respiratory electron-transport chains, and high levels of intracellular ATP. These phenotypic features, characteristic of the viable but nonculturable (VBNC) adaptation strategy, appear to be associated with upregulation of the stress-response factor sigB. In contrast, in TF-treated exponential-phase cells, membrane integrity was severely compromised and membrane potential showed hyperpolarization, suggesting sublethal injury accompanied by downregulation of sigB. Furthermore, the TF treatment enhanced the formation of extracellular vesicles in L. innocua and the deposition of extracellular polymeric substances in stationary-phase cells. The overall findings of this work demonstrate that sensitivity of Listeria cells to nature-based antimicrobials depends on their growth phase and, therefore, modulating their growth-phase-associated physiological status may counteract their VBNC defense strategy and improve biocidal efficacy.IMPORTANCEIn this work, we examined the efficacy of our novel, nature-based green sanitizer as a means to improve microbiological food safety without health and environmental risks, using gram-positive Listeria innocua as a model organism. L. innocua serves as a surrogate of the foodborne pathogen L. monocytogenes for evaluating sanitation efficacy and may also be involved in virulence and multidrug resistance transfer between the Listeria species. We demonstrate that the sanitizer effect depends on the physiological state of bacterial cells, which is affected by their growth phase. The treatment caused membrane damage to L. innocua in its exponential phase but induced the viable but nonculturable state during its stationary phase. Thus, this study demonstrates a way to overcome bacterial defense mechanisms and improve sanitizer efficacy by modulating the growth-phase-associated physiological status of targeted bacteria.

Antimicrobial resistance in gram-negative bacteria has renewed reliance on colistin, yet resistance to this last-line agent is increasing. In Klebsiella pneumoniae, activation of the PmrA/PmrB two-component system remodels lipid A and reduces colistin binding, motivating the sensor kinase PmrB as an adjuvant target. Here, we combined computational drug repurposing with experimental validation to identify colistin potentiators. High-throughput virtual screening of DrugBank using integrated ligand- and structure-based approaches shortlisted five approved drugs (mebendazole, flurbiprofen, tirbanibulin, flufenamic acid, and netarsudil) for experimental evaluation. In vitro assays against colistin-resistant K. pneumoniae showed flufenamic acid most robustly resensitized resistant isolates and suppressed the emergence of additional resistance under combination exposure. Mechanistic studies indicated that the colistin-flufenamic acid combination increased membrane permeability and significantly downregulated PmrA/PmrB-regulated genes (pmrC and arnT). Molecular docking and molecular dynamics simulations further supported a plausible interaction of flufenamic acid with the PmrB ATPase region, and representative PmrB substitutions (D150Y, T157P, and R256G) did not alter the predicted binding mode. Together, these data suggest that modulation of the PmrA/PmrB pathway may contribute to colistin potentiation and demonstrate a target-guided repurposing-to-validation framework for antibiotic adjuvant discovery in colistin-resistant K. pneumoniae.IMPORTANCEColistin is one of the last remaining treatment options for multidrug-resistant Klebsiella pneumoniae infections, but resistance to this drug is rising worldwide. In this study, we used a PrmB-informed, computer-guided drug repurposing workflow followed by in vitro validation to identify approved compounds that can potentiate colistin activity against colistin-resistant K. pneumoniae. This approach independently prioritized flufenamic acid as the most robust colistin enhancer among the screened candidates, restoring colistin activity and reducing the emergence of additional resistance under combination exposure. Mechanistically, our findings are consistent with modulation of the PmrB/PmrA resistance pathway, supporting bacterial histidine-kinase signaling as a promising direction for antibiotic adjuvant development. Overall, this study highlights a practical and scalable framework for discovering resistance-targeted adjuvants to help protect last-resort antibiotics and improve treatment options for difficult-to-treat infections.

Biomonitoring is a widely used strategy for evaluating chemical contamination. Two species are used in particular in French rivers: Dreissena polymorpha and Gammarus fossarum. Although biomonitoring of fecal contamination has been initiated in the freshwater mussel D. polymorpha, relatively few studies are available for crustaceans. This study aimed to determine, for the first time, the value of the freshwater crustacean G. fossarum for monitoring viral contamination in comparison with D. polymorpha. Laboratory exposure was first performed to determine their ability to accumulate infectious F-specific RNA bacteriophages (FRNAPH). In situ exposure was then conducted to confirm this ability in the field, and also to study the bioaccumulation of viral genomes (FRNAPH and norovirus), along a continuum of fecal contamination. Laboratory exposure showed a significant difference in accumulation, with D. polymorpha (bioaccumulation factor [BAF] ~ 1) accumulating six times more infectious FRNAPH than G. fossarum (BAF ~ 0.3). In situ exposure confirmed that G. fossarum (BAF ~ 17) accumulated less infectious FRNAPH compared with mussels (BAF ~ 3,500). Moreover, FRNAPH and norovirus genomes were only quantified in mussels. Finally, the concentrations measured in D. polymorpha evidenced a fecal contamination gradient. D. polymorpha therefore appears to be more relevant than G. fossarum for monitoring viral contamination of fecal origin in freshwater environments.

Importance: Biomonitoring is a common method for assessing chemical contamination. In France, Dreissena polymorpha (zebra mussel) and Gammarus fossarum (a freshwater crustacean) are widely used species. This study aimed to compare, for the first time, their effectiveness in monitoring viral contamination, specifically F-specific RNA bacteriophages (FRNAPH) and noroviruses. Laboratory experiments showed that D. polymorpha accumulated six times more infectious FRNAPH than G. fossarum. Field exposures confirmed these findings, revealing that only mussels accumulated detectable levels of FRNAPH and norovirus genomes. Additionally, the viral concentrations in D. polymorpha reflected a gradient of fecal contamination across sites. These results demonstrate that D. polymorpha is significantly more effective than G. fossarum for monitoring viral contamination of fecal origin in freshwater environments, making it the more suitable species for such biomonitoring efforts.

Intensive pesticide use drives antimicrobial resistance (AMR) in agriculture, yet the effects of specific practices remain poorly understood. This study evaluated the impact of dairy manure and agrochemicals (glyphosate, copper, streptomycin, and propiconazole) on the composition of culturable AMR bacteria (CARB), AMR genes (ARGs; n = 87), and the microbiome in a processing tomato field (n = 64 experimental plots). Glyphosate-treated plots harbored the lowest levels of CARB, but the highest prevalence of ARGs (especially tetA, tetB, OXA-50, and OXA-58) in the tomato leaves (P < 0.05). Manure-treated plots had the highest levels of CARB and ARGs in the soil and in tomato leaves (especially ACT-1, LAT, MIR, aadA1, and aphA6). The prevalence of multiple ARGs (IMP-12, ACT-1, DHA, MIR, MOX, OXA-58, OXA-60, ermB, oprj, and oprm) was lower in streptomycin- or propiconazole-treated plots compared to non-treated plots. Shifts in the soil and leaf microbiome correlated with changes in ARG composition, particularly aminoglycoside-, fluoroquinolone-, and beta-lactamase-associated genes. These findings show that dairy manure, glyphosate, and propiconazole significantly alter the tomato field microbiome and ARG landscape, indicating that fungicide and herbicide applications may contribute to AMR development and dissemination similar to conventional antibacterial agents in agricultural ecosystems.IMPORTANCEPlant agricultural practices are commonly used by farmers to assure the yield and quality of crops; however, they are also associated with the emergence and dissemination of antimicrobial-resistant (AMR) pathogens. AMR is a critical concern in plant agriculture, as it can affect food safety, security, and sustainability. To combat this issue, it is critical to understand the impact of agricultural practices on AMR. Our study demonstrated that biological amendment (dairy manure) and pesticides (glyphosate, copper, streptomycin, and propiconazole) significantly exacerbated the AMR burden in the applied tomato field, which could increase the food safety risk of the fruit. Findings from this study will raise awareness among farmers, policymakers, and consumers, promote responsible and judicious use of antimicrobial agents in plant agriculture, and prioritize the development of sustainable practices to mitigate current and future AMR challenges.