There is now an urgent need to develop reliable, rapid, and cost-effective methods for bacterial detection, particularly for point-of-care applications. This study explores the unique properties of silicon nanowire (SiNW) arrays as a resistive sensing platform for detecting Listeria innocua. Vertically aligned SiNWs, fabricated via metal-assisted chemical etching, exhibited high sensitivity to bacterial adsorption. Conductance measurements revealed a more than 10-fold increase as bacterial concentrations rose from 10(5) to 10(7) CFU/mL, with clear saturation at higher levels. The study employed both direct current (DC) and alternating current (AC) methodologies, with AC conductance consistently outperforming DC due to reduced potential barrier effects. An equivalent circuit model was developed to describe the impedance behavior of the SiNW-bacteria system, offering valuable insights into charge transport mechanisms. These results demonstrate the potential of SiNW-based sensors as robust, scalable, and high-performance diagnostic tools. Beyond bacterial detection, the proposed platform offers promising applications in clinical diagnostics, environmental monitoring, and food safety.

Wild aquatic birds are a major reservoir of the influenza A virus in natural ecosystems, facilitating its entry into the aquatic microbial food web through their feces. Free-living protozoa and particularly bacterivorous ciliates are essential players of the microbial food web. This study investigates the interactions between the Influenza A(H1N1)pdm09 virus and the ciliated protozoan Tetrahymena pyriformis at the population and ultrastructural levels. Co-cultivation of Influenza A(H1N1)pdm09 and T. pyriformis resulted in a decline and eventual complete elimination of the viral population. The inactivation of the virus was not mediated by products excreted by T. pyriformis but required A(H1N1)pdm09 endocytosis. Viruses ingested by protozoa lost their virulence within 48 hours post infection (hpi) and, as determined by hemagglutination assays, were entirely inactivated within 72 hpi. When lysates infected with A(H1N1)pdm09 T. pyriformis were applied to MDCK cells 1.5 and 24 hpi the undamaged part of ingested virions caused a cytopathic effect. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) of infected T. pyriformis cells revealed large food vacuoles, including multiple undamaged and partly processed virus particles, at 1.5 and 24 hpi. Furthermore, TEM identified coated and half-coated small one-virus endosomes that predominated at 48 hpi. These results demonstrated that A(H1N1)pdm09 inactivation by T. pyriformis includes two types of endosomes that dominated at different periods of interpopulation interactions. The process of A(H1N1)pdm09 inactivation in protozoan cells occurs rapidly, but not instantaneously, that suggesting a dual role of protozoa in the fate of influenza A viruses in natural ecosystems, both as predators and as potential vectors.

In this study, a novel approach for simultaneous interferometric and surface-enhanced Raman spectroscopy (SERS) detection of bacteria utilizing porous silicon nanowires (pSi NWs) modified with silver and gold nanoparticles is reported. The pSi NWs were fabricated through gold-assisted chemical etching of p-type, single-crystal silicon wafers with a (100) crystallographic orientation and resistivity between 1 and 5 mΩ·cm. Following etching, the nanowires' surfaces were modified with silver, then gold nanoparticles by reduction from their respective salts in the presence of 5 M HF. This dual-mode sensing platform was tested with Listeria innocua, a nonpathogenic strain of Listeria, demonstrating significant interferometric fringe shifts after bacterial adsorption due to changes in the samples' effective optical thickness. This interferometric method achieved detection limits of L. innocua down to 6.4 × 106 CFU/mL. Additionally, the plasmonic nanoparticle-modified nanowires exhibited strong SERS activity, enabling Raman spectral detection of adsorbed bacteria with a sensitivity limit of 3.2 × 106 CFU/mL. This work demonstrates the potential of AuAg-modified pSi NWs as a versatile and highly sensitive dual-mode optical sensor for rapid bacterial detection, offering both real-time refractive index-based interferometric monitoring and molecularly specific SERS capabilities.

Biofilm formation by pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Escherichia coli, represents a major clinical challenge due to the high resistance of biofilms to conventional antimicrobial therapy. In this in vitro study, we investigated the antimicrobial and antibiofilm activity of synthetic peptides R23IT, R23LP, V31KT, R44KS, R44KP, V31KS, and I31KP against methicillin-resistant S. aureus (MRSA, SA180-F strain), S. aureus (129B), P. aeruginosa (2943), and E. coli (MG1655). In liquid medium, peptides R23LP and R44KS exhibited the broadest and most potent antimicrobial activity against all tested strains. On solid agar, these peptides demonstrated comparable activity, with notable effects particularly against E. coli. We further assessed the peptides’ impact on both early-stage and mature biofilms using crystal violet staining for total biomass and the MTT assay for cellular metabolic activity. Peptide R44KS showed a strong dose-dependent inhibitory effect on early MRSA biofilm formation, while most peptides unexpectedly enhanced biofilm formation by S. aureus and E. coli. Peptides R44KP and V31KS at 10 mg/mL significantly reduced both biomass and metabolic activity of early P. aeruginosa biofilms. None of the peptides inhibited mature biofilm biomass across species; however, several, particularly I31KP, significantly reduced the metabolic activity of MRSA within mature biofilms. These findings underscore the strain- and stage-specific effects of antimicrobial peptides and highlight R23LP, R44KS, R44KP, V31KS, and I31KP as promising candidates for targeted biofilm control in vitro, especially against MRSA.

Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157) strains do not produce curli and do not form biofilms, but they retain their ability for autoaggregation. In our study, we investigated whether curli expression would impact E. coli O157 autoaggregation. Curli-expressing strain CPM1 was derived from E. coli O157 strain ATCC 43890 as a clone forming red colonies on Congo red (CR)-agar. To quantitatively evaluate autoaggregation we applied a recently developed experimental system based on magnetic levitation. The efficiency of autoaggregation was evaluated by the geometry of macroautoaggregates and by relative amounts of aggregated and free-swimming bacteria. The curli producing CPM1 strain's autoaggregates had a volume 3.4 times smaller than that of ATCC 43890, despite the number of autoaggregated CPM1 bacteria being higher. Curli proteins were incorporated into matrix, making CPM1 autoaggregate more compact. Curliated CPM1 bacteria adhered better to vertical surfaces reducing the number of free swimmers. The Ser206Phe substitution in the transcriptional regulator RcsB was responsible for CPM1 curli-expressing phenotype. The mutation affected RcsB interactions with the accessory protein RscA. RcsA hyperexpression inhibited curli production and decreased efficiency of autoaggregation. Taken together, the obtained results demonstrated that removal of RcsB/RcsA-dependent inhibition of curli expression improves autoaggregation in the E. coli O157.

Rapid antibiotic susceptibility testing (AST) is crucial for selecting appropriate antibiotic treatments and customizing empirical therapy to effectively manage serious bacterial infections. This study focuses on developing and characterizing surface-enhanced Raman scattering (SERS)-active nanostructured composite substrates designed for swift and highly sensitive bacterial detection, followed by accelerated AST. The substrates were fabricated by depositing noble metal nanoparticles (Au, Ag) onto porous silicon nanowires (pSiNWs) formed through metal-assisted chemical etching (MACE) of crystalline silicon. Scanning electron microscopy (SEM) images depict Listeria innocua bacteria localized in close proximity to AuAg nanoparticles atop pSiNWs. SERS spectra of L.innocua were acquired using a 633 nm laser beam, enabling rapid label-free bacterial detection. Comparisons with the traditional disk diffusion method show that the developed SERS approach allows for real-time monitoring of bacterial antibiotic susceptibility within 3 h, in contrast to the standard 24-h test duration. These findings underscore the potential of AuAg@pSiNWs substrates to expedite the AST process, offering a significant advantage in clinical diagnostics and antimicrobial resistance monitoring.

The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23FS*, V31KS*, and R44KS*, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus, along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23FS*, V31KS*, and R44KS* exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 μM. The peptides showed a stronger antimicrobial effect against B. cereus. Notably, peptide R44KS* displayed high efficacy compared to peptides R23FS* and V31KS*, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31KS* and R44KS* demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31KS and R44KS. These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens.

—Rapid and accurate bacterial identification plays a crucial role across diverse sectors, offering valuable applications. While traditional culture and molecular techniques maintain a high standard, the contemporary demand for diagnostics centers on swiftly delivering dependable insights into bacterial infections, directly at the site, within a short timeframe. In this study, we present a novel approach utilizing surface-enhanced Raman scattering (SERS)-active nanostructured substrates in the form of gold- and silver-coated porous silicon nanowires (AuAg@pSiNWs) for rapid and highly sensitive bacterial detection. The porous silicon nanowires are fabricated using a straightforward method known as metal-assisted chemical etching. Subsequently, silver and gold decoration is achieved through chemical reduction of metal salts, imbuing the substrates with SERS-active properties. Scanning electron microscopy data reveals that upon incubation with AuAg@pSiNWs, bacteria are localized amidst clusters and on the surface of the nanowires, particularly in the vicinity of bimetallic gold and silver nanoparticles. Illustrated through the utilization of Listeria innocua bacteria as a model, the SERS efficacy of AuAg@pSiNWs is highlighted, enabling rapid label-free bacterial diagnosis with a limit of detection of 1.14 × 10 4 CFU/mL.

Antibiotic resistance is one of the most serious global health threats. Therefore, there is a need to develop antimicrobial agents with new mechanisms of action. Targeting of bacterial cystathionine γ-lyase (bCSE), an enzyme essential for bacterial survival, is a promising approach to overcome antibiotic resistance. Here, we described a series of (heteroarylmethyl)benzoic acid derivatives and evaluated their ability to inhibit bCSE or its human ortholog hCSE using known bCSE inhibitor NL2 as a lead compound. Derivatives bearing the 6-bromoindole group proved to be the most active, with IC50 values in the midmicromolar range, and highly selective for bCSE over hCSE. Furthermore, none of these compounds showed significant toxicity to HEK293T cells. The obtained data were rationalized by ligand-based and structure-based molecular modeling analyses. The most active compounds were also found to be an effective adjunct to several widely used antibacterial agents against clinically relevant antibiotic-resistant strains of such bacteria as Staphylococcus aureus, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The most potent compounds, 3h and 3i, also showed a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. Finally, compound 3i manifested potentiating activity in pneumonia, sepsis, and infected-wound in vivo models.

This paper explores the potential for diagnosing bacterial presence in biological fluids using Fabry-Perot interference in silicon nanostructures with different morphologies. It compares the bacterial detection capabilities of porous silicon films and silicon nanowires, specifically focusing on the detection of Escherichia coli bacteria. The reflectance spectra of these nanostructures exhibit interference fringes resulting from Fabry-Perot interference. By analyzing changes in the effective optical thickness of the silicon nanostructures after bacterial deposition, the study concludes that silicon nanostructures with varying morphologies can be effectively used for bacterial detection.

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 μM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development.

Here we investigated whether it is possible to use magnetic levitation as a model of microgravity. The proteome of E. coli grown under spaceflight or combined spaceflight and magnetic force conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic levitation conditions.

Magnetic force and gravity are two fundamental forces affecting all living organisms, including bacteria. On Earth, experimentally created magnetic force can be used to counterbalance gravity and place living organisms in conditions of magnetic levitation. Under conditions of microgravity, magnetic force becomes the only force that moves bacteria, providing an acceleration towards areas of the lowest magnetic field and locking cells in this area. In this review, we consider basic principles and experimental systems used to create a magnetic force strong enough to balance gravity. Further, we describe how magnetic levitation is applied in on-Earth microbiological studies. Next, we consider bacterial behavior under combined conditions of microgravity and magnetic force onboard a spacecraft. At last, we discuss restrictions on applications of magnetic force in microbiological studies and the impact of these restrictions on biotechnological applications under space and on-Earth conditions.

Surface-enhanced Raman Scattering (SERS) is a powerful vibrational optical spectroscopy method that allows highly sensitive detection of molecules at very low concentrations of matter by amplifying the electromagnetic fields created by excitation of localized surface plasmons on the surface of noble metal nanostructures. In the presented work a method of manufacturing composite nanostructures of silicon nanowires decorated with silver (AgSiNWs) has been developed. The SERS activity of AgSiNWs for protein detection was investigated using human serum albumin as an example. For the first time, the possibility of rapid diagnosis of internalin B (InlB) protein of pathogenic bacteria Listeria monocytogenes by SERS using the obtained nanostructures was shown. In the spectra of InlB adsorbed on AgSiNWs at different concentrations, distinct peaks corresponding to Raman scattering on protein molecules are observed. Based on the experimental data obtained, the detection limit of InlB was calculated to be 4.8· 10-9 M. The results presented in this work demonstrate the high potential of the obtained composite nanostructures for the diagnosis of various proteins by SERS. Keywords: Surface-enhanced Raman scattering, silicon nanowires, composite nanostructures, albumin, listeria, internalin B.

Listeria monocytogenes is motile at 22°C and non-motile at 37°C. In contrast, expression of L . monocytogenes virulence factors is low at 22°C and up-regulated at 37°C. Here, we studied a character of L . monocytogenes near surface swimming (NSS) motility and its effects on adhesion patterns and invasion into epithelial cells. L . monocytogenes and its saprophytic counterpart L . innocua both grown at 22°C showed similar NSS characteristics including individual velocities, trajectory lengths, residence times, and an asymmetric distribution of velocity directions. Similar NSS patterns correlated with similar adhesion patterns. Motile bacteria, including both pathogenic and saprophytic species, showed a preference for adhering to the periphery of epithelial HEp-2 cells. In contrast, non-motile bacteria were evenly distributed across the cell surface, including areas over the nucleus. However, the uneven distribution of motile bacteria did not enhance the invasion into HEp-2 cells unless virulence factor production was up-regulated by the transient shift of the culture to 37°C. Motile L . monocytogenes grown overnight at 22°C and then shifted to 37°C for 2 h expressed invasion factors at the same level and invaded human cells up to five times more efficiently comparatively with non-motile bacteria grown overnight at 37°C. Taken together, obtained results demonstrated that (i) NSS motility and correspondent peripheral location over the cell surface did not depend on L . monocytogenes virulence traits; (ii) motility improved L . monocytogenes invasion into human HEp-2 cells within a few hours after the transition from the ambient temperature to the human body temperature.

Objective. To evaluate the potential of magnetic levitation systems when studying the autoaggregation of gram-negative and gram-positive pathogenic bacteria and elucidating mechanisms controlling autoaggregation. Materials and methods. Escherichia coli O157:H7, Pseudomonas aeruginosa , Staphylococcus aureus , and Listeria monocytogenes were used. The number of alive bacteria was determined using a Live/Dead® dye. E. coli curli were stained with Congo red. Results. All four tested bacterial species formed autoaggregates that levitated within the liquid volume for up to 72 hours (observation time). After 72 hours, the number of alive bacteria in the autoaggregates ranged from 82% ( E. coli ) to 99% ( L. monocytogenes ). The formation of E. coli autoaggregates was shown to depend on the production of curli, which represent surface structures playing an important role in biofilm formation. Conclusion. The proposed system of magnetic levitation can be used to study molecular mechanisms of bacterial autoaggregation and flocculation.

Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms on L. monocytogenes uptake and intracellular proliferation in human macrophages. Three isoforms of the receptor binding domain (idInlB) were derived from phylogenetically distinct L. monocytogenes strains belonging to the highly virulent CC1 (idInlBCC1), medium-virulence CC7 (idInlBCC7), and low-virulence CC9 (idInlBCC9) clonal complexes. The constant dissociation increased in the order idInlBCC1 << idInlBCC7 < idInlBCC9 for interactions with c-Met, and idInlBCC1 ≈ idInlBCC7 < idInlBCC9 for interactions with gC1q-R. The comparison of uptake and intracellular proliferation of isogenic recombinant strains which expressed full-length InlBs revealed that the strain expressing idInlBCC1 proliferated in macrophages twice as efficiently as other strains. Macrophage pretreatment with idInlBCC1 followed by recombinant L. monocytogenes infection disturbed macrophage functions decreasing pathogen uptake and improving its intracellular multiplication. Similar pretreatment with idInlBCC7 decreased bacterial uptake but also impaired intracellular multiplication. The obtained results demonstrated that InlB impaired macrophage functions in an idInlB isoform-dependent manner. These data suggest a novel InlB function in L. monocytogenes virulence.

The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.

Aeromonas spp. are gram-negative facultatively anaerobic bacilli recovered mainly from aquatic environments. Aeromonas spp. were reported to be associated with infections primarily in aquatic and to a lesser extent in terrestrial animals as well as in humans. Up-to-date little is known about aeromonads associated with wild animals, especially with rodents. This study reported the first isolation and characterization of two Aeromonas spp. from internal organs of apparently healthy wild rodents Apodemus uralensis and Apodemus flavicollis captured in the wild environment in the European part of Russia. Isolates were identified as A. hydrophila M-30 and A. encheleia M-2 using the multilocus sequence analysis (MLSA) approach. The isolation of the A. encheleia from rodents is the first described case. Both strains demonstrated beta-hemolytic activity towards human erythrocytes. Antimicrobial susceptibility testing showed that both Aeromonas strains were resistant and intermediate to carbapenems and piperacillin-tazobactam, which was caused by the expression of the genus-specific CphA carbapenemases. A. hydrophila M-30 also demonstrated trimethoprim resistant phenotype. This is usually caused by the carriage of the dfrA or dfrB genes in aeromonads which are frequently associated with integron class I. The latter however was absent in both isolates. Our results expand our understanding of possible aeromonad reservoirs and demonstrate the likelihood of the formation of natural foci of Aeromonas infection and a new link in the chain of the spread of antimicrobial resistance as well.

The paper compares the autoaggregation ability of the enteropathogenic bacterium Escherichia coli O157:H7 and its transcription factor RcsB mutant using a new system based on magnetic levitation. It has been found that the transcription factor RcsB affects the process of autoaggregation through the control of curli-fimbria.