Optimizing Antimicrobial Peptide Design: Integration of Cell-Penetrating Peptides, Amyloidogenic Fragments, and Amino Acid Residue Modifications.

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.

(Heteroarylmethyl)benzoic Acids as a New Class of Bacterial Cystathionine γ-Lyase Inhibitors: Synthesis, Biological Evaluation, and Molecular Modeling.

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.

Enhancing the Antimicrobial Properties of Peptides through Cell-Penetrating Peptide Conjugation: A Comprehensive Assessment.

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.

Motility provides specific adhesion patterns and improves Listeria monocytogenes invasion into human HEp-2 cells.

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.

Natural Isoforms of Listeria monocytogenes Virulence Factor Inlb Differ in c-Met Binding Efficiency and Differently Affect Uptake and Survival Listeria in Macrophage.

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 first detection of two Aeromonas strains in mice of the genus Apodemus.

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.

Combination of growth conditions and InlB-specific dot-immunoassay for rapid detection of Listeria monocytogenes in raw milk.

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.

Experimentally Created Magnetic Force in Microbiological Space and On-Earth Studies: Perspectives and Restrictions.

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.

Amyloidogenic Peptides: New Class of Antimicrobial Peptides with the Novel Mechanism of Activity.

Antibiotic-resistant bacteria are recognized as one of the leading causes of death in the world. We proposed and successfully tested peptides with a new mechanism of antimicrobial action "protein silencing" based on directed co-aggregation. The amyloidogenic antimicrobial peptide (AAMP) interacts with the target protein of model or pathogenic bacteria and forms aggregates, thereby knocking out the protein from its working condition. In this review, we consider antimicrobial effects of the designed peptides on two model organisms, E. coli and T. thermophilus, and two pathogenic organisms, P. aeruginosa and S. aureus. We compare the amino acid composition of proteomes and especially S1 ribosomal proteins. Since this protein is inherent only in bacterial cells, it is a good target for studying the process of co-aggregation. This review presents a bioinformatics analysis of these proteins. We sum up all the peptides predicted as amyloidogenic by several programs and synthesized by us. For the four organisms we studied, we show how amyloidogenicity correlates with antibacterial properties. Let us especially dwell on peptides that have demonstrated themselves as AMPs for two pathogenic organisms that cause dangerous hospital infections, and in which the minimal inhibitory concentration (MIC) turned out to be comparable to the MIC of gentamicin sulfate. All this makes our study encouraging for the further development of AAMP. The hybrid peptides may thus provide a starting point for the antibacterial application of amyloidogenic peptides.

Antimicrobial Resistance and Comparative Genomic Analysis of Elizabethkingia anophelis subsp. endophytica Isolated from Raw Milk.

Elizabethkingia anophelis is an emerging multidrug-resistant pathogen that causes severe nosocomial and community-acquired infections worldwide. We report the first case of E. anophelis isolation in Russia and the first isolation from raw cow's milk. The ML-44 demonstrated resistance to 28 antimicrobials of 33 tested in the disk-diffusion test. Whole genome-based phylogeny showed ML-44 strain clustered together with the F3201 strain isolated from a human patient in Kuwait in 1982. Both strains were a part of the "endophytica" clade. Another clade was formed by subsp. anophelis strains. Each of the E. anophelis compared genomes carried 18 to 21 antibiotic resistance determinants. The ML-44 chromosome harbored nine efflux system genes and three beta-lactamase genes, along with six other antimicrobial resistance genes. In total, 72 virulence genes were revealed. The set of virulence factors was quite similar between different E. anophelis strains and included LPS and capsule encoded genes, type IV pili, oxidative stress response genes, and genes encoding TIVSS and TVISS effectors. The particular interest caused the mip and zmp1 gene homologs, which can be essential for intracellular survival. In sum, our findings suggest that raw milk might be a source of E. anophelis harboring a set of virulence factors and a broad resistance to generally used antimicrobials.

Combined Impact of Magnetic Force and Spaceflight Conditions on Escherichia coli Physiology.

Changes in bacterial physiology caused by the combined action of the magnetic force and microgravity were studied in Escherichia coli grown using a specially developed device aboard the International Space Station. The morphology and metabolism of E. coli grown under spaceflight (SF) or combined spaceflight and magnetic force (SF + MF) conditions were compared with ground cultivated bacteria grown under standard (control) or magnetic force (MF) conditions. SF, SF + MF, and MF conditions provided the up-regulation of Ag43 auto-transporter and cell auto-aggregation. The magnetic force caused visible clustering of non-sedimenting bacteria that formed matrix-containing aggregates under SF + MF and MF conditions. Cell auto-aggregation was accompanied by up-regulation of glyoxylate shunt enzymes and Vitamin B12 transporter BtuB. Under SF and SF + MF but not MF conditions nutrition and oxygen limitations were manifested by the down-regulation of glycolysis and TCA enzymes and the up-regulation of methylglyoxal bypass. Bacteria grown under combined SF + MF conditions demonstrated superior up-regulation of enzymes of the methylglyoxal bypass and down-regulation of glycolysis and TCA enzymes compared to SF conditions, suggesting that the magnetic force strengthened the effects of microgravity on the bacterial metabolism. This strengthening appeared to be due to magnetic force-dependent bacterial clustering within a small volume that reinforced the effects of the microgravity-driven absence of convectional flows.

Listeria monocytogenes Invasion Into Sheep Kidney Epithelial Cells Depends on InlB, and Invasion Efficiency Is Modulated by Phylogenetically Defined InlB Isoforms.

The facultative intracellular pathogen Listeria monocytogenes is of major veterinary importance in small ruminants. Nevertheless, details of L. monocytogenes interactions with cells of small ruminants are not fully established. To study the potential of L. monocytogenes to infect sheep cells, we used the finite sheep kidney cell line (shKEC), which was infected with the wild-type L. monocytogenes strain EGDe. The invasion efficiency was 0.015 ± 0.004%. The invasion factor InlB was critically important for invasion, and inlB gene deletion almost prevented L. monocytogenes invasion into shKEC cells. Comparison of the potential of phylogenetically defined InlB isoforms to restore the invasive phenotype of the EGDeΔinlB strain demonstrated that although all InlB isoforms restored invasion of the EGDeΔinlB strain into shKEC cells, the InlB isoforms typical of highly virulent ruminant strains of the clonal complexes CC1 and CC7 were more efficient than isoforms typical of CC2 and CC9 strains (which are less virulent toward ruminants) in supporting invasion. Listeria monocytogenes effectively multiplied with a doubling of time in about 90 min after they entered the sheep cells. Intracellular bacteria moved using the well-known actin polymerization mechanism. Cell-to-cell spreading was restricted to the infection of a few tens of neighboring cells for 7 days. Overall, the obtained results demonstrated that (i) InlB is required for invasion into sheep cells, (ii) InlB isoforms might be important for hypervirulence of certain clonal groups toward ruminants, and (iii) L. monocytogenes effectively multiplies in ovine cells once entered.

Multiple Antimicrobial Effects of Hybrid Peptides Synthesized Based on the Sequence of Ribosomal S1 Protein from Staphylococcus aureus.

The need to develop new antimicrobial peptides is due to the high resistance of pathogenic bacteria to traditional antibiotics now and in the future. The creation of synthetic peptide constructs is a common and successful approach to the development of new antimicrobial peptides. In this work, we use a simple, flexible, and scalable technique to create hybrid antimicrobial peptides containing amyloidogenic regions of the ribosomal S1 protein from Staphylococcus aureus. While the cell-penetrating peptide allows the peptide to enter the bacterial cell, the amyloidogenic site provides an antimicrobial effect by coaggregating with functional bacterial proteins. We have demonstrated the antimicrobial effects of the R23F, R23DI, and R23EI hybrid peptides against Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa, Escherichia coli, and Bacillus cereus. R23F, R23DI, and R23EI can be used as antimicrobial peptides against Gram-positive and Gram-negative bacteria resistant to traditional antibiotics.

Diversity of Listeria monocytogenes Strains Isolated from Food Products in the Central European Part of Russia in 2000-2005 and 2019-2020.

Totally, 45 L. monocytogenes strains isolated from meat, poultry, dairy, and fish products in the Central European part of Russia in 2001-2005 and 2019-2020 were typed using a combined MLST and internalin profile (IP) scheme. Strains belonged to 14 clonal complexes (CCs) of the phylogenetic lineages I and II. Almost half of the strains (20 of 45) belonged to six CCs previously recognized as epidemic clones (ECs). ECI and ECV strains were isolated during both studied periods, and ECII, ECIV, ECVI, and ECVII strains were isolated in 2001-2005, but not in 2019-2020. ECI, ECIV, ECV, and ECVII strains were isolated from products of animal origin. ECII and ECVI were isolated from fish. Testing of invasion efficiencies of 10 strains isolated in different years and from different sources and belonging to distinct CCs revealed a statistically significant difference between phylogenetic lineage I and II strains but not between ECs and non-EC CCs or strains differing by year and source of isolation. Strains isolated in 2001-2005 were characterized by higher phylogenetic diversity and greater presentation of ECs and CCs non-typical for natural and anthropogenic environments of the European part of Russia comparatively to isolates obtained in 2019-2020.Closing of the Russian market in 2019-2020 for imported food might be responsible for these differences.

Antimicrobial Resistance of Listeria monocytogenes Strains Isolated from Humans, Animals, and Food Products in Russia in 1950-1980, 2000-2005, and 2018-2021.

Susceptibility of 117 L. monocytogenes strains isolated during three time periods (1950-1980; 2000-2005, and 2018-2021) to 23 antibiotics was tested by the disk diffusion method. All strains were sensitive to aminoglycosides (gentamicin, kanamycin, neomycin, streptomycin), glycopeptides (vancomycin and teicoplanin), clarithromycin, levofloxacin, amoxicillin/clavulanic acid, and trimethoprim/sulfamethoxazole. Resistance to clindamycin was observed in 35.5% of strains. Resistance to carbapenems, imipenem and meropenem was found in 4% and 5% of strains, respectively. Resistance to erythromycin, penicillin G, trimethoprim, and ciprofloxacin was found in 4%, 3%, 3%, and 2.5% of strains, respectively. Resistance to tylosin, ampicillin, enrofloxacin, linezolid, chloramphenicol, and tetracycline was found in less than 2%. Three strains with multiple antibiotic resistance and 12 strains with resistance to two antibiotics were revealed. Comparison of strains isolated in different time periods showed that the percentage of resistant strains was the lowest among strains isolated before 1980, and no strains with multiple antibiotic resistance were found among them. Statistical analysis demonstrated that the temporal evolution of resistance in L. monocytogenes has an antibiotic-specific character. While resistance to some antibiotics such as ampicillin and penicillin G has gradually decreased in the population, resistance to other antibiotics acquired by particular strains in recent years has not been accompanied by changes in resistance of other strains.

Is It Possible to Create Antimicrobial Peptides Based on the Amyloidogenic Sequence of Ribosomal S1 Protein of P. aeruginosa?

The development and testing of new antimicrobial peptides (AMPs) represent an important milestone toward the development of new antimicrobial drugs that can inhibit the growth of pathogens and multidrug-resistant microorganisms such as Pseudomonas aeruginosa, Gram-negative bacteria. Most AMPs achieve these goals through mechanisms that disrupt the normal permeability of the cell membrane, which ultimately leads to the death of the pathogenic cell. Here, we developed a unique combination of a membrane penetrating peptide and peptides prone to amyloidogenesis to create hybrid peptide: "cell penetrating peptide + linker + amyloidogenic peptide". We evaluated the antimicrobial effects of two peptides that were developed from sequences with different propensities for amyloid formation. Among the two hybrid peptides, one was found with antibacterial activity comparable to antibiotic gentamicin sulfate. Our peptides showed no toxicity to eukaryotic cells. In addition, we evaluated the effect on the antimicrobial properties of amino acid substitutions in the non-amyloidogenic region of peptides. We compared the results with data on the predicted secondary structure, hydrophobicity, and antimicrobial properties of the original and modified peptides. In conclusion, our study demonstrates the promise of hybrid peptides based on amyloidogenic regions of the ribosomal S1 protein for the development of new antimicrobial drugs against P. aeruginosa.

Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model.

Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

Evaluation of Antibiotic Resistance of Salmonella Serotypes and Whole-Genome Sequencing of Multiresistant Strains Isolated from Food Products in Russia.

Food products may be a source of Salmonella, one of the main causal agents of food poisoning, especially after the emergence of strains resistant to antimicrobial preparations. The present work dealt with investigation of the occurrence of resistance to antimicrobial preparations among S. enterica strains isolated from food. The isolates belonged to 11 serovars, among which Infantis (28%), Enteritidis (19%), and Typhimurium (13.4%) predominated. The isolates were most commonly resistant to trimethoprim/sulfamethoxazole (n = 19, 59.38%), cefazolin (n = 15, 46.86%), tetracycline (n = 13, 40.63%), and amikacin (n = 9, 28.13%). Most of the strains (68.75%) exhibited multiple resistance to commonly used antibiotics. High-throughput sequencing was used to analyse three multidrug-resistant strains (resistant to six or more antibiotics). Two of them (SZL 30 and SZL 31) belonged to S. Infantis, while one strain belonged to S. Typhimurium (SZL 38). Analysis of the genomes of the sequenced strains revealed the genes responsible for antibiotic resistance. In the genomes of strains SZL 30 and SZL 31 the genes of antibiotic resistance were shown to be localized mostly in integrons within plasmids, while most of the antibiotic resistance genes of strain SZL 38 were localized in a chromosomal island (17,949 nt). Genomes of the Salmonella strains SZL 30, SZL 31, and SZL 38 were shown to contain full-size pathogenicity islands: SPI-1, SPI-2, SPI-4, SPI-5, SPI-9, SPI-11, SPI-13, SPI-14, and CS54. Moreover, the genome of strain SZL 38 was also found to contain the full-size pathogenicity islands SPI-3, SPI-6, SPI-12, and SPI-16. The emergence of multidrug-resistant strains of various Salmonella serovars indicates that further research on the transmission pathways for these genetic determinants and monitoring of the distribution of these microorganisms are necessary.

Listeria monocytogenes Infection of Bat Pipistrellus nathusii Epithelial cells Depends on the Invasion Factors InlA and InlB.

L. monocytogenes is a widespread facultative intracellular pathogen. The range of natural hosts that supporting L. monocytogenes persistence in the environment has not been fully established yet. In this study, we were interested in the potential of L. monocytogenes to infect cells of bats, which are being increasingly recognized as a reservoir for microorganisms that are pathogenic to humans and domestic animals. A stable epithelial cell line was developed from the kidneys of Pipistrellus nathusii, a small bat widely distributed across Europe. The wild-type L. monocytogenes strain EGDe infected this cell line with an invasion efficiency of 0.0078 ± 0.0009%. Once it entered bat cells, L. monocytogenes doubled within about 70 minutes. When L. monocytogenes lacked either of the major invasion factors, InlA and InlB, invasion efficiency decreased by a factor of 10 and 25 respectively (p < 0.000001). The obtained results suggest that bat epithelial cells are susceptible to L. monocytogenes infection and that L. monocytogenes invasion of bat cells depends on the major invasion factors InlA and InlB. These results constitute the first report on in vitro studies of L. monocytogenes infection in bats.

An In Vitro Model of Nonattached Biofilm-Like Bacterial Aggregates Based on Magnetic Levitation.

Chronic infections are associated with the formation of nonattached biofilm-like aggregates. In vitro models of surface-attached biofilms do not always accurately mimic these processes. Here, we tested a new approach to create in vitro nonattached bacterial aggregates using the principle of magnetic levitation of biological objects placed into a magnetic field gradient. Bacteria grown under magnetic levitation conditions formed nonattached aggregates that were studied with confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) and characterized quantitatively. Nonattached aggregates consisted of bacteria submerged into an extracellular matrix and demonstrated features characteristic of biofilms, such as a polymeric matrix that binds Ruby Red and Congo red dyes, a prerequisite of bacterial growth, and increased resistance to gentamicin. Three quantitative parameters were explored to characterize strain-specific potential to form nonattached aggregates: geometric sizes, relative quantities of aggregated and free-swimming bacteria, and Congo red binding. Among three tested Escherichia coli strains, one strain formed nonattached aggregates poorly, and for this strain, all three of the considered parameters were different from those of the other two strains (P < 0.05). Further, we characterized biofilm formation on plastic and agar surfaces by these strains and found that good biofilm formation ability does not necessarily indicate good nonattached aggregate formation ability, and vice versa. The model and quantitative methods can be applied for in vitro studies of nonattached aggregates and modeling bacterial behavior in chronic infections, as it is important to increase our understanding of the role that nonattached bacterial aggregates play in the pathogenesis of chronic diseases.IMPORTANCE An increasing amount of evidence indicates that chronic infections are associated with nonattached biofilm-like aggregates formed by pathogenic bacteria. These aggregates differ from biofilms because they form under low-shear conditions within the volume of biological fluids and they do not attach to surfaces. Here, we describe an in vitro model that provides nonattached aggregate formation within the liquid volume due to magnetic levitation. Using this model, we demonstrated that despite morphological and functional similarities of nonattached aggregates and biofilms, strains that exhibit good biofilm formation might exhibit poor nonattached aggregate formation, suggesting that mechanisms underlying the formation of biofilms and nonattached aggregates are not identical. The magnetic levitation approach can be useful for in vitro studies of nonattached aggregate formation and simulation of bacterial behavior in chronic infections.