Bentonite in Korea: A Resource and Research Focus for Biomedical and Cosmetic Industries.

This study provides an in-depth review of bentonite, focusing on its applications in Korea's biomedical and cosmetic sectors. It delves into bentonite's chemical properties, which make it a valuable resource in various industries, particularly in the health and beauty industries. We discuss bentonite's antimicrobial properties, showcasing its effectiveness against a wide range of pathogens and its potential as a biomedicine adjuvant to boost immune responses. Despite its benefits, the review also addresses the need for caution due to its possible side effects when used in human therapy. In the cosmetics industry, bentonite is prized for its ability to absorb impurities, making it a popular ingredient in products from leading brands. The review highlights the ongoing research and development efforts aiming to further explore bentonite's capabilities and applications, underlining the material's significant contribution to advancing Korea's innovation in the biomedical and cosmetic fields. This review suggests that with more research, bentonite's full potential can be unlocked, offering new opportunities for these industries.

PmrAB controls virulence-associated traits and outer membrane vesicle biogenesis in Acinetobacter baumannii.

The PmrAB two-component system modulates colistin resistance in Acinetobacter baumannii, but its association with the virulence traits of this bacterium remains uncharacterized. This study explored the role of A. baumannii PmrAB in surface motility, biofilm formation, and outer membrane vesicle (OMV) biogenesis using wild-type (WT) A. baumannii 17978 and ΔpmrA and ΔpmrB mutant strains. The two mutant strains exhibited significantly decreased surface motility compared with that of WT strain by the low expression of abaI, abaR, A1S_0113, A1S_0115, and A1S_0116. Biofilm mass also significantly decreased in the two mutant strains at 12 h of incubation, but restored at 24 h. Under static culture conditions for 12 h, the two mutant strains showed low pgaA expression. However, the other biofilm-associated genes, such as csuC, csuE, ompA, and bap, showed different expression between the two mutant strains. Although the size of OMVs was similar among the three strains, the number of OMVs secreted from the two mutant strains slightly decreased compared with that secreted from the WT strain. Protein concentrations in the OMVs of ΔpmrA mutant significantly decreased compared with those in the OMVs of WT and ΔpmrB strains. Overall, PmrAB modulates virulence traits and OMV biogenesis in A. baumannii.

First Report of Plasmid-Mediated Macrolide-Clindamycin-Tetracycline Resistance in a High Virulent Isolate of Cutibacterium acnes ST115.

Cutibacterium acnes, a prevalent skin commensal, has emerged as a significant global challenge due to its widespread antibiotic resistance. To investigate the antibiotic resistance mechanisms and clinical characterization of C. acnes in Korea, we collected 22 clinical isolates from diverse patient specimens obtained from the National Culture Collection for Pathogens across Korea. Among the isolates, KB112 isolate was subjected to whole genome sequencing due to high resistance against clindamycin, erythromycin, tetracycline, doxycycline, and minocycline. The whole genome analysis of KB112 isolate revealed a circular chromosome of 2,534,481 base pair with an average G + C content of 60.2% with sequence type (ST) 115, harboring the potential virulent CAMP factor pore-forming toxin 2 (CAMP2), the multidrug resistance ABC transporter ATP-binding protein YknY, and the multidrug efflux protein YfmO. The genomic sequence also showed the existence of a plasmid (30,947 bp) containing the erm(50) and tet(W) gene, which confer resistance to macrolide-clindamycin and tetracycline, respectively. This study reports plasmid-mediated multi-drug resistance of C. acnes for the first time in Korea.

In Vitro Biofilm Formation on Zirconia Implant Surfaces Treated with Femtosecond and Nanosecond Lasers.

(1) Background: The purpose of this study was to evaluate how a zirconia implant surface treated with laser technology affects the degree of biofilm formation. (2) Methods: Experimental titanium (Ti) disks were produced that were sandblasted with large grit and acid-etched (T), and they were compared with zirconia (ZrO2) discs with a machined (M) surface topography; a hydrophilic surface topography with a femtosecond laser (HF); and a hydrophobic surface topography with a nanosecond laser (HN) (N = 12 per surface group). An in vitro three-species biofilm sample (Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi)) was applied to each disc type, and bacterial adhesion was assessed after 48 and 72 h of incubation using an anaerobic flow chamber model. Statistical significance was determined using the Kruskal-Wallis H test, with Bonferroni correction used for the post-hoc test (α = 0.05). (3) Results: Compared to the T group, the M group exhibited more than twice as many viable bacterial counts in the three-species biofilm samples (p < 0.05). In comparison to the T group, the HF group had significantly higher viable bacterial counts in certain biofilm samples at 48 h (Aa and Pi) and 72 h (Pi) (p < 0.05). The HN group had higher viable bacterial counts in Pi at 48 h (5400 CFU/mL, p < 0.05) than the T group (4500 CFU/mL), while showing significantly lower viable bacterial counts in Pg at both 48 (3010 CFU/mL) and 72 h (3190 CFU/mL) (p < 0.05). (4) Conclusions: The surface treatment method for zirconia discs greatly influences biofilm formation. Notably, hydrophobic surface treatment using a nanosecond laser was particularly effective at inhibiting Pg growth.

Genome insight and probiotic potential of three novel species of the genus Corynebacterium.

Three bacterial strains, B5-R-101T, TA-R-1T, and BL-R-1T, were isolated from the feces of a healthy Korean individual. Cells of these strains were Gram-stain-positive, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped, and non-motile. They were able to grow within a temperature range of 10-42°C (optimum, 32-37°C), at a pH range of 2.0-10.0 (optimum, pH 5.5-8.0), and at NaCl concentration of 0.5-10.5% (w/v). All the three strains exhibited 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities ranging from 58 ± 1.62 to 79 ± 1.46% (% inhibition). These strains survived in lower pH (2.0) and in 0.3% bile salt concentration for 4 h. They did not show hemolytic activity and exhibited antimicrobial activity against pathogenic bacteria, such as Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Salmonella enterica. The genomic analysis presented no significant concerns regarding antibiotic resistance or virulence gene content, indicating these strains could be potential probiotic candidates. Phylogenetic analysis showed that they belonged to the genus Corynebacterium, with 98.5-99.0% 16S rRNA gene sequence similarities to other members of the genus. Their major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The abundant cellular fatty acids were C16:0, C18:1ω9c, and anteiso-C19:0. Genomic analysis of these isolates revealed the presence of genes necessary for their survival and growth in the gut environment, such as multi-subunit ATPases, stress response genes, extracellular polymeric substance biosynthesis genes, and antibacterial genes. Furthermore, the genome of each strain possessed biosynthetic gene clusters with antioxidant and antimicrobial potentials, including terpenes, saccharides, polyketides, post-translationally modified peptides (RIPPs), and non-ribosomal peptides (NRPs). In silico DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were lower than the thresholds to distinguish novel species. Based on phenotypic, genomic, phylogenomic, and phylogenetic analysis, these potential probiotic strains represent novel species within the genus Corynebacterium, for which the names Corynebacterium intestinale sp. nov. (type strain B5-R-101T = CGMCC 1.19408T = KCTC 49761T), Corynebacterium stercoris sp. nov. (type strain TA-R-1T = CGMCC 1.60014T = KCTC 49742T), and Corynebacterium faecium sp. nov. (type strain BL-R-1T = KCTC 49735T = TBRC 17331T) are proposed.

Influence of Dental Titanium Implants with Different Surface Treatments Using Femtosecond and Nanosecond Lasers on Biofilm Formation.

This study aimed to evaluate the impact of different surface treatments (machined; sandblasted, large grit, and acid-etched (SLA); hydrophilic; and hydrophobic) on dental titanium (Ti) implant surface morphology, roughness, and biofilm formation. Four groups of Ti disks were prepared using distinct surface treatments, including femtosecond and nanosecond lasers for hydrophilic and hydrophobic treatments. Surface morphology, wettability, and roughness were assessed. Biofilm formation was evaluated by counting the colonies of Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Prevotella intermedia (Pi) at 48 and 72 h. Statistical analysis was conducted to compare the groups using the Kruskal-Wallis H test and the Wilcoxon signed-rank test (α = 0.05). The analysis revealed that the hydrophobic group had the highest surface contact angle and roughness (p < 0.05), whereas the machined group had significantly higher bacterial counts across all biofilms (p < 0.05). At 48 h, the lowest bacterial counts were observed in the SLA group for Aa and the SLA and hydrophobic groups for Pg and Pi. At 72 h, low bacterial counts were observed in the SLA, hydrophilic, and hydrophobic groups. The results indicate that various surface treatments affect implant surface properties, with the hydrophobic surface using femtosecond laser treatment exerting a particularly inhibitory effect on initial biofilm growth (Pg and Pi).

Development of a Magnetic Bead-Based Method for Specific Detection of Enterococcus faecalis Using C-Terminal Domain of ECP3 Phage Endolysin.

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

Insight into gut dysbiosis of patients with inflammatory bowel disease and ischemic colitis.

The collection of whole microbial communities (bacteria, archaea, fungi, and viruses) together constitutes the gut microbiome. Diet, age, stress, host genetics, and diseases cause increases or decreases in the relative abundance and diversity of bacterial species (dysbiosis). We aimed to investigate the gut microbial composition at different taxonomic levels of healthy controls (HCs) with active Crohn's disease (CD), ulcerative colitis (UC), and ischemic colitis (IC) using culture- and non-culture-based approaches and identify biomarkers to discriminate CD, UC, or IC. We determined the specific changes in the gut microbial profile using culture-independent (16S rRNA gene amplicon sequencing) as well as culture-based (culturomic) approaches. Biomarkers were validated using quantitative Real-Time PCR (qPCR). In both methods, bacterial diversity and species richness decreased in disease-associated conditions compared with that in HCs. Highly reduced abundance of Faecalibacterium prausnitzii and Prevotella sp. and an increased abundance of potentially pathogenic bacteria such as Enterococcus faecium, Enterococcus faecalis, and Escherichia coli in all CD, UC, or IC conditions were observed. We noted a high abundance of Latilactobacillus sakei in CD patients; Ligilactobacillus ruminis in UC patients; and Enterococcus faecium, Escherichia coli, and Enterococcus faecalis in IC patients. Highly reduced abundance of Faecalibacterium prausnitzii in all cases, and increased abundance of Latilactobacillus sakei and Enterococcus faecium in CD, Ligilactobacillus ruminis and Enterococcus faecium in UC, and Enterococcus faecium, Escherichia coli, and Enterococcus faecalis in IC could be biomarkers for CD, UC, and IC, respectively. These biomarkers may help in IBD (CD or UC) and IC diagnosis.

Complete genome sequence of the multidrug-resistant clinical isolate Acinetobacter baumannii KBN10P05679: Insights into antimicrobial resistance genotype and potential virulence traits.

OBJECTIVES: Acinetobacter baumannii, a nosocomial pathogen, exhibits multidrug resistance and is a major concern worldwide. We therefore aimed to evaluate the genomic features of the clinical strain A. baumannii KBN10P05679 to elucidate its antibiotic resistance mechanisms and virulence factors. METHODS: In silico multilocus sequence typing, phylogenetic identification, genome annotation, genome analysis, antibiotic susceptibility testing, and biofilm formation assay were performed, and the expression levels of antibiotic resistance- and biofilm-related genes were investigated. RESULTS: The complete genome of KBN10P05679 comprises a circular chromosome of 3 990 428 bp and two plasmids (74 294 and 8731 bp) and was assigned to the ST451 sequence type. Clusters of Orthologous Gene annotation identified 3810 genes, including those involved in amino acid transport and metabolism, transcription, inorganic ion transport, energy production and conversion, replication, recombination and repair, and carbohydrate and protein metabolism. The antibiotic resistance genes were investigated by searching the Comprehensive Antibiotic Resistance Database, and the genome was found to harbour 30 different antibiotic resistance genes. Analysis of the Virulence Factor Database revealed 86 virulence factor genes in the KBN1005679 genome. The KBN10P05679 strain was found to have a higher capacity for biofilm formation and expressed biofilm-related genes at a higher level than the other tested strains. CONCLUSIONS: The antibiotic resistance genotype and potential virulence factor-related data obtained in this study would help direct future studies for developing the control measures for this multidrug-resistant pathogen.

Differentiation of Escherichia fergusonii and Escherichia coli Isolated from Patients with Inflammatory Bowel Disease/Ischemic Colitis and Their Antimicrobial Susceptibility Patterns.

Genotypically, 16S rRNA gene sequence analysis clearly differentiates between species. However, species delineation between Escherichia fergusonii and Escherichia coli is much more difficult and cannot be distinguished by 16S rRNA gene sequences alone. Hence, in this study, we attempted to differentiate E. fergusonii and E. coli isolated from faecal samples of disease-associated Korean individuals with inflammatory bowel disease (IBD)/ischemic colitis (IC) and test the antimicrobial susceptibility patterns of isolated strains. Phylogenetic analysis was performed using the adenylate kinase (adk) housekeeping gene from the E. coli multi locus sequence typing (MLST) scheme. Antimicrobial susceptibility and minimum inhibitory concentration (MIC) of all disease-associated strains in addition to healthy control isolates to 14 antibiotics were determined by broth microdilution-based technique. Next, 83 isolates from 11 disease-associated faecal samples were identified as E. fergusonii using 16S rRNA gene sequence analysis. Phylogenetic analysis using the adk gene from E. coli MLST scheme revealed that most of the strains (94%) were E. coli. A total of 58 resistance patterns were obtained from 83 strains of disease-associated (IBD/IC) isolates. All isolates were resistant to at least one tested antimicrobial agent, with the highest resistance against erythromycin (88.0%), ampicillin (86.7%), ciprofloxacin (73.5%), cephalothin (72.3%), gentamicin (59%), trimethoprim-sulfamethoxazole (53%), cefotaxime (49.4%), and ceftriaxone (48.2%). A total of 90.7% of isolates were extended-spectrum beta-lactamase (ESBL)-producers among the resistant strains to third-generation cephalosporins (cefotaxime or ceftriaxone). ESBL-producing E. coli isolates from patients with Crohn's disease (CD), ulcerative colitis (UC), and ischemic colitis (IC) were 92.3%, 82.4%, and 100%, respectively. In conclusion, adk-based phylogenetic analysis may be the most accurate method for distinguishing E. coli and E. fergusonii from Escherichia genus. We identified four loci in adk gene sequences which makes it easier to discriminate between E. coli and E. fergusonii. Additionally, we believe that gut colonization by multidrug-resistant ESBL-producing E. coli may play a significant role in IBD/IC pathogenesis.

Engineering of lysin by fusion of antimicrobial peptide (cecropin A) enhances its antibacterial properties against multidrug-resistant Acinetobacter baumannii.

Most clinical isolates of Acinetobacter baumannii, a nosocomial pathogen, are multidrug-resistant (MDR), fueling the search for alternative therapies. Bacteriophage-derived endolysins have potent antibacterial activities and are considered as alternatives to antibiotics against A. baumannii infection. Gram-negative bacteria possess outer lipid membrane that prevents direct contact between the endolysins and the cell wall. We hypothesized that the fusion of antimicrobial peptide (AMP) with endolysin could help to reduce bacterial endolysin resistance and increase antimicrobial activity by membrane permeability action. Accordingly, we fused cecropin A, a commonly used AMP, with the N-terminus of AbEndolysin, which enhances the bactericidal activity of the chimeric endolysin. The bactericidal activity of cecropin A-fused AbEndolysin increased by at least 2-8 fold for various MDR A. baumannii clinical isolates. The in vitro bactericidal activity results also showed higher bacterial lysis by the chimeric endolysin than that by the parental lysin. The engineered AbEndolysin (eAbEndolysin) showed synergistic effects with the beta-lactam antibiotics cefotaxime, ceftazidime, and aztreonam, and an additive effect with meropenem and imipenem. eAbEndolysin had no cytotoxic effect on A549 cell line and rescued mice (40% survival rate) from systemic A. baumannii infection. Together, these findings suggest the potential of lysin therapy and may prompt its use as an alternative to antibiotics.

Genomic and Phenotypic Characterization of Cutibacterium acnes Bacteriophages Isolated from Acne Patients.

Cutibacterium acnes is a pathogen that can cause acne vulgaris, sarcoidosis, endodontic lesions, eye infections, prosthetic joint infections, and prostate cancer. Recently, bacteriophage (phage) therapy has been developed as an alternative to antibiotics. In this study, we attempted to isolate 15 phages specific to C. acnes from 64 clinical samples obtained from patients with acne vulgaris. Furthermore, we sequenced the genomes of these three phages. Bioinformatic analysis revealed that the capsid and tape measure proteins are strongly hydrophobic. To efficiently solubilize the phage particles, we measured the adsorption rate, one-step growth curve, and phage stability using an SMT2 buffer containing Tween 20. Here, we report the genotypic and phenotypic characteristics of the novel C. acnes-specific phages.

Production of Membrane Vesicles in Listeria monocytogenes Cultured with or without Sub-Inhibitory Concentrations of Antibiotics and Their Innate Immune Responses In Vitro.

Listeriosis is a food-borne illness caused by Listeria monocytogenes. Ampicillin (AMP) alone or in combination with gentamicin (GEN) is the first-line treatment option. Membrane vesicle (MV) production in L. monocytogenes under antibiotic stress conditions and pathologic roles of these MVs in hosts have not been reported yet. Thus, the aim of this study was to investigate the production of MVs in L. monocytogenes cultured with sub-minimum inhibitory concentrations (MICs) of AMP, GEN, or trimethoprim/sulfamethoxazole (SXT) and determine pathologic effects of these MVs in colon epithelial Caco-2 cells. L. monocytogenes cultured in tryptic soy broth with 1/2 MIC of AMP, GEN, or SXT produced 6.0, 2.9, or 1.5 times more MV particles, respectively, than bacteria cultured without antibiotics. MVs from L. monocytogenes cultured with AMP (MVAMP), GEN (MVGEN), or SXT (MVSXT) were more cytotoxic to Caco-2 cell than MVs obtained from cultivation without antibiotics (MVTSB). MVAMP induced more expression of tumor necrosis factor (TNF)-α gene than MVTSB, MVGEN and MVSXT, whereas MVTSB induced more expression of interleukin (IL)-1ß and IL-8 genes than other MVs. Expression of pro-inflammatory cytokine genes by L. monocytogenes MVs was significantly inhibited by proteinase K treatment of MVs. In conclusion, antibiotic stress can trigger the biogenesis of MVs in L. monocytogenes and MVs produced by L. monocytogenes exposed to sub-MIC of AMP can induce strong pro-inflammatory responses by expressing TNF-α gene in host cells, which may contribute to the pathology of listeriosis.

Antibacterial Effects of Recombinant Endolysins in Disinfecting Medical Equipment: A Pilot Study.

Nosocomial infections caused by multidrug-resistant (MDR) bacteria are severe life-threatening factors. Endolysins (lysins) degrade the bacterial cell wall peptidoglycan and may help control pathogens, especially MDR bacteria prevalent in hospital settings. This study was conducted to verify the potential of lysin as disinfectant to kill bacteria contaminating medical devices that cause hospital infections. Eight catheters removed from hospitalized patients were collected and tested for their ability to kill bacteria contaminating the catheters using two lysins, LysSS and CHAP-161. Catheter-contaminating bacterial species were isolated and identified by 16s rRNA sequencing. From the eight catheters, bacteria were cultured from seven catheters, and five bacterial species (Bacillus megaterium, Bacillus muralis, Corynebacterium striatum, Enterococcus faecium, and Staphylococcus epidermidis) were identified. LysSS could inhibit catheter-contaminating bacteria, including C. striatum and S. epidermidis, compared with untreated controls but could not inhibit the growth of E. faecium. CHAP-161 showed more bactericidal effects than LysSS, but could not inhibit the growth of S. epidermidis. This study showed the potential of lysin as an alternative disinfectant for hazardous chemical disinfectants used in hospitals.

LysSAP26, a New Recombinant Phage Endolysin with a Broad Spectrum Antibacterial Activity.

Multidrug-resistant (MDR) bacteria are a major threat to public health. Bacteriophage endolysins (lysins) are a promising alternative treatment to traditional antibiotics. However, the lysins currently under development are still underestimated. Herein, we cloned the lysin from the SAP-26 bacteriophage genome. The recombinant LysSAP26 protein inhibited the growth of carbapenem-resistant Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, oxacillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with minimum inhibitory concentrations of 5~80 µg/mL. In animal experiments, mice infected with A. baumannii were protected by LysSAP26, with a 40% survival rate. Transmission electron microscopy analysis confirmed that LysSAP26 treatment resulted in the destruction of bacterial cell walls. LysSAP26 is a new endolysin that can be applied to treat MDR A. baumannii, E. faecium, S. aureus, K. pneumoniae, P. aeruginosa, and E. coli infections, targeting both Gram-positive and Gram-negative bacteria.

Antibacterial activities of and biofilm removal by Ablysin, an endogenous lysozyme-like protein originated from Acinetobacter baumannii 1656-2.

OBJECTIVES: Multidrug-resistant (MDR) Acinetobacter baumannii as well as MDR Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and other Enterobacteriaceae ('ESKAPE' pathogens) currently present a major public-health problem. These bacteria are associated with opportunistic infections in intensive care units as well as in immunocompromised patients. There is an urgent need for new alternative antibacterials to control these MDR bacteria. Here we describe the antibacterial action of a novel peptidoglycan hydrolase that targets the bacterial cell wall, identified in the genome of clinical isolate A. baumannii 1656-2. METHODS: We generated a recombinant protein from a sequence encoding a lysozyme-like protein identified in the genome of A. baumannii 1656-2. We named it Ablysin and tested its antibacterial activity and biofilm removal ability targeting ESKAPE pathogens. RESULTS: In vitro application of Ablysin resulted in growth inhibition of the six aforementioned bacterial species, with a highest activity against A. baumannii. Electron microscopy revealed the concentration-dependent (250-2000 µg/mL) rupture of A. baumannii bacterial cells accompanied by elimination of the associated biofilm. CONCLUSIONS: Ablysin represents a potential new class of antibacterial proteins that can be used to target MDR A. baumannii as well as other bacterial species.

Outer membrane vesicles produced by Burkholderia cepacia cultured with subinhibitory concentrations of ceftazidime enhance pro-inflammatory responses.

BURKHOLDERIA CEPACIA: is an opportunistic pathogen that infects patients with debilitating underlying diseases. This study investigated the production of outer membrane vesicles (OMVs) by B. cepacia cultured with sub-minimum inhibitory concentrations (MICs) of antibiotics and examined their pathogenic roles both in vitro and in vivo. B. cepacia ATCC 25416 produced more OMVs under antibiotic stress conditions than controls. OMVs isolated from B. cepacia cultured in Luria-Bertani (LB) broth (OMVs/LB) induced cytotoxicity and the expression of pro-inflammatory cytokine genes in A549 cells in a dose-dependent manner. Host cell cytotoxicity and pro-inflammatory responses were significantly higher in A549 cells treated with B. cepacia OMVs cultured with 1/4 MIC of ceftazidime (OMVs/CAZ) than in the cells treated with OMVs/LB, OMVs cultured with 1/4 MIC of trimethoprim/sulfamethoxazole (OMVs/SXT), or OMVs cultured with 1/4 MIC of meropenem. Intratracheal injection of B. cepacia OMVs also induced histopathology in vivo in mouse lungs. Expressions of IL-1ß and TNF-α genes were significantly up-regulatedin the lungs of mice treated with OMVs/CAZ compared to mice administered other OMVs; the expression of the GRO-α gene, however, was significantly up-regulated in OMVs/SXT. In conclusion, OMVs produced by B. cepacia under different antibiotic stress conditions induce different host responses that may contribute to the pathogenesis of B. cepacia.

Synergy between Florfenicol and Aminoglycosides against Multidrug-Resistant Escherichia coli Isolates from Livestock.

The increasing prevalence of antimicrobial resistance and the laborious development of novel antimicrobial agents have limited the options for effective antimicrobial therapy. The combination of previously used antimicrobial agents represents an alternative therapy for multidrug-resistant (MDR) pathogens. The objective of this study was to investigate the synergistic effect of a florfenicol (FFL)-based combination with other antimicrobial agents against MDR Escherichia coli isolates from livestock using checkerboard assays and murine infection models. The FFL/amikacin (AMK) and FFL/gentamicin (GEN) combinations showed synergy against 10/11 and 6/11 MDR E. coli isolates in vitro, respectively. The combination of FFL with aminoglycosides (AMK or GEN) exhibited a better synergistic effect against MDR E. coli isolates than the cephalothin (CEF)/GEN or FFL/CEF combinations. The combination of FFL with AMK or GEN could reduce the emergence of resistant mutants in vitro. The FFL/AMK combination showed a higher survival rate of mice infected with MDR E. coli isolates than FFL or AMK alone. In summary, the combination of FFL with aminoglycosides (AMK or GEN) is highly effective against MDR E. coli isolates both in vitro and in vivo. Our findings may contribute to the discovery of an effective combination regimen against MDR E. coli infections in veterinary medicine.

Antimicrobial activity of LysSS, a novel phage endolysin, against Acinetobacter baumannii and Pseudomonas aeruginosa.

OBJECTIVES: Multidrug-resistant (MDR) bacteria are a major public-health concern. Bacteriophage endolysins (lysins) can be used as novel antimicrobial agents against bacterial infections. In this study, a novel endolysin (LysSS) containing a lysozyme-like domain was evaluated for its antibacterial activity against various species of bacteria. METHODS: The LysSS-encoding gene was analyzed and cloned and the LysSS recombinant protein was expressed and purified. Purified LysSS was used to determine its antimicrobial activity against various bacterial species in vitro and to measure its protection rate against Acinetobacter baumannii systemic infection in an in vivo murine model. RESULTS: Recombinant LysSS showed activity against MDR A. baumannii, MDR Escherichia coli, MDR Klebsiella pneumoniae, MDR Pseudomonas aeruginosa and Salmonella sp. without pre-treatment with an outer membrane permeabiliser. Moreover, LysSS inhibited the growth of methicillin-resistant Staphylococcus aureus (MRSA). The minimum inhibitory concentration (MIC) of LysSS against 16 MDR A. baumannii strains ranged from 0.063-0.25mg/mL. LysSS had no cytotoxic effect on A549 human lung cells below 250µg/mL. In an animal model, mice infected with A. baumannii were protected (40% survival rate with 125µg LysSS) by intraperitoneal injection of LysSS. CONCLUSION: The current results demonstrate that LysSS may be a novel and promising antimicrobial agent against MRSA and MDR Gram-negative bacteria, including A. baumannii and P. aeruginosa.

The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an important opportunistic pathogen responsible for various nosocomial infections. The BfmRS two-component system plays a role in pathogenesis and antimicrobial resistance of A. baumannii via regulation of bacterial envelope structures. This study investigated the role of the sensor kinase, BfmS, in localization of outer membrane protein A (OmpA) in the outer membrane and production of outer membrane vesicles (OMVs) using wild-type A. baumannii ATCC 17978, ΔbfmS mutant, and bfmS-complemented strains. RESULTS: The ΔbfmS mutant showed hypermucoid phenotype in the culture plates, growth retardation under static culture conditions, and reduced susceptibility to aztreonam and colistin compared to the wild-type strain. The ΔbfmS mutant produced less OmpA in the outer membrane but released more OmpA via OMVs than the wild-type strain, even though expression of ompA and its protein production were not different between the two strains. The ΔbfmS mutant produced 2.35 times more OMV particles and 4.46 times more OMV proteins than the wild-type stain. The ΔbfmS mutant OMVs were more cytotoxic towards A549 cells than wild-type strain OMVs. CONCLUSIONS: The present study demonstrates that BfmS controls production of OMVs in A. baumannii. Moreover, BfmS negatively regulates antimicrobial resistance of A. baumannii and OMV-mediated host cell cytotoxicity. Our results indicate that BfmS negatively controls the pathogenic traits of A. baumannii via cell envelope structures and OMV production.