Biofilm Synthesis Research Articles

Novel Strain Bacillus velezensis LAFUEL 03: Activity Against Xanthomonas vasicola pv. vasculorum, Control of Bacterial Leaf Streak of Corn and Genome Insights into Its Antagonistic Activity

The main objective of this study was to investigate the antimicrobial activity of three putative antagonist bacterial strains of Bacillus spp. against Xanthomonas vasicola pv. vasculorum (Xvv) and their potential to control bacterial leaf streak (BLS) of corn. Additionally, the study included investigations on the genome of one of these antagonist bacteria, such as genome sequencing and mining of genes involved in biofilm formation, swarming motility, and synthesis of secondary metabolites. The growth of Xvv was inhibited by both cell suspensions and cell-free supernatants of the bacterial strains LAFUEL 01, LAFUEL 02, and LAFUEL 03 in agar diffusion tests. All three antagonist strains significantly reduced the severity of BLS in the 3rd and 4th leaves of corn plants that were artificially inoculated at the V3 growth stage under greenhouse conditions. The 16S rRNA sequencing confirmed that the antagonistic bacterial strains belong to the genus Bacillus, with LAFUEL 03 having approximately 97% similarity to B. velezensis. B. velezensis LAFUEL 03 harbors genes related to the biosynthesis of secondary metabolites, biofilm formation/regulation, and swarming motility that enhances its potential for controlling BLS in corn and suggests a promising candidate for the development of a commercial biocontrol agent.

DUF1127-containing protein and ProQ had opposite effects on biofilm formation in Vibrio alginolyticus

The RNA binding protein is crucial for gene regulation at the post transcription level. In this study, functions of the DUF1127-containing protein and ProQ, which are RNA-binding proteins, were revealed in Vibrio alginolyticus. DUF1127 deletion increased the ability of biofilm formation, whereas ProQ deletion reduced the amount of biofilm. Moreover, extracellular proteinase secretion was significantly reduced in the DUF1127 deletion strain. ProQ, not DUF1127-containing protein, can help the cell to defense oxidative stress. Deletion of DUF1127 resulted in a higher ROS level in the cell, however, ProQ deletion showed no difference. RNA-seq unveiled the expression of genes involved in extracellular protease secretion were significantly downregulated and biofilm synthesis-related genes, such as rbsB and alsS, were differentially expressed in the DUF1127 deletion strain. ProQ affected the expression of genes involved in biofilm synthesis (flgC and flgE), virulence (betB and hutG), and oxidative stress. Moreover, the DUF1127-containing and ProQ affected the mRNA levels of various regulators, such as LysR and BetI. Overall, our study revealed that the DUF1127-containing protein and ProQ have crucial functions on biofilm formation in V. alginolyticus.

Gallic acid as biofilm inhibitor can improve transformation efficiency of Ruminiclostridium papyrosolvens.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its genetic transformation has been severely impeded by extracellular biofilm. Here, we analyzed the effects of five different inhibitors with gradient concentrations on R. papyrosolvens growth and biofilm formation. Gallic acid was proved to be a potent inhibitor of biofilm synthesis of R. papyrosolvens. Furthermore, the transformation efficiency of R. papyrosolvens was significantly increased when the cells were treated by the gallic acid, and the mutant strain was successfully obtained by the improved transformation method. Thus, inhibition of biofilm formation of R. papyrosolvens by using gallic acid will contribute to its genetic transformation and efficient metabolic engineering.

Transcriptomic investigations of polymyxins and colistin/sulbactam combination against carbapenem-resistant Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a Priority 1 (Critical) pathogen urgently requiring new antibiotics. Polymyxins are a last-line option against CRAB-associated infections. This transcriptomic study utilized a CRAB strain to investigate mechanisms of bacterial killing with polymyxin B, colistin, colistin B, and colistin/sulbactam combination therapy. After 4 h of 2 mg/L polymyxin monotherapy, all polymyxins exhibited common transcriptomic responses which primarily involved disruption to amino acid and fatty acid metabolism. Of the three monotherapies, polymyxin B induced the greatest number of differentially expressed genes (DEGs), including for genes involved with fatty acid metabolism. Gene disturbances with colistin and colistin B were highly similar (89 % common genes for colistin B), though effects on gene expression were generally lower (0–1.5-fold in most cases) with colistin B. Colistin alone (2 mg/L) or combined with sulbactam (64 mg/L) resulted in rapid membrane disruption as early as 1 h. Transcriptomic analysis of this combination revealed that the effects were driven by colistin, which included disturbances in fatty acid synthesis and catabolism, and inhibition of nutrient uptake. Combination therapy produced substantially higher fold changes in 72 % of DEGs shared with monotherapy, leading to substantially greater reductions in fatty acid biosynthesis and increases in biofilm, cell wall, and phospholipid synthesis. This indicates synergistic bacterial killing with the colistin/sulbactam combination results from a systematic increase in perturbation of many genes associated with bacterial metabolism. These mechanistic insights enhance our understanding of bacterial responses to polymyxin mono- and combination therapy and will assist to optimize polymyxin use in patients.

A rare case of TFEB/6p21/VEGFA-amplified renal cell carcinoma diagnosed by whole-exome sequencing: clinicopathological and genetic feature report and literature review

BackgroundTFEB/6p21/VEGFA-amplified renal cell carcinoma (RCC) is rare and difficult to diagnose, with diverse histological patterns and immunohistochemical and poorly defined molecular genetic characteristics.Case presentationWe report a case of a 63-year-old male admitted in 2017 with complex histomorphology, three morphological features of clear cell, eosinophilic and papillary RCC and resembling areas of glomerular and tubular formation. The immunophenotype also showed a mixture of CD10 and P504s. RCC with a high suspicion of collision tumors was indicated according to the 2014 WHO classification system; no precise diagnosis was possible. The patient was diagnosed at a different hospital with poorly differentiated lung squamous cell carcinoma one year after RCC surgery. We exploited molecular technology advances to retrospectively investigate the patient’s molecular genetic alterations by whole-exome sequencing. The results revealed a 6p21 amplification in VEGFA and TFEB gene acquisition absent in other RCC subtypes. Clear cell, papillary, chromophobe, TFE3-translocation, eosinophilic solid and cystic RCC were excluded. Strong TFEB and Melan-A protein positivity prompted rediagnosis as TFEB/6p21/VEGFA-amplified RCC as per 2022 WHO classification. TMB-L (low tumor mutational load), CCND3 gene acquisition and MRE11A and ATM gene deletion mutations indicated sensitivity to PD-1/PD-L1 inhibitor combinations and the FDA-approved targeted agents Niraparib (Grade C), Olaparib (Grade C), Rucaparib (Grade C) and Talazoparib (Class C). GO (Gene Ontology) and KEGG enrichment analyses revealed major mutations and abnormal CNVs in genes involved in biological processes such as the TGF-β, Hippo, E-cadherin, lysosomal biogenesis and autophagy signaling pathways, biofilm synthesis cell adhesion substance metabolism regulation and others. We compared TFEB/6p21/VEGFA-amplified with TFEB-translocated RCC; significant differences in disease onset age, histological patterns, pathological stages, clinical prognoses, and genetic characteristics were revealed.ConclusionWe clarified the patient’s challenging diagnosis and discussed the clinicopathology, immunophenotype, differential diagnosis, and molecular genetic information regarding TFEB/6p21/VEGFA-amplified RCC via exome analysis and a literature review.

Construction and characterization of a modA gene mutant strain of Klebsiella pneumoniae

To construct a mutant strain of Klebsiella pneumoniae NTUH- K2044 with modA gene deletion and its complementary strain and explore the role of modA gene in modulating anaerobic nitrate respiratory growth and phenotypes of K. pneumoniae. The modA deletion mutant K. pneumoniae strain was constructed by homologous recombination using the suicide vector pKO3-Km. To obtain the complementary strain C-modA, the whole sequence fragment containing the promoter, open reading frame and terminator regions of modA was cloned into pGEM-T-easy and electrically transformed into the modA deletion mutant. The NTUH-K2044 wild-type strain, modA gene deletion mutant and complementary strain were compared by measuring in vitro anaerobic nitrate respiration growth, competitiveness index, biofilm quantification, mucoviscosity assay and morphological measurement using Image J. The modA deletion mutant strain ΔmodA and the complementary strain C-modA were successfully constructed. The modA gene knockout strain showed inhibited anaerobic nitrate respiratory growth compared with the wild- type and C-modA strains with significantly weakened competitiveness, reduced capacity of biofilm synthesis during anaerobiosis, and lowered mucoviscosity under anaerobic conditions. The ΔmodA strain showed a spherical morphology in anaerobic conditions as compared with the normal short rod-like morphology of K. pneumoniae, with also distinctly shorter length than the wild-type and C-modA strains. The molybdate transport system encoding gene modA is associated with the pathogenic capacity of K. pneumoniae by modulating its anaerobic nitrate respiration, competitiveness, biofilm formation, hypermucoviscous phenotype and morphology.

Deletion of speA and aroC genes impacts the pathogenicity of Vibrio anguillarum in spotted sea bass

Vibrio anguillarum is one of the major pathogens responsible for bacterial infections in marine environments, causing significant impacts on the aquaculture industry. The misuse of antibiotics leads to bacteria developing multiple drug resistances, which is detrimental to the development of the fisheries industry. In contrast, live attenuated vaccines are gradually gaining acceptance and widespread recognition. In this study, we constructed a double-knockout attenuated strain, V. anguillarum ΔspeA-aroC, to assess its potential for preparing a live attenuated vaccine. The research results indicate a significant downregulation of virulence-related genes, including Type VI secretion system, Type II secretion system, biofilm synthesis, iron uptake system, and other related genes, in the mutant strain. Furthermore, the strain lacking the genes exhibited a 67.47% reduction in biofilm formation ability and increased sensitivity to antibiotics. The mutant strain exhibited significantly reduced capability in evading host immune system defenses and causing in vivo infections in spotted sea bass (Lateolabrax maculatus), with an LD50 that was 13.93 times higher than that of the wild-type V. anguillarum. Additionally, RT-qPCR analysis of immune-related gene expression in spotted sea bass head kidney and spleen showed a weakened immune response triggered by the knockout strain. Compared to the wild-type V. anguillarum, the mutant strain caused reduced levels of tissue damage. The results demonstrate that the deletion of speA and aroC significantly reduces the biosynthesis of biofilms in V. anguillarum, leading to a decrease in its pathogenicity. This suggests a crucial role of biofilms in the survival and invasive capabilities of V. anguillarum.

Investigating the Role of OrbF in Biofilm Biosynthesis and Regulation of Biofilm-Associated Genes in Bacillus cereus BC1.

Bacillus cereus (B. cereus), a prevalent foodborne pathogen, constitutes a substantial risk to food safety due to its pronounced resilience under adverse environmental conditions such as elevated temperatures and ultraviolet radiation. This resilience can be attributed to its capacity for biofilm synthesis and sustained high viability. Our research aimed to elucidate the mechanisms governing biofilm biosynthesis in B. cereus. To this end, we constructed a 5088-mutant library of the B. cereus strain BC1 utilizing the transposon TnYLB-1. Systematic screening of this library yielded mutants exhibiting diminished biofilm formation capabilities. Twenty-four genes associated with the biofilm synthesis were identified by reverse PCR in these mutants, notably revealing a significant reduction in biofilm synthesis upon disruption of the orbF gene in B. cereus BC1. Comparative analysis between the wild type and orbF-deficient BC1 strains (BC1ΔorbF) indicated a marked downregulation (decreased by 11.7% to 96.7%) in the expression of genes implicated in biofilm formation, flagellar assembly, and bacterial chemotaxis in the BC1ΔorbF. Electrophoretic mobility shift assay (EMSA) further corroborated the role of OrbF, demonstrating its binding to the promoter region of the biofilm gene cluster, subsequently leading to the suppression of transcriptional activity of biofilm-associated genes in B. cereus BC1. Our findings underscore the pivotal role of orbF in biofilm biosynthesis in B. cereus, highlighting its potential as a target for strategies aimed at mitigating biofilm formation in this pathogen.

Characterization of Virulence Genotypes, Antimicrobial Resistance Patterns, and Biofilm Synthesis in Salmonella spp Isolated from Foodborne Outbreaks.

Salmonella is the main bacterial pathogen that causes foodborne disease, particularly in developing countries. Nontyphoidal Salmonella (NTS) include Enteritidis and Typhimurium as the most prevalent strains which are one of the significant causes of acute gastroenteritis in children. Therefore, identifying the most predominant serovars, types of common contaminated food, and paying attention to their antibiotic resistance are the main factors in the prevention and control strategy of salmonellosis. This study was undertaken to evaluate the prevalence rate of serovars, the biofilm formation, antimicrobial resistance (AMR) status, and phenotypic virulence factors of Salmonella strains isolated from diarrhea samples in some cities of Iran. A total of 40 (10.41%) Salmonella isolates were recovered from 384 diarrhea samples processed and the most common serovar was Salmonella serovar Typhimurium (82.5). Also, all isolates belonging to serovar Typhimurium showed more virulence factors compared to other serovars. The isolates showed a high resistance rate to ampicillin (95%) and nalidixic acid (87.5%), while a low resistance rate was found for chloramphenicol (2.5%). Moreover, significant variances in the capacity of biofilm formation were found between different Salmonella serotypes. The resistance of NTS to extant choice drugs is a potential public health problem. Constant monitoring of AMR pattern and virulence profile of NTS serovars is suggested for the prevention of salmonellosis in humans.

Effects of exposure to combined acid-cold stress and repair processing on the heat tolerance of Lactobacillus paracasei LP4

To enhance the heat tolerance of lactobacillus in the process of industrial application, the effects of combined acid-cold stress and bacterial repair processes were investigated in Lactobacillus paracasei LP4. The results showed that compared with the control group without stress (10.36% ± 1.75%), the survival rate of the strain after heat treatment was significantly increased after treatment with combined acid-cold stress at pH 3.5 and 20 °C for 1.5 h followed by repair at 35 °C for 15 min (50.68% ± 1.95%, p < 0.05). Analysis of the underlying mechanism showed that compared with single stress, the combined acid-cold stress promoted biofilm synthesis, thus establishing a protective layer surrounding the cell and reducing heat damage to the cell membrane. It was also found to induce the synthesis of peptidoglycans and surface proteins to reduce cell wall damage and improve survival under high temperatures. Additionally, repair processes after combined acid-cold stress were found to reduce DNA damage caused by acid stress, further enhancing heat tolerance. These findings suggest means of improving the heat tolerance and commercial application of lactobacillus through the use of combined acid-cold stress and repair processes.

Synergistic antibacterial effects of exopolysaccharides/nickel-nanoparticles composites against multidrug-resistant bacteria

The need for an alternative treatment to fight infectious diseases caused by antibiotic-resistant bacteria is increasing. A possible way to overcome bacterial resistance to antibiotics is by reintroducing commonly used antibiotics with a sensitizer capable of enhancing their antimicrobial effect in resistant bacteria. Here, we use a composite composed of exopolysaccharide capped-NiO NPs, with antimicrobial effects against antibiotic-resistant Gram-positive and Gram-negative bacteria. It potentiated the antimicrobial effects of four different antibiotics (ampicillin, kanamycin, chloramphenicol, and ciprofloxacin) at lower concentrations than their minimal inhibitory concentrations. We observed that the Ni-composite synergistically enhanced, fourfold, the antibacterial effect of kanamycin and chloramphenicol against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa, as well as ampicillin against multidrug-resistant Staphylococcus aureus, and ciprofloxacin against multidrug-resistant Pseudomonas aeruginosa by eightfold. We also found that Ni-composite could not inhibit biofilm synthesis on the tested bacterial strains. Our results demonstrated the possibility of using metal nanoparticles, like NiO, as a sensitizer to overcome bacterial antibiotic resistance.

Preparation of Thin Films Containing Modified Hydroxyapatite Particles and Phospholipids (DPPC) for Improved Properties of Biomaterials

The main aims of thin biofilm synthesis are to either achieve a new form to promote the transport of drugs in oral delivery systems or as a coating to improve the biocompatibility of the implant’s surface. In this study, the Langmuir monolayer technique was employed to obtain films containing Mg-doped hydroxyapatite with 0.5%, 1.0%, and 1.5% Mg(II). The obtained modified HA particles were analysed via the FT-IR, XRD, DLS, and SEM methods. It was shown that the modified hydroxyapatite particles were able to form thin films at the air/water interface. BAM microscopy was employed to characterized the morphology of these films. In the next step, the mixed films were prepared using phospholipid (DPPC) molecules and modified hydroxyapatite particles (HA-Mg(II)). We expected that the presence of phospholipids (DPPC) in thin films improved the biocompatibility of the preparing films, while adding HA-Mg(II) particles will promote antibacterial properties and enhance osteogenesis processes. The films were prepared in two ways: (1) by mixing DPPC and HA-Mg (II) and spreading this solution onto the subphase, or (2) by forming DPPC films, dropping the HA-Mg (II) dispersion onto the phospholipid monolayer. Based on the obtained π–A isotherms, the surface parameters of the achieved thin films were estimated. It was observed that the HA-Mg(II) films can be stabilized with phospholipid molecules, and a more stable structure was obtained from films synthesied via method (2).

Chitosan- and gallic acid-based (NPF) displayed antibacterial activity against three Pseudomonas spp. plant pathogens and boosted systemic acquired resistance in kiwifruit and olive plants.

Pseudomonas syringae pv. actinidiae (Psa), P. syringae pv. tomato (Pst) and P. savastanoi pv. savastanoi (Psav) are bacterial plant pathogens with worldwide impact that are mainly managed by the preventive application of cupric salts. These are dangerous for ecosystems and have favoured the selection of resistant strains, so they are candidates to be replaced in the next few years. Thus, there is an urgent need to find efficient and bio-based solutions to mitigate these bacterial plant diseases. Nanotechnology could represent an innovative way to control plant diseases, providing alternative solutions to the agrochemicals traditionally employed, thanks to the formulation of the so-called third-generation and nanotechnology-based agrochemicals. In this work, a novel nanostructured formulation (NPF) composed of cellulose nanocrystals (CNC) as carrier, high amylose starch (HAS) as excipient, and chitosan (CH) and gallic acid (GA) as antimicrobials, was tested at 2% in vitro and in vivo with respect to the three different Pseudomonas plant pathogens. In vitro agar assays demonstrated that the NPF inhibited ≤80% Psa, Pst and Psav. Moreover, the NPF did not decrease biofilm synthesis and it did not influence bacterial cells flocculation and adhesion. On plants, the NPF displayed complete biocompatibility and boosted the transcript levels of the major systemic acquired resistance responsive genes in kiwifruit and olive plants. This works provides novel and valuable information regarding the several modes-of-action of the novel NPF, which could potentially be useful to mitigate Psa, Pst and Psav infections even in organic agriculture. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Erythritol alters gene transcriptome signatures, cell growth, and biofilm formation in Staphylococcus pseudintermedius

BackgroundErythritol was found to inhibit the growth of microorganisms. The present study aimed to demonstrate the growth inhibition of Staphylococcus pseudintermedius by erythritol and to define the changes in gene transcription signatures induced by erythritol. Changes in the gene transcription profiles were analysed by RNA sequencing and quantitative reverse transcription PCR. Gene ontology analysis was performed to assign functional descriptions to the genes.ResultsErythritol inhibited S. pseudintermedius growth in a dose-dependent manner. We then performed a transcriptome analysis of S. pseudintermedius with and without 5% (w/w) erythritol exposure to validate the mechanism of growth inhibition. We revealed that erythritol induced up-regulation of three genes (ptsG, ppdK, and ppdkR) that are related to the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS). Glucose supplementation restored the up-regulation of the PTS-related genes in response to erythritol. In addition, erythritol down-regulated eleven genes that are located in a single pur-operon and inhibited biofilm formation of S. pseudintermedius.ConclusionsThese findings indicated that erythritol antagonistically inhibits PTS-mediated glucose uptake, thereby exerting a growth inhibitory effect on S. pseudintermedius. Moreover, erythritol inhibits the ‘de novo’ IMP biosynthetic pathway that may contribute to biofilm synthesis in S. pseudintermedius.

Synergistic Role of Biofilm-Associated Genes and Efflux Pump Genes in Tigecycline Resistance of Acinetobacter baumannii.

BACKGROUND Previous research reported that the resistance mechanism of Acinetobacter baumannii resistance to tigecycline was mainly related to the overexpression of the AdeABC efflux pump system. Biofilm formation is a notable pathogenesis of A. baumannii infections and antibiotic resistance. Our study explores the latent relevance of biofilm-associated genes and efflux pump genes in A. baumannii tigecycline resistance. MATERIAL AND METHODS A total of 78 clinical samples were collected from October 2018 to October 2019. Seventy-two clinically isolated A. baumannii strains were divided into a tigecycline-resistant Acinetobacter baumannii (TR-AN) group and tigecycline-sensitive Acinetobacter baumannii (TS-AN) group by tigecycline minimum inhibitory concentration tests. The biofilm formation of the 2 groups was observed using crystal violet staining. Furthermore, biofilm-related genes and efflux pump genes were analyzed by RT-PCR. RESULTS The biofilm-forming rate of the TR-AN group was 82.2%, and that of the TS-AN group was 14.8%. The biofilm synthesis gene bfs was 91.3% positive in the TR-AN group, significantly higher than in the TS-AN group at the transcription level (P<0.05). The minimum inhibitory concentration of tigecycline was higher in the TR-AN group with biofilm formation than in the TR-AN group without biofilm formation (P<0.05). The efflux pump AdeB gene was 95.2% positive in the TR-AN group with biofilm formation and 38.7% positive in the TR-AN group without biofilm formation. CONCLUSIONS The biofilm formation of A. baumannii may be positively related to tigecycline resistance ability because of the co-expression of the bfs gene and the AdeB efflux pump gene. The enhanced transcription level of bfs and AdeB promotes biofilm formation to improve the resistance of A. baumannii to tigecycline.

Hydrogen peroxide from l-amino acid oxidase of king cobra (Ophiophagus hannah) venom attenuates Pseudomonas biofilms

Because of the high incidence of Pseudomonas aeruginosa biofilms-related nosocomial infections, venoms from common Thai snakes were tested. Although venoms from king cobra (Ophiophagus hannah; OH) and green pit viper (Trimeresurus albolabris) showed the broadest antibacterial spectrum, OH venom demonstrated more profound anti-biofilm activities against P. aeruginosa. Additionally, purified l-amino acid oxidase from OH venom (OH-LAAO), using a three-step chromatography and protein identification, reduced biofilm mass as indicated by the downregulation of several genes, including the genes for biofilm synthesis (algD and pslB) and biofilm regulators (algU, gacA, and siaD). Moreover, OH-LAAO disrupted Pseudomonas-preformed biofilms via upregulation of several genes for biofilm dispersion (nbdA, bdlA, and dipA) and biofilm degradation (endA and pslG), resulting in a reduction of the biofilm biomass. Due to the antimicrobial effects and anti-biofilm activities (reduced production plus increased dispersion) neutralized by catalase, a hydrogen peroxide (H2O2)-degrading enzyme, the enhanced H2O2 by OH venom might be one of the anti-biofilm mechanisms. Hence, OH-LAAO was proposed as a novel agent against Pseudomonas biofilms for either treatment or prevention. More studies are interesting.

1H, 13C, and 15N resonance assignments of SarA monomer from Staphylococcus aureus in complex with DNA.

SarA is a global transcription regulator in S. aureus which regulates the expression of over 120 genes related to quorum sensing, biofilm synthesis, drug resistance and many other important physiological processes during host infection. SarA can bind to the promoter region of agr and other target genes to activate or repress the transcription. The crystal structure of SarA uncovered a MarR protein-like conformation with two symmetrical winged helix domains, while its DNA binding mechanism is still unknown. We have constructed a monomeric DNA binding domain of SarA (SarAΔN19) for the study of the interaction between SarA and DNA with NMR spectroscopy. Here, we report the 1H, 13C and 15N NMR assignment of SarAΔN19/DNA complex which is the first step towards further structure and function analysis.

First insights into antimicrobial resistance, toxigenic profiles, and genetic diversity in Bacillus cereus isolated from Chinese sausages

Bacillus cereus is a common food-borne bacterial pathogen. This study was to designed the prevalence of B. cereus in Chinese sausages and evaluate its genetic diversity, toxigenic profiles, and antimicrobial resistance. Using bacteriological and biochemical detection tests, B. cereus was isolated from 12.63% (N = 49) of the total 388 samples. However, only 37 of the 49 isolates were simultaneously classified as the strictly B. cereus based on the classification of their genomes. Multi-locus sequence typing showed that the clonal complex was highly concentrated in CC142. Enterotoxin-encoding genes (hblABCD, nheABC, entFM, and cytK) were detected in all the strains. A total of 25 genes, associated with biofilm synthesis, were confirmed according to a computerized approach. In total, 89.19% of the isolates possessed only four resistance genes (BcⅠ, BcⅡ, fosB, and vanZF). Nevertheless, all isolates displayed resistance to three or more kinds of antibiotics. Moreover, three isolates were discovered to have tet(45) gene variants and were resistant to tigecycline. This study revealed the powerful potential of B. cereus to cause food poisoning in humans. The presence of multidrug-resistant B. cereus, especially with resistance to tigecycline, mediated by the tet(45) variant, poses a major threat to food safety and public health.

Antimicrobial susceptibility and biofilm forming ability of staphylococci from subclinical buffalo mastitis.

The starting objective of this research communication was to determine the prevalence of subclinical mastitis in buffalo in Turkey. We also seeked to isolate and identify staphylococci, determine their antimicrobial susceptibilities and biofilm-forming abilities as well as investigating the presence of biofilm-related genes and microbial surface components recognizing adhesive matrix molecules. A total of 107 (66.9%) staphylococci (28 S. aureus and 79 coagulase-negative staphylococci, CoNS) were isolated from 160 mastitic milk samples collected from 200 lactating water buffalos. The staphylococci were especially resistant to beta-lactams except for cefoxitin but were less resistant to the other antimicrobials that were tested. Based on the Congo red agar method, 92.9% of the S. aureus and 70.9% of the CoNS isolates were positive for biofilm-forming ability, while all S. aureus and 97.5% of CoNS isolates were positive by a microtiter plate analysis. The presence of icaA and icaD genes was not always correlated with biofilm synthesis, and even in the absence of these genes, the isolates were able to synthesize biofilm.

Anodized Nanostructured 316L Stainless Steel Enhances Osteoblast Functions and Exhibits Anti-Fouling Properties.

Poor osseointegration and infection are among the major challenges of 316L stainless steel (SS) implants in orthopedic applications. Surface modifications to obtain a nanostructured topography seem to be a promising method to enhance cellular interactions of 316L SS implants. In this study, arrays of nanodimples (NDs) having controlled feature sizes between 25 and 250 nm were obtained on 316L SS surfaces by anodic oxidation (anodization). Results demonstrated that the fabrication of NDs increased the surface area and, at the same time, altered the surface chemistry of 316L SS to provide chromium oxide- and hydroxide-rich surface oxide layers. In vitro experiments showed that ND surfaces promoted up to a 68% higher osteoblast viability on the fifth day of culture. Immunofluorescence images confirmed a well-spread cytoskeleton organization on the ND surfaces. In addition, higher alkaline phosphate activity and calcium mineral synthesis were observed on the ND surfaces compared to non-anodized 316L SS. Furthermore, a 71% reduction in Staphylococcus aureus (S. aureus) and a 58% reduction in Pseudomonas aeruginosa (P. aeruginosa) colonies were observed on the ND surfaces having a 200 nm feature size compared to non-anodized surfaces at 24 h of culture. Cumulatively, the results showed that a ND surface topography fabricated on 316L SS via anodization upregulated the osteoblast viability and functions while preventing S. aureus and P. aeruginosa biofilm synthesis.