Determination of an effective agent combination using nisin against Salmonella biofilm.

This present study aims to determine the lowest concentration effects of the assayed different antibiotics; antimicrobial agents alone and their combinations with nisin were investigated to prevent the biofilm formation and break down the biofilm structure of Salmonella. While the combination of nisin and EDTA showed a synergistic effect against Salmonella strain, chlorhexidine digluconate and streptomycin with nisin showed a partial synergetic effect; citric acid and sulfonamides with nisin showed an indifferent effect. The use of citric acid and chlorhexidine digluconate alone was very effective in Salmonella inhibition. While the citric acid combined with other agents had not much effect, the use of chlorhexidine digluconate combined with nisin and EDTA inactivated the total initial count within 24 h. Significantly, when citric acid and sulfonamides are used alone, they reduce by 64% and 44%, respectively. When they used nisin + EDTA, this ratio increased to 83% and 84%, respectively. For the prevention of biofilm, the most suitable conditions were determined as 97% biofilm inhibition. The results of this study can be used as a guide for the emergence of new approaches to ensure the food safety and quality of the food industry.

Genetic Diversity and Characterization of Plant Growth-Promoting Effects of Bacteria Isolated from Rhizospheric Soils.

Plant growth-promoting rhizobacteria (PGPR) have the potential to make a significant contribution to the development of sustainable agricultural systems. Generally, PGPRs function in three different ways, summarized as the synthesis of certain compounds for plants, facilitating the uptake of certain nutrients from the soil and protecting plants from diseases. This study aims to isolate plant growth-promoting bacteria from different plant rhizospheres from Ankara province, to reveal their genetic diversity, and to determine their plant growth-promoting properties. The identification of the 69 isolates was made according to the 16S rDNA sequence results and ARDRA analyses were also performed using AluI, HeaIII, and MspI enzymes. Nitrogen fixation, phosphate dissolving, IAA (indole-3-acetic acid) and siderophore production capacities of the 69 bacterial strains including 12 different genera (30 Pseudomonas, 13 Arthrobacter, 7 Bacillus, 4 Phyllobacter, 4 Variovorax, 3 Olivibacter, 3 Enterobacter, 2 Paenarthrobacter, 1 Stenotrophomonas, 1 Flavobacterium, 1 Caulobacter, 1 Paenibacillus) were evaluated in in vitro conditions. Nitrogen fixation capacities of 55 isolates varied between 2.29 and 46.11 µg mL-1 according to micro-kjeldahl method. Among the strains studied, nifH gene was detected only in Paenibacillus polymyxa H8/2 strain. The highest Phosphorus dissolving and IAA production capacity (in tryptophan-added medium) of isolates were  186.52 µg mL-1, and 50.05 µg mL-1  respectively, and 31 of 69 isolates were able to produce siderophore. Regarding antifungal activities, results showed that 31 bacterial isolates had antagonistic activities against at least one of the tested pathogens. Nitrogen fixation and phosphate solubilizing potential of the promising bacterial strains were determined through two-independent pot experiments with wheat and it has been found that they have positive effects on the yield parameters of wheat.

What makes another life possible in bacteria? Global regulators as architects of bacterial biofilms.

Biofilm structures are the main mode of evolutionary reproductive adaptation of bacteria, and even these features alone, are sufficient to make them the focus of genetic and physiological studies. As this life form is a multicellular-like life form coordinated by genetic and physiological programming, it is quite different from the planktonic form. In bacterial biofilms, which are often composed of more than one species in nature, there is a clear division of labor, nutrient channels, and a language (signaling) established between the cells forming the biofilm. On the other hand, biofilms, especially formed by pathogens, cause important industrial and clinical problems due to their high resistance to environmental stress conditions. Obtaining new data on the molecular basis of bacterial evolution and understanding the intra- and inter-species ecosystem relations in this context, as well as finding permanent solutions to the serious problems they create, are directly related to a detailed understanding of the genetic regulation of bacterial biofilm structures. Today, it is becoming increasingly certain that environmental signals effective in the transition from planktonic form to biofilm form and their receptor/response molecules are generally managed by similar systems and global regulator molecules in bacteria. In this sense; Besides the quorum sensing (QS) systems, cyclic adenosine monophosphate-catabolite suppressor protein (cAMP-CRP) and bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) signaling molecules are of critical importance. In this review article, current information on bacterial biofilms is summarized and interpreted based on this framework.

[The Role of eDNA in Biofilm Structure of Enterococcus faecalis and Investigation of the Efficiency of Enzyme and Antibiotic Application in Biofilm Eradication]. / Enterococcus faecalis'in Biyofilm Yapisinda eDNA'nin Rolü ve Biyofilm Eradikasyonunda Enzim ve Antibiyotik Uygulamasinin Etkinliginin Arastirilmasi.

Biofilm structures, which are the predominant form of microbial life, are a formation that allows pathogenic microorganisms to remain alive by colonizing in different tissues and organs in the human body, as well as on inanimate surfaces. One of the important criteria in the fight against biofilm structures is the weakening of the exopolymeric matrix (EPS). Although it is known that extracellular DNA (eDNA) is one of the most abundant macromolecules in EPS in enterococcal biofilms, its function in biofilm structure is controversial. Since biofilm-forming enterococci are more resistant to antibiotics, the use of antibiotics together with agents that will damage the biofilm structure is being investigated. In this study, it was aimed to target eDNA with enzyme application for the eradication of Enterococcus faecalis biofilm structures and to investigate the increase of the effectiveness of antibiotic therapy on it. The amount of eDNA and optimal production time in biofilm structures of four different strains and isolates of E.faecalis (two clinical isolates coded 74 and 114, and two control strains coded ATCC 29212 and ATCC OG1RF) were determined by spectrophotometric measurement of PicoGreen fluorescence. For the eradication of biofilm; the effects of kanamycin, tetracycline, nalidixic acid, and ampicillin alone and in combination with Benzonase® and DNase I enzymes were investigated by viable cell count on Tryptic Soy Agar. It was determined that optimum biofilm production of E.faecalis strains and isolates occurred at 37°C for 12 hours. E.faecalis 114 isolate was identified as the strongest biofilm producer among the tested bacteria and the isolate containing the highest amount of eDNA (286 98 ng/µl) in the biofilm structure. While the tested antibiotics did not show significant antibiofilm activity against E.faecalis biofilm structures alone, strong antibiofilm activity was detected when ampicillin and tetracycline were applied together with DNase I enzyme. In this study, the use of DNA-degrading enzyme/antibiotic combinations in the eradication of enterococcal biofilms and the effectiveness of these combinations against eDNA were investigated for the first time in the literature. As a result, supportive results were obtained that the use of antibiotics together with the DNase I enzyme targeting the DNA molecule in the EPS structure will be more successful in the fight against the biofilm structures of E.faecalis, which is an important cause of nosocomial infection. These results need to be supported by further clinical studies.

Regulation of biofilm formation by marT in Salmonella Typhimurium.

In this study, we aimed at identifying the regulatory role of marT gene, known as the regulator of misL, on 15 different biofilm-related genes in S. Typhimurium 14028 strain. We also tested the strains for their ability to form biofilm and determined the adherence characteristics of the wild type and the mutant strains of the organism on Caco-2 and HEp-2 cells. For comparative analyses of the candidate genes, individual gene mutations were created via antibiotic gene cassette insertion into each gene of interest. marT gene was cloned behind an arabinose inducible BAD promoter in order to control marT expression. This recombinant plasmid was transfer into each of the 15 mutant strains to investigate the level of expression of each single gene in the presence and absence of marT induction. Besides determination of variations in biofilm formation by each mutant strain, the attachment characteristics of them onto Caco-2 and HEp-2 cell lines were also reported. As a result of attachments experiments on polystyrene surfaces, it was determined that the biofilm production capacity of each mutant strain decreased in a statistically significant manner (p < 0.05). QRT-PCR trials indicated that the marT gene regulates the expression of 14 genes, namely fimA, fimD, fimF, fimH, stjB, stjC, csgA, csgD, ompC, sthB, sthE, rmbA, fliZ and yaiC, in a positive manner. QRT-PCR studies were also revealed that the MarT protein positively regulates its own promoter. When the adherence characteristics of the mutant strains and the wild-type were investigated by using Caco-2 and HEp-2 cells, it was determined that the single gene mutations did have no effect on bacterial adhesion. In view of our mutational analyses and biofilm formation studies, it was concluded that fliZ, ompC, rmbA, stjB and stjC genes are related with biofilm formation in Salmonella, besides other cellular functions of them. Taken together, our data suggested that the regulatory role of MarT protein is not only restricted to the regulation of misL gene expression, but it rather acts as a general regulator on the biofilm-related genes in Salmonella.

Investigation of the effect of different environmental conditions on biofilm structure of Salmonella enterica serotype Virchow via FTIR spectroscopy.

This study aims to describe the content of polymeric matrix components under different incubation temperatures and pH levels. Optimal biofilm production of 15 S. Virchow isolates occurred following the incubation in LB-NaCl for 72 h, at pH 6.6 and 20 °C. The expression of csgA, csgD, adrA and bcsA genes at 20 °C, 25 °C and 30 °C in S. Virchow DMC18 was analyzed, and it was discovered that the maximum production of cellulose and curli fimbriae occurred at 20 °C. The physical characteristics of pellicle structure of S. Virchow DMC18 was determined as rigid at 20 °C, while becoming fragile at higher temperatures. FTIR analyses confirmed the obtained molecular findings. The intensities of the 16 different peaks originating from carbohydrate, protein, and nucleic acid in the spectra of biofilm samples significantly diminished (p < 0.05) with the increasing temperature. The highest intensities of lipids and carbohydrates were observed at 20 °C indicating the changes in cell surface properties.

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage, EDTA and nisin.

P22 phage >105 PFU ml-1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55-80%. Concentrations of EDTA >1.25 mM and concentrations of nisin >1,200 µg ml-1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (102 PFU ml-1), and low EDTA (1.25 mM) and nisin (9.375 µg ml-1) concentrations. A reduction of 70% in the mature biofilm was possible when 107 PFU ml-1 of P22 phage, 20 mM of EDTA and 150 µg ml-1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.

Modeling and predicting the biofilm formation of Salmonella Virchow with respect to temperature and pH.

Biofilm formation of Salmonella Virchow was monitored with respect to time at three different temperature (20, 25 and 27.5 °C) and pH (5.2, 5.9 and 6.6) values. As the temperature increased at a constant pH level, biofilm formation decreased while as the pH level increased at a constant temperature, biofilm formation increased. Modified Gompertz equation with high adjusted determination coefficient (Radj(2)) and low mean square error (MSE) values produced reasonable fits for the biofilm formation under all conditions. Parameters of the modified Gompertz equation could be described in terms of temperature and pH by use of a second order polynomial function. In general, as temperature increased maximum biofilm quantity, maximum biofilm formation rate and time of acceleration of biofilm formation decreased; whereas, as pH increased; maximum biofilm quantity, maximum biofilm formation rate and time of acceleration of biofilm formation increased. Two temperature (23 and 26 °C) and pH (5.3 and 6.3) values were used up to 24 h to predict the biofilm formation of S. Virchow. Although the predictions did not perfectly match with the data, reasonable estimates were obtained. In principle, modeling and predicting the biofilm formation of different microorganisms on different surfaces under various conditions could be possible.

Identification of genomic heterogeneity among Lactococcus lactis strains by plasmid profiling, PFGE and 16S rDNA sequence analysis.

Lactococcus lactis strains are used commonly as starters, which contribute to desirable flavour and texture properties known as strain-specific, in dairy industry. Genomic heterogeneity of 30 L. lactis strains originating from Turkey and characterized phenotypically were investigated in this study. Plasmid profiling, PFGE and 16S rDNA sequence analyses were performed to determine the genetic variability of strains. High degree of heterogeneity was detected among the L. lactis strains. Plasmid profiles of strains showed that compared to the plasmid free control strains, namely; L. lactis subsp. lactis IL1403 and L. lactis subsp. cremoris MG1614, all tested strains carried one to ten plasmids with molecular size ranging from 1.5 to 41.5kb. The fingerprints of strains obtained by PFGE from digestion with ApaI, SmaI and I-CeuI restriction endonucleases of chromosomal DNA's were compared with each other. All strains out of four were grouped into a large cluster A with at least 44% similarity level. The other four strains formed a minor duster B, distinctively different from major cluster A. PFGE results were confirmed by 16S rDNA sequence analysis and strains included in cluster B were identified as members of different species. These results suggested that morphologic and biochemical methods should be verified by reliable molecular approaches for the purpose of strain typing. Also, PFGE was found suitable to determine genomic differentiations among inter- and intra species.

Characteristics of Bacterial Biofilm Formation in Nasolacrimal Silicone Tubes Post-dacryocystorhinostomy.

PURPOSE: To examine the biofilm formation characteristics of bacteria identified at the genus level in samples obtained from silicone tubes after dacryocystorhinostomy surgery. METHODS: In the study involving consecutive patients who underwent dacryocystorhinostomy surgery at Ankara Bilkent City Hospital and whose silicone tubes were removed six months after surgery, between January 2023 and May 2023; the tubes were placed in glycerol-PBS (phosphate buffered saline) solution and cultured on descriptive selective media at the genus level. The biofilm-forming properties of the obtained isolates were examined in solid-air and liquid-air interphases. Salmonella Typhimurium ATCC SL1344 strain was used as the control bacterium. RESULTS: As a result of the analysis of the samples taken from the patients, Pseudomonas spp. was identified in three of the samples, Staphylococcus spp. in five of the samples, and Streptococcus spp. in one of the samples. Among these samples, except for the bacteria identified in samples one and five, the rest were found to be strong biofilm producers. In all strong biofilm producers, the maximum biofilm production time was determined as 72 h and the incubation temperature was 37°C. The presence of cellulose and amyloid proteins in biofilm matrix structures is identified. Swimming and swarming motilities were observed in all bacterial samples. CONCLUSION: Since biofilms are considered potential factors in the pathogenesis of infectious and inflammatory diseases, they are a subject that needs to be thoroughly investigated. In our study, although there were no clinical infections in any of the patients, biofilm formation was detected in the patient samples. The fact that the bacteria exhibited moderate to strong biofilm formation characteristics suggests that these microorganisms could be persistent infectious agents.

Comparative global gene expression analysis of biofilm forms of Salmonella Typhimurium ATCC 14028 and its seqA mutant.

In this study, comparative transcriptomic analyzes (mRNA and miRNA) were performed on the biofilm forms of S. Typhimurium ATCC 14028 wild-type strain and its seqA gene mutant in order to determine the regulation characteristics of the seqA gene in detail. The results of global gene expression analyses showed an increase in the expression level of 54 genes and a decrease in the expression level of 155 genes (p < 0.05) in the seqA mutant compared to the wild-type strain. 10 of the 48 miRNAs identified on behalf of sequence analysis are new miRNA records for Salmonella. Transcripts of 14 miRNAs differed between wild-type strain and seqA mutant (p < 0.05), of which eight were up-regulated and six were down-regulated. Bioinformatic analyzes showed that differentially expressed genes in the wild-type strain and its seqA gene mutant play a role in different metabolic processes as well as biofilm formation, pathogenicity and virulence. When the transcriptomic data were interpreted together with the findings obtained from phenotypic tests such as motility, attachment to host cells and biofilm morphotyping, it was determined that the seqA gene has a critical function especially for the adhesion and colonization stages of biofilm formation, as well as for biofilm stability. Transcriptomic data pointing out that the seqA gene is also a general positive regulator of T3SS effector proteins active in cell invasion in S. Typhimurium wild-type biofilm, proves that this gene is involved in Salmonella host cell invasion.

Effect of nisin and p-coumaric acid on autoinducer-2 activity, biofilm formation, and sprE expression of Enterococcus faecalis.

Quorum sensing (QS) is an inter- and intracellular communication mechanism that regulates gene expression in response to population size. Autoinducer-2 (AI-2) signaling is a QS signaling molecule common to both Gram-negative and Gram-positive bacteria. Enterococcus faecalis is one of the leading causes of nosocomial infections worldwide. There has been an increasing interest in controlling infectious diseases through targeting the QS mechanism using natural compounds. This study aimed to investigate the effect of nisin and p-coumaric acid (pCA), on biofilm formation and AI-2 signaling in E. faecalis. Their effect on the expression of the QS-regulated virulence encoding gene sprE was also investigated. Nisin exhibited a MIC ranging from 0.25 to 0.5 mg/mL, while the MIC of pCA was 1 mg/mL. The luminescence-based response of the reporter strain Vibrio harveyi BB170 was used to determine AI-2 activity in E. faecalis strains. Nisin was not effective in inhibiting AI-2 activity, while pCA reduced AI-2 activity by ≥ 60%. Moreover, pCA and nisin combination showed higher inhibitory effect on biofilm formation of E. faecalis, compared to the treatment of pCA or nisin alone. qRT-PCR analysis showed that nisin alone and the combination of nisin and pCA, at their MIC values, led to a 32.78- and 40.22-fold decrease in sprE gene expression, respectively, while pCA alone did not have a significant effect. Considering the demand to explore new therapeutic avenues for infectious bacteria, this study was the first to report that pCA can act like a quorum sensing inhibitor (QSI) against AI-2 signaling in E. faecalis.

Phenotypic impacts and genetic regulation characteristics of the DNA adenine methylase gene (dam) in Salmonella Typhimurium biofilm forms.

In this study, transcriptional level gene expression changes in biofilm forms of Salmonella Typhimurium ATCC 14028 and its dam mutant were investigated by performing RNAseq analysis. As a result of these analyzes, a total of 233 differentially expressed genes (DEGs) were identified in the dam mutant, of which 145 genes were downregulated and 88 genes were upregulated compared to the wild type. According to data from miRNA sequence analysis, of 13 miRNAs differentially expressed in dam mutant, 9 miRNAs were downregulated and 4 miRNAs were upregulated. These data provide the first evidence that the dam gene is a global regulator of biofilm formation in Salmonella. In addition, phenotypic analyses revealed that bacterial swimming and swarming motility and cellulose production were highly inhibited in the dam mutant. It was determined that bacterial adhesion in Caco-2 and HEp-2 cell lines was significantly reduced in dam mutant. At the end of 90 min, the adhesion rate of wild type strain was 43.3% in Caco-2 cell line, while this rate was 14.9% in dam mutant. In the HEp-2 cell line, while 45.5% adherence was observed in the wild-type strain, this rate decreased to 15.3% in the dam mutant.

Identification and characterization of lytic bacteriophages specific to foodborne pathogenic Escherichia coli O157:H7.

The objective of this study was to identify and characterize five different lytic bacteriophages specific to Escherichia coli O157:H7. vB_EcoM-P12, vB_EcoM-P13, vB_EcoM-P23, and vB_EcoM-P34 phages belonged to the Myoviridae family and vB_EcoS-P24 phage was in the Siphoviridae family. Their plaque sizes changed between 0.48 ± 0.03 and 0.90 ± 0.03 mm in diameter. stx1 and stx2 virulent gene regions were absent in the genome of five Eco-phages and their genome size was 33 kbp. The protein band profiles of the five phages were found to be different from each other. Their latent period, burst size, and burst time changed between 10-15 min, 72-144 PFU/cell and 20-35 min, respectively. Multiplicity of infection values and mutant frequency of the phages were among 0.1-0.001 and 1.14 × 10-7-3.69 × 10-8, respectively. The phages had strong lytic activity against their host bacteria (E. coli NCTC 12900, ATCC 43888, and ATCC 35150) at 5-37 ℃ and adsorbed to their host cells by 92.7-97.5% in the first five minutes of incubation. These phages are thought to be good candidates as therapeutic and biocontrol agents against E. coli O157:H7 in the veterinary science and food industry due to short latent period, high burst size, rapid development in host cells, high lytic activity, high adsorption rate, stability over a wide pH range and high temperature, and absence of stx1 and stx2 genes.

The role of bcsE gene in the pathogenicity of Salmonella.

The effects of the bcsE gene and BcsE protein on bacterial physiology and pathogenicity in SalmonellaTyphimurium and Salmonella Group C1 were investigated. It was observed that biofilm and pellicle formation did not occur in the bcsE gene mutants of wild-type strains. Besides, the 'rdar' (red, dry, rough) biofilm morphotype in wild-type strains changed significantly in the mutants. In terms of the bcsE gene, the swimming and swarming motility in mutant strains showed a dramatic increase compared to the wild-type strains. The Salmonella bcsE gene was cloned into Escherichia coli BL21, and the his-tagged protein produced in this strain was purified to obtain polyclonal antibodies in BALB/c mice. The antibodies were showed labeled antigen specificity to the BscE protein. As a result of immunization and systemic persistence tests carried out with BALB/c mice, BscE protein was determined to trigger high levels of humoral and cellular responses (Th1 cytokine production, IgG2a/IgG1 > 1). Systemic persistence in the liver and spleen samples decreased by 99.99% and 100% in the bcsE mutant strains. Finally, invasion abilities on HT-29 epithelial cells of wild-type strains were utterly disappeared in their bcsE gene mutant strains.

Physicochemical and Microbiological Characterization of Protected Designation of Origin Ezine Cheese: Assessment of Non-starter Lactic Acid Bacterial Diversity with Antimicrobial Activity.

Ezine cheese is a non-starter and long-ripened cheese produced in the Mount of Ida region of Çanakkale, Turkey, with a protected designation of origin status. Non-starter lactic acid bacteria (NSLAB) have a substantial effect on the quality and final sensorial characteristics of long-ripened cheeses. The dominance of NSLAB can be attributed to their high tolerance to the hostile environment in cheese during ripening relative to many other microbial groups and to its ability to inhibit undesired microorganisms. These qualities promote the microbiological stability of long-ripened cheeses. In this study, 144 samples were collected from three dairies during the ripening period of Ezine cheese. Physicochemical composition and NSLAB identification analyses were performed using both conventional and molecular methods. According to the results of a 16S rRNA gene sequence analysis, 13 different species belonging to seven genera were identified. Enterococcus faecium (38.42%) and E. faecalis (18.94%) were dominant species during the cheese manufacturing process, surviving 12 months of ripening together with Lactobacillus paracasei (13.68%) and Lb. plantarum (11.05%). The results indicate that NSLAB contributes to the microbiological stability of Ezine cheese over 12 months of ripening. The isolation of NSLAB with antimicrobial activity, potential bacteriocin producers, yielded defined collections of natural NSLAB isolates from Ezine cheese that can be used to generate specific starter cultures for the production of Ezine cheese (PDO).

Effects of dam and seqA genes on biofilm and pellicle formation in Salmonella.

In this study, the effects of dam and seqA genes on the formation of pellicle and biofilm was determined using five different Salmonella serovars S. Group C1 (DMC2 encoded), S. Typhimurium (DMC4 encoded), S. Virchow (DMC11 encoded), S. Enteritidis (DMC22 encoded), and S. Montevideo (DMC89 encoded). dam and seqA mutants in Salmonella serovars were performed by the single step lambda red recombination method. The mutants obtained were examined according to the properties of biofilm on the polystyrene surfaces and the pellicle formation on the liquid medium. As a result of these investigations, it was determined that the biofilm formation properties on polystyrene surfaces decreased significantly (p < 0.05) in all tested dam and seqA mutants, while the pellicle formation properties were lost in the liquid medium. When pBAD24 vector, containing the dam and seqA genes cloned behind the inducible arabinose promoter, transduced into dam and seqA mutant strains, it was determined that the biofilm formation properties on the polystyrene surfaces reached to the natural strains' level in all mutant strains. Also, the pellicle formation ability was regained in the liquid media. All these data demonstrate that dam and seqA genes play an important role in the formation of biofilm and pellicle structures in Salmonella serovars.