The stability of Staphylococcal bacteriophage in presence of local vancomycin concentrations used in clinical practice.

PURPOSE: To evaluate the stability of a clinically used Staphylococcal bacteriophage with doses of vancomycin that are encountered with local administration of vancomycin for musculoskeletal infections. METHODS: A Staphylococcal bacteriophage was evaluated for stability in different pH ranges. Then that same bacteriophage was evaluated for stability with different concentrations of vancomycin and with vancomycin biodegradable antibiotic beads. RESULTS: The bacteriophage had stability within a pH range of 4-10. There was a statistically significant (P < 0.05) decrease in the amount of bacteriophage over 24 h for vancomycin concentrations of 10 mg/mL and 100 mg/mL compared to lower vancomycin concentrations (1 mg/mL, 0.1 mg/mL and normal saline). However, no statistically significant decrease in the amount of bacteriophage was seen with biodegradable vancomycin beads over 24 h. CONCLUSION: These findings have important clinical ramifications in that they show local administration of bacteriophages with concomitant local vancomycin powder therapy should be avoided. Moreover, these findings should spearhead further research into bacteriophage stability in in vivo environments.

Study on the virulome and resistome of a vancomycin intermediate-resistance Staphylococcus aureus.

Methicillin-resistant S. aureus (MRSA) has been considered a potential "Super Bugs", responsible for various infectious diseases. Vancomycin has been the most effective antibitic to treat MRSA originated infections. In this study, we aimed at investigating the genomic features of a vancomycin intermediate-resistance S. aureus strain Guangzhou-SauVS2 isolated from a female patient suffering from chronic renal function failure, emphasizing on its antimicrobial resistance and virulence determinants. The genome has a total length of 2,605,384 bp and the G+C content of 33.21%, with 2,239 predicted genes annotated with GO terms, COG categories, and KEGG pathways. Besides the carriage of vancomycin b-type resistance protein responsible for the vancomycin intermediate-resistance, S. aureus strain Guangzhou-SauVS2 showed resistance to ß-lactams, quinolones, macrolide, and tetracycline, due to the acquisition of corresponding antimicrobial resistance genes. In addition, virulence factors including adherence, antiphagocytosis, iron uptake, and toxin were determined, indicating the pathogenesis of the strain.

Do bacteriophages have activity in synovial fluid and against synovial fluid induced bacterial aggregates?

Bacteriophage therapy is a promising adjuvant therapy for the treatment of periprosthetic joint infections. However, there is a paucity of knowledge about the activity of bacteriophages in synovial fluid. Therefore, this study evaluated the activity of a clinically used bacteriophage in synovial fluid as well as the ability of that bacteriophage to prevent the formation of and eradicate bacteria in synovial fluid induced aggregates. The results of this study reinforce that synovial fluid induced aggregates form rapidly in numerous synovial fluid concentrations. More importantly, there was a statistically significant reduction in bacteriophage activity in synovial fluid compared to tryptic soy broth (p < 0.05) and the bacteriophage could not prevent the formation synovial fluid induced aggregates. Also the bacteriophage could not significantly reduce the amount of bacteria in the synovial fluid induced aggregates when compared to controls, and this was not secondary to resistance. Rather the reduced activity seems to be caused by bacteriophages being hindered in the ability to attach to bacterial receptors. We hypothesize this occurred because the viscosity of synovial fluid slowed bacteriophage interactions with planktonic bacteria and the synovial fluid polymers obstructed the bacteriophage attachment receptors thereby preventing attachment to bacteria in the aggregates. These findings have clinical ramifications, supporting the use of bacteriophage therapy as an adjunct to surgical interventions and not in isolation, at the nascent stage. While these findings show a shortcoming of bacteriophage therapy in periprosthetic joint infections, the knowledge gained should spearhead further research to ultimately devise effective and reproducible bacteriophage therapeutics.

Differential alteration in Lactiplantibacillus plantarum subsp. plantarum quorum-sensing systems and reduced Candida albicans yeast survival and virulence gene expression in dual-species interaction.

Candida albicans (C. albicans) and Lactiplantibacillus plantarum subsp. plantarum (L. plantarum) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans, including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS. Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans, indicating the potential interruption of pathogenic activities by L. plantarum. Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans. The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.IMPORTANCEThe anti-Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans.

Biofilm formation of two genetically diverse Staphylococcus aureus isolates under beta-lactam antibiotics.

Purpose: Our aim was to evaluate the biofilm formation of 2 genetically diverse Staphylococcus aureus isolates, 10379 and 121940, under different concentrations of beta-lactam antibiotics on biomass content and biofilm viability. Methods: Biofilm formation and methicillin resistance genes were tested using PCR and multiplex PCR. PCR was combined with bioinformatics analysis to detect multilocal sequence typing (MLST) and SCCmec types, to study the genetical correlation between the tested strains. Then, the crystal violet (CV) test and XTT were used to detect biomass content and biofilm activity. Antibiotic susceptibility was tested using a broth dilution method. According to their specific MIC, different concentrations of beta-lactam antibiotics were used to study its effect on biomass content and biofilm viability. Results: Strain 10379 carried the icaD, icaBC, and MRSA genes, not the icaA, atl, app, and agr genes, and MLST and SCCmec typing was ST45 and IV, respectively. Strain 121940 carried the icaA, icaD, icaBC, atl, and agr genes, not the aap gene, and MLST and SCCmec typed as ST546 and IV, respectively. This suggested that strains 10379 and 121940 were genotypically very different. Two S. aureus isolates, 10379 and 121940, showed resistance to beta-lactam antibiotics, penicillin, ampicillin, meropenem, streptomycin and kanamycin, some of which promoted the formation of biofilm and biofilm viability at low concentrations. Conclusion: Despite the large differences in the genetic background of S. aureus 10379 and 121940, some sub-inhibitory concentrations of beta-lactam antibiotics are able to promote biomass and biofilm viability of both two isolates.

Sub-MIC streptomycin and tetracycline enhanced Staphylococcus aureus Guangzhou-SAU749 biofilm formation, an in-depth study on transcriptomics.

Staphylococcus aureus is a major human pathogen, a potential "Super-bug" and a typical biofilm forming bacteria. With usage of large amount of antibiotics, the residual antibiotics in clinical settings further complicate the colonization, pathogenesis and resistance of S. aureus. This study aimed at investigating the phenotypical and global gene expression changes on biofilm formation of a clinical S. aureus isolate treated under different types of antibiotics. Firstly, an isolate Guangzhou-SAU749 was selected from a large sale of previously identified S. aureus isolates, which exhibited weak biofilm formation in terms of biomass and viability. Secondly, 9 commonly prescribed antibiotics for S. aureus infections treatment, together with 10 concentrations ranging from 1/128 to 4 minimum inhibitory concentration (MIC) with 2-fold serial dilution, were used as different antibiotic stress conditions. Then, biofilm formation of S. aureus Guangzhou-SAU749 at different stages including 8 h, 16 h, 24 h, and 48 h, was tested by crystal violet and MTS assays. Thirdly, the whole genome of S. aureus Guangzhou-SAU749 was investigated by genome sequencing on PacBio platform. Fourthly, since enhancement of biofilm formation occurred when treated with 1/2 MIC tetracycline (TCY) and 1/4 MIC streptomycin (STR) since 5 h, the relevant biofilm samples were selected and subjected to RNA-seq and bioinformatics analysis. Last, expression of two component system (TCS) and biofilm associated genes in 4 h, 8 h, 16 h, 24 h, and 48 h sub-MIC TCY and STR treated biofilm samples were performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Although most antibiotics lowered the biomass and cell viability of Guangzhou-SAU749 biofilm at concentrations higher than MIC, certain antibiotics including TCY and STR promoted biofilm formation at sub-MICs. Additionally, upon genome sequencing, RNA-seq and RT-qPCR on biofilm samples treated with sub-MIC of TCY and STR at key time points, genes lytR, arlR, hssR, tagA, clfB, atlA and cidA related to TCS and biofilm formation were identified to contribute to the enhanced biofilm formation, providing a theoretical basis for further controlling on S. aureus biofilm formation.

Xylitol and Maltitol Improve the Rheological Property of Kappa-Carrageenan.

To further extend the use of κ-carrageenan (κ-C) in real food systems (such as beverages), the understanding of gelation properties of κ-C with the presence of food ingredients is critical. The effects of xylitol and maltitol (up to 30 wt %) on the rheological and structural properties of κ-C were inspected by means of rheometer and Fourier transform infrared (FTIR). With the addition of xylitol, the gelation temperature increased from 44.1 to 57.3 °C, while the gelation temperature increased from 44.1 to 61.4 °C in maltitol systems. With the increasing concentration of both xylitol and maltitol, the values of fractal dimension df and complex modulus G* of κ-C increased, while the relaxation exponent n decreased from 0.87 to 0.39 of xylitol and 0.87 to 0.78 of maltitol, respectively. These indicated that the gel networks of aqueous κ-C were improved by the addition of xylitol and maltitol. The FTIR results showed that the interaction between κ-C and these polyols contributed to the increase of hydrogen bonds. The effects of maltitol on κ-C were stronger than those of xylitol because of more equatorial-OH bonds in maltitol. These findings contribute to a better understanding of the gelation processes of κ-C/polyols systems.

Kappa-carrageenan enhances the gelation and structural changes of egg yolk via electrostatic interactions with yolk protein.

The effect of κ-carrageenan (κ-C) on yolk over heat-induced gelation at natural yolk pH (6.2) and natural whole egg pH (7.5) was studied. The results showed the zeta potential values changed from -2.3 to -31.3 mV, from -8.6 to -28.6 mV for native pH yolk and pH 7.5 yolk because of the κ-C addition, respectively. These results indicated electrostatic interactions formed between protein and κ-C. The average area of holes formed by yolk gelation increased by κ-C addition. The addition of 1.0% κ-C decreased the gelling points from 62.1 to 54.4 °C, from 64.5 to 61. 6 °C for native pH and pH 7.5 yolk, respectively. A schematic model was established to show that κ-C enhances the yolk properties via electrostatic interactions. And the Fourier transform infrared (FTIR) spectroscopy verified the formation of κ-C-protein interactions. This study provides a guidance for designing novel food systems containing yolk and κ-C.

Antimicrobial susceptibility and genetic features of a heterogeneous vancomycin intermediate-resistant Staphylococcus aureus strain.

This study aimed to characterize the antimicrobial susceptibility and genetic features of a heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) strain Guangzhou-SauVS2 recovered from a female patient in Guangzhou, representative of southern China. The genome of Guangzhou-SauVS2 was sequenced using Illumina HiSeq 2500 platform and assembled de novo using Velvet v1.2.08. Annotations and bioinformatics analysis were further performed. Results showed that Guangzhou-SauVS2 was susceptible and resistant to 7 and 11 antibiotic drugs, respectively, and exhibited hVISA with a minimum inhibitory concentration of vancomycin as 4 µg/mL. Its genome is 2,883,941 bp in length and contains 2934 predicted genes with an average G + C content of 32.9%. Besides, a total of 38 virulence factors and 4 antibiotic-resistant genes were identified. These results can be employed to further study the pathogenic and antimicrobial mechanisms of hVISA.

Study on the Viable but Non-culturable (VBNC) State Formation of Staphylococcus aureus and Its Control in Food System.

A Viable but non-culturable (VBNC) state is a bacterial survival strategy under reverse conditions. It poses a significant challenge for public health and food safety. In this study, the effect of external environmental conditions including acid, nutrition, and salt concentrations on the formation of S. aureus VBNC states at low temperatures were investigated. Different acidity and nutritional conditions were then applied to food products to control the VBNC state formation. Four different concentration levels of each factor (acid, nutrition, and salt) were selected in a total of 16 experimental groups. Nutrition showed the highest influence on the VBNC state formation S. aureus, followed by acid and salt. The addition of 1% acetic acid could directly kill S. aureus cells and inhibit the formation of the VBNC state with a nutrition concentration of 25, 50, and 100%. A propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied and considered as a rapid and sensitive method to detect S. aureus in VBNC state with the detection limit of 104 CFU/mL.

Effect of Environmental Conditions on the Formation of the Viable but Nonculturable State of Pediococcus acidilactici BM-PA17927 and Its Control and Detection in Food System.

Objective: This study aimed to investigate the effect of environmental conditions including nutrient content, acetic acid concentration, salt concentration, and temperature on the formation of viable but nonculturable (VBNC) state of Pediococcus acidilactici, as well as its control and detection in food system. Methods: Representing various environmental conditions in different food systems, 16 induction groups were designed for the formation of VBNC state of P. acidilactici. Traditional plate counting was applied to measure the culturable cell numbers, and Live/Dead Bacterial Viability Kit combined with fluorescent microscopy was used to identify viable cells numbers. The inhibition of bacterial growth and VBNC state formation by adjusting the environmental conditions were investigated, and the clearance effect of VBNC cells in crystal cake system was studied. In addition, a propidium monoazide-polymerase chain reaction (PMA-PCR) assay was applied to detect the VBNC P. acidilactici cells in crystal cake food system. Results: Among the environmental conditions included in this study, acetic acid concentration had the greatest effect on the formation of VBNC state of P. acidilactici, followed by nutritional conditions and salt concentration. Reducing nutrients in the environment and treating with 1.0% acetic acid can inhibit P. acidilactici from entering the VBNC state. In the crystal cake system, the growth of P. acidilactici and the formation of VBNC state can be inhibited by adding 1.0% acetic acid and storing at -20°C. In crystal cake system, the PMA-PCR assay can be used to detect VBNC P. acidilactici cells at a concentration higher than 104 cells/ml. Conclusion: The VBNC state of P. acidilactici can be influenced by the changing of environmental conditions, and PMA-PCR assay can be applied in food system for the detection of VBNC P. acidilactici cells.

Spoilage Lactic Acid Bacteria in the Brewing Industry.

Lactic acid bacteria (LAB) have caused many microbiological incidents in the brewing industry, resulting in severe economic loss. Meanwhile, traditional culturing method for detecting LAB are time-consuming for brewers. The present review introduces LAB as spoilage microbes in daily life, with focus on LAB in the brewing industry, targeting at the spoilage mechanism of LAB in brewing industry including the special metabolisms, the exist of the viable but nonculturable (VBNC) state and the hop resistance. At the same time, this review compares the traditional and novel rapid detection methods for these microorganisms which may provide innovative control and detection strategies for preventing alcoholic beverage spoilage, such as improvement of microbiological quality control using advanced culture media or different isothermal amplification methods.

Inhibitory effects of two types of food additives on biofilm formation by foodborne pathogens.

The inhibition of microbial biofilms is a significant concern in food safety. In the present study, the inhibitory effect of sodium citrate and cinnamic aldehyde on biofilm formation at minimum inhibitory concentrations (MICs) and sub-MICs was investigated for Escherichia coli O157:H7 and Staphylococcus aureus. The biofilm inhibition rate was measured to evaluate the effect of sodium citrate on S. aureus biofilms at 24, 48, 72, and 96 hr. According to the results, an antibiofilm effect was shown by both food additives, with 10 mg/ml of sodium citrate exhibiting the greatest inhibition of S. aureus biofilms at 24 hr (inhibition rate as high as 77.51%). These findings strongly suggest that sodium citrate exhibits a pronounced inhibitory effect on biofilm formation with great potential in the extension of food preservation and storage.