Crystal Violet Staining Method Research Articles

Biofilm-forming ability of Salmonella enterica strains of different serotypes isolated from multiple sources in China

Salmonella is an important zoonotic and foodborne pathogen that can infect humans and animals, causing severe concerns about food safety and a heavy financial burden worldwide. The pathogen can adhere to living and abiotic surfaces by forming biofilms, which increases the risk of transmission and infection. In this study, we investigated the biofilm-forming ability of 243 Salmonella strains of 36 serotypes from different sources in China using microplate crystal violet staining method. The results showed that 99.6% tested strains, with the exception of one strain of S. Thompson, were capable of forming biofilms. The strains with the biofilm-forming ability of strong, medium and weak accounted for 2.88%, 24.28% and 72.43%, respectively. The strains of S. Havana and S. Hvittingfoss had the strongest biofilm-forming ability, with the OD570 of 0.81 ± 0.02 and 0.81 ± 0.38, respectively, while the strains of S. Agona and S. Bovismorbificans had the weakest biofilm-forming ability, with the OD570 of 0.16 ± 0.02 and 0.15 ± 0.00, respectively. Furthermore, statistical analysis results demonstrated that isolation of source had no effect on the biofilm formation ability, while the detection rates of pefABCD and ddhC were positively correlated with the biofilm formation ability of Salmonella. In particular, the detection rate of ddhC gene was more than 60% in the biofilm forming strains. These findings have important guiding significance for the investigation of pathogenesis, as well as the prevention and control of salmonellosis.

Effects of Repeated in-vitro Exposure to Saudi Honey on Bacterial Resistance to Antibiotics and Biofilm Formation.

Although Sumra and Sidr Saudi honey is widely used in traditional medicine due to its potent activity, it is unknown whether its prolonged usage has impact upon bacterial virulence or leading to reduced antibiotic sensitivity. Thus, the study aims to investigate the effect of prolonged (repeated) in-vitro exposure to Saudi honey on the antibiotic susceptibility profiles and biofilm formation of pathogenic bacteria. Several bacteria, including Staphylococcus aureus, Escherichia coli, and Acinetobacter baumannii, were in-vitro exposed ten times [passaged (P10)]to Sumra and Sider honey individually to introduce adapted bacteria (P10). Antibiotic susceptibility profiles of untreated (P0) and adapted (P10) bacteria were assessed using disc diffusion and microdilution assays. The tendency regarding biofilm formation following in-vitro exposure to honey (P10) was assessed using the Crystal violet staining method. Adapted (P10) bacteria to both Sumra and Sidr honey showed an increased sensitivity to gentamicin, ceftazidime, ampicillin, amoxycillin/clavulanic acid, and ceftriaxone, when compared with the parent strains (P0). In addition, A. baumannii (P10) that was adapted to Sidr honey displayed a 4-fold increase in the minimal inhibitory concentration of the same honey following in-vitro exposure. 3-fold reduction in the tendency toward biofilm formation was observed for the Sumra-adapted (P10) methicillin resistant S. aureus strain, although there was a lower rate of reduction (1.5-fold) in biofilm formation by both the Sumra- and Sidr-adapted A. baumannii (P10) strains. The data highlight the positive impact of prolonged in-vitro exposure to Saudi honey (Sumra and Sider) for wound-associated bacteria since they displayed a significant increase in their sensitivity profiles to the tested antibiotic and a reduction in their ability to form biofilm. The increased bacterial sensitivity to antibiotics and a limited tendency toward biofilm formation would suggest the great potential therapeutic use of this Saudi honey (Sumra and Sidr) to treat wound infections.

Untargeted Metabolomics for Unraveling the Metabolic Changes in Planktonic and Sessile Cells of Salmonella Enteritidis ATCC 13076 after Treatment with Lippia origanoides Essential Oil.

Nontyphoidal Salmonella species are one of the main bacterial causes of foodborne diseases, causing a public health problem. In addition, the ability to form biofilms, multiresistance to traditional drugs, and the absence of effective therapies against these microorganisms are some of the principal reasons for the increase in bacterial diseases. In this study, the anti-biofilm activity of twenty essential oils (EOs) on Salmonella enterica serovar Enteritidis ATCC 13076 was evaluated, as well as the metabolic changes caused by Lippia origanoides thymol chemotype EO (LOT-II) on planktonic and sessile cells. The anti-biofilm effect was evaluated by the crystal violet staining method, and cell viability was evaluated through the XTT method. The effect of EOs was observed by scanning electron microscopy (SEM) analysis. Untargeted metabolomics analyses were conducted to determine the effect of LOT-II EO on the cellular metabolome. LOT-II EO inhibited S. Enteritidis biofilm formation by more than 60%, without decreasing metabolic activity. Metabolic profile analysis identified changes in the modulation of metabolites in planktonic and sessile cells after LOT-II EO treatment. These changes showed alterations in different metabolic pathways, mainly in central carbon metabolism and nucleotide and amino acid metabolism. Finally, the possible mechanism of action of L. origanoides EO is proposed based on a metabolomics approach. Further studies are required to advance at the molecular level on the cellular targets affected by EOs, which are promising natural products for developing new therapeutic agents against Salmonella sp. strains.

The Anti-Biofilm Activity and Mechanism of Apigenin-7-O-Glucoside Against Staphylococcus aureus and Escherichia coli.

This study aimed to examine the anti-biofilm activity and mechanism of gallic acid (GA), kaempferol-7-O-glucoside (K7G) and apigenin-7-O-glucoside (A7G) against Staphylococcus aureus and Escherichia coli. The antibacterial activity of the natural compounds was determined by serial dilution method. The inhibitory activity of natural compounds on biofilms was determined by crystal violet staining method. The effects and mechanisms of natural compounds on bacterial biofilms were analyzed by atomic force microscopy. In our study, compared with GA and K7G, A7G was found to exhibit the strongest anti-biofilm and antibacterial activities. The minimum biofilm inhibitory concentration (MBIC) of A7G against S. aureus and E. coli was 0.20 mg/mL and 0.10 mg/mL, respectively. The inhibition rates of 1/2 MIC of A7G on biofilms of S. aureus and E. coli were 88.9%, and 83.2% respectively. Moreover, atomic force microscope (AFM) images showed the three-dimensional biofilm morphology of S. aureus and E. coli, and the results indicated that A7G was highly effective in biofilm inhibition. It was found that the inhibition of A7G on biofilm was achieved through inhibiting on exopolysaccharides (EPS), quorum sensing (QS), and cell surface hydrophobicity (CSH). A7G exerted strong anti-biofilm activities by inhibiting EPS production, QS, and CSH. Hence, A7G, as a natural substance, could be a promising novel antibacterial and anti-biofilm agent for control of biofilm in food industry.

Isolation and characterization of a lytic Salmonella Typhimurium-specific phage as a potential biofilm control agent

This study aimed to characterize a lytic Salmonella Typhimurium-specific (ST) phage and its biofilm control capability against S. Typhimurium biofilm on polypropylene surface. ST phage was isolated, propagated, and purified from water used in a slaughterhouse. The morphology of ST phage was observed via transmission electron microscopy. Its bactericidal effect was evaluated by determining bacterial concentrations after the phage treatment at various multiplicities of infection (MOIs) of 0.01, 1.0, and 100. Once the biofilm was formed on the polypropylene tube after incubation at 37°C for 48 h, the phage was treated and its antibiofilm capability was determined using crystal violet staining and plate count method. The phage was isolated and purified at a final concentration of ∼11 log PFU/mL. It was identified as a myophage with an icosahedral head (∼104 nm) and contractile tail (∼90-115 nm). ST phage could significantly decrease S. Typhimurium population by ∼2.8 log CFU/mL at an MOI of 100. After incubation for 48 h, biofilm formation on polypropylene surface was confirmed with a bacterial population of ∼6.9 log CFU/cm2. After 1 h treatment with ST phage, the bacterial population in the biofilm was reduced by 2.8 log CFU/cm2. Therefore, these results suggest that lytic ST phage as a promising biofilm control agent for eradicating S. Typhimurium biofilm formed on food contact surfaces.

Antimicrobial and Antibiofilm Effect of Commonly Used Disinfectants on Salmonella Infantis Isolates.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

IN VITRO EVALUATION OF THE ANTIOXIDANT, ANTIBACTERIAL, ANTIBIOFILM, AND CYTOTOXIC ACTIVITY OF MENTHA PIPERITA ESSENTIAL OIL

Nowadays, antibacterial resistance to synthetic antibiotics has become a big issue worldwide. Mentha piperita essential oil (EO) and its bioactive components with antibacterial, antibiofilm, and antioxidant properties are recommended for the treatment of microbial infections in traditional medicine. This study aimed to detect phytochemical components, antioxidant and cytotoxic activity; to evaluate antibacterial and antibiofilm effects of M. piperita EO on strains. Gas chromatography/mass spectrometry (GC-MS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) tests were used to determine chemical composition, antioxidant and cytotoxic potentials of EO, respectively. Antibacterial activity was carried out through disc diffusion and minimum inhibitory concentration (MIC) assays. The biofilm biomass of bacteria was determined by the crystal violet (CV) staining method. Menthone (27.28%), L-menthol (23.71%), and limonene (7.48%) were the main components. M. piperita EO was found to have strong antibacterial activity with the MIC-MBC values in the range of 0.125-16.00% (v/v) and moderate antioxidant activity. The peppermint EO showed an antibiofilm effect on all strains and a cytotoxic effect on NIH 3T3 cells. M. piperita EO may be used alone or in combination with antibiotics or carrier oils as a therapeutic agent against bacterial infections. Further in vivo experiments should be carried out to detect a safe dosage of EO.

Combined effect of apigenin and reduced graphene oxide against Enterococcus faecalis biofilms.

Enterococcus faecalis (E. faecalis) is one of the major microorganisms that causes failure of endodontic treatment. This study aimed to investigate the antibacterial activity of apigenin and its synergistic effect with reduced graphene oxide (RGO) in treating E. faecalis biofilms. The antibacterial activities were characterized by viability analysis including colony forming units and confocal laser scanning microscopy (CLSM) analyses. The effect on biofilm biomass was measured using a crystal violet staining method. Live and dead bacteria bio-volumes were determined by CLSM analysis, and the morphology of E. faecalis biofilm after treatment with apigenin and apigenin + RGO was observed by scanning electron microscopy (SEM). The viability of E. faecalis in biofilms decreased by apigenin treatment in a dose-dependent manner. While apigenin alone did not significantly affect the biofilm biomass, apigenin + RGO reduced the biomass in an apigenin concentration-dependent manner. Likewise, the bio-volume of live bacteria decreased and the bio-volume of dead bacteria increased in apigenin-treated biofilms. According to SEM images, apigenin + RGO-treated samples showed less E. faecalis in biofilms than apigenin-only treated samples. The results suggested that the combined use of apigenin and RGO could be a potential strategy for effective endodontic disinfection.

Phytochemical analysis and biological activities of Salvia candidissima subsp. candidissima mericarps

The sterol, amino acid, mineral, total phenolic, and total flavonoid contents, and the antioxidant, antimicrobial, and antibiofilm activities of Salvia candidissima subsp. candidissima mericarps were investigated. The mericarps were collected in the province of Bayburt, Turkey. Gas chromatography was used for sterol analysis, Prominence ultra-fast liquid chromatography for amino acid analysis, and ICPMS for mineral analysis. Folin-Ciocalteu, Al(NO3)3, and DPPH radical scavenging activity assays were performed on ethanol extracts of the mericarps to evaluate the total phenolic and flavonoid contents, and antioxidant potential, respectively. The ethanol and hexane extracts of the mericarps were tested for their antimicrobial activity against seven bacterial and three fungal strains using the microdilution method and for antibiofilm activity against Pseudomonas aeruginosa biofilm using the crystal violet staining method. The sterol, amino acid, and mineral contents and antibiofilm activity of the mericarps were studied for the first time. The most abundant components of the mericarps are determined as ?-sitosterol (69.8%), glutamic acid (4895 mg/100 g), and potassium (11474 ?g/g). The results of total phenolic and flavonoid contents and DPPH free radical scavenging activity were 3.47 ?g GAE/mg extract, 0.35 ?g QE/mg extract, and 79.79%, respectively. Both of the tested extracts exhibited antimicrobial activity against all the tested microorganisms, however, the efficiency of the extracts was not as strong as the reference drugs ampicillin and fluconazole. While both extracts were effective in preventing biofilm formation, the ethanol extract was found to be more effective than the hexane extract in preformed biofilm inhibition. In conclusion, S. candidissima subsp. candidissima mericarps have good nutritional potential with high amounts of amino acids, sterols (especially ?-sitosterol), minerals, and phenolics and flavonoids. Additionally, our findings provide important preliminary data for the literature in terms of the antibiofilm activity of Salvia candidissima subsp. candidissima mericarps.

Catheter-associated urinary tract biofilms: can achyranthes aspera extract work against them?

Catheter-associated urinary tract infection is mainly related to biofilm on the catheter surface which provides the opportunity for colonization and attachment resulting complex structured biofilm formation makes development of antibiotic resistant organism creates management of CAUTI is more critical. Therefore, there is an alarming call for drug development against multidrug resistant organisms. Hence, our study attempted the antibacterial activity of methanolic extract of Achyranthes aspera against Staphylococcus aureus one of the prevalent organisms involved in CAUTI. The A. aspera antibacterial activity was determined against S. aureus and minimum inhibitory concentration of A. aspera against S. aureus calculated was as 3.6 mg/ml which needed to inhibit the growth of tested microbe. The colony forming ability of S. aureus was studied in the presence of A. aspera methanolic extract using crystal violet staining method and the colony formation was observed after the MIC level indicates colony formation increases when decreasing the concentration of A. aspera. To ascertain the effect of A. aspera on biofilm formation after treatment with various concentrations, the biofilm formation assay performed. The A. aspera can able to reduce the biofilm formation as 80 and 88% for 1X MIC and 2X MIC against S. aureus indicating the antibiofilm forming ability. To prevent the microbial colonization on catheter surface, the coating of catheter with methanolic extract of A. aspera was investigated for its antibacterial activity against S. aureus using in vitro bladder model. The clear zone formations around the catheter piece indicating the anti-adhesive property of A. aspera against S. aureus. Collectively, methanolic extract of A. aspera can pave the way for new antibacterial agent against S. aureus.

Development of a robust and quantitative high-throughput screening method for assessing phenotypic variation in large Neisseria meningitidis isolate collections

Genome-wide association studies are a powerful approach for identifying determinants of disease. For infectious diseases, high throughput assays are required for measuring the variance in multiple virulence-related phenotypes of large bacterial isolate collections and for association of this phenotypic variance with genotype. The primary limiting factors are cost, effectiveness and a standardized inoculum. A method was developed to create an inoculum array of multiple isolates that could be used for a series of high-throughput multi-isolate phenotypic investigations in a laboratory setting. A key starting point was the standardisation of the inoculum by production of identical batches of each isolate from cells grown to mid-log phase. Cultures with pre-determined optical densities were aliquoted in set patterns into multiple multi-well plates containing 50% glycerol and stored at -80 °C. Prior to a specific assay, an inoculum plate was defrosted and subjected to a brief period of incubation. Control strains can be placed on each plate in order to control for intra-assay variability. A high throughput screen is described in detail for quantification of biofilm formation. This example utilised the crystal violet staining method and multi-assay stock plates containing 16 meningococcal isolates.•Multi-assay stock plate of exponentially growing isolates is cost-effective and simple to implement in a laboratory setting.•This method would predict realistic standard deviations for multiple isolates in phenotypic assays and generate data for performance of power calculations for genotyping.•This method has the potential to identify both known and unknown genetic determinants of phenotypic variability for each tested isolate when paired with genetic analysis of whole genome sequencing data.

Phlomoides molucelloides (Bunge) Salmaki'nin Antibakteriyel Etkileri

The insufficiency of existing antibiotics in the combat against antibiotics-resistant bacteria has necessitated the discovery of new and effective antibacterial drugs. The discovery that bacteria synthesize various virulence factors by the quorum sensing system has suggested that quorum sensing inhibitors may be used in the fight against infectious diseases. This study aimed to determine the antibacterial and anti-quorum sensing activities of methanol and water extracts of aerial and root parts of Phlomoides molucelloides (Bunge) Salmaki. The minimal inhibitory concentration (MIC) values of the extracts were investigated against reference bacterial strains using the broth microdilution method. Anti-quorum sensing activities were examined by violacein and pyocyanin pigments inhibition and swarming motility inhibition assays against Chromobacterium violaceum ATCC 12472 and Pseudomonas aeruginosa PAO1 bioreporter strains. The antibiofilm activities of the extracts were tested against P. aeruginosa PAO1 using the crystal violet staining method. The MIC value (> 2000 µg/mL) of all extracts against the tested bacteria could not be determined at the concentrations studied. All extracts partially inhibited the swarming motility of P. aeruginosa. Methanol extract of the aerial part inhibited pyocyanin production by 81.7% without interfering with P. aeruginosa growth. The extracts had no significant inhibitory activity on biofilm formation and violacein pigment production. These results showed that the extracts of P. molucelloides may be good anti-quorum sensing agents. Further research can be done to elucidate the mechanisms underlying these biological activities.

New Strategies for Inhibition of Listeria monocytogenes and Klebsiella pneumoniae Biofilm Formation and Persistence

Objective: This study aimed to find new strategies for the prevention of bacterial biofilms and investigate the effect of some plant extracts on the biofilm formation by certain pathogenic bacterial strains in-vitro.
 Methods: Fourteen different biofilm forming bacterial isolates were collected and their biofilm were quantitatively measured under different temperature, pH and growth medium using Crystal violet staining method. Exopolysaccharides (EPS) produced by the isolates were estimated and a comparison between the tested isolates was made. The effect of some plant extracts on bacterial growth, biofilm formation and exopolysaccharide quantity was determined.
 Results: The two isolates, Listeria monocytogenes (ATCC13932) and Klebsiella pneumoniae (ATCC700613) were among the most active biofilm forming bacterial isolates. The optimum temperature, pH and media for EPS production and biofilm formation were determined. The effect of some plant extracts of Cranberry; Pomegranate peel and, Arak on growth and formation of biofilm and EPS were recorded. Moreover, minimum inhibition concentration of each plant extract was performed.
 Conclusion: EPS quantity produced from the tested isolates depends on some effective factors such temperature, media contents and pH. Aqueous-Cranberry, aqueous-Pomegranate Peel and methanolic-Arak extracts have antibacterial and antibiofilm activities on L. monocytogenes and K. pneumonia. Thus, application of new natural approaches for inhibiting bacterial biofilm is important to prevent persistent and recurrent biofilm related infections.

The biofilm formation and antibiotic resistance of bacterial profile from endotracheal tube of patients admitted to intensive care unit in southwest of Iran.

Ventilator-associated pneumonia (VAP) is a prevalent nosocomial illness in mechanically ventilated patients. Hence, the aim of this study was to investigate the pattern of antibiotic resistance and biofilm formation of bacterial profiles from Endotracheal Tubes of patients hospitalized in an intensive care unit in southwest Iran. According to the standard operating method, the microbiological laboratory conducts bacteria culture and susceptibility testing on endotracheal Tube samples suspected of carrying a bacterial infection. The Clinical and laboratory standards institute (CLSI) techniques are used to determine the Antimicrobial resistance (AMR) of bacterial isolates to antibiotics using the disk diffusion method. The crystal violet staining method was used to assess the biofilm-forming potential of isolates in a 96-well microtiter plate. In total, (51%) GPBs were included in this study. The isolated GPB were coagulase-negative Staphylococcus (16%), S. aureus (14%). In total, (40%) of GNB were included in this study. The isolated GNB were Klebsiella spp. (36%), A. baumannii (22%), P. aeruginosa (35%). (32%) bacterial strains were MDR and (29%) strains were XDR. The results of biofilm formation showed (72%) were biofilm producers. VAP is a common and severe nosocomial infection in mechanically ventilated patients. Controlling biofilm formation, whether on the ET or in the oropharyngeal cavity, is thus an important technique for treating VAP. Colistin and linezolid are antibiotics that are effective against practically all resistant GNB and GPB isolates.

Viral Titer Quantification of West Nile Virus by Immunostaining Plaque Assay.

Immunostained plaque assay based on the specific antibody binding to viral antigen enables the detection and titration of virus infectivity, especially for viruses that could not form plaques using the classical crystal violet or neutral red staining methods. Here we describe the application of this method to quantify viral titers of wild-type West Nile virus (WNV-WT) and replication-defective WNV-ΔNS1 virus.

Propionate and Butyrate Inhibit Biofilm Formation of Salmonella Typhimurium Grown in Laboratory Media and Food Models.

Salmonella is among the most frequently isolated foodborne pathogens, and biofilm formed by Salmonella poses a potential threat to food safety. Short-chain fatty acids (SCFAs), especially propionate and butyrate, have been demonstrated to exhibit a beneficial effect on promoting intestinal health and regulating the host immune system, but their anti-biofilm property has not been well studied. This study aims to investigate the effects of propionate or butyrate on the biofilm formation and certain virulence traits of Salmonella. We investigated the effect of propionate or butyrate on the biofilm formation of Salmonella enterica serovar Typhimurium (S. Typhimurium) SL1344 grown in LB broth or food models (milk or chicken juice) by crystal violet staining methods. Biofilm formation was significantly reduced in LB broth and food models and the reduction was visualized using a scanning electron microscope (SEM). Biofilm metabolic activity was attenuated in the presence of propionate or butyrate. Meanwhile, both SCFAs decreased AI-2 quorum sensing based on reporter strain assay. Butyrate, not propionate, could effectively reduce bacterial motility. Bacterial adhesion to and invasion of Caco-2 cells were also significantly inhibited in the presence of both SCFAs. Finally, two SCFAs downregulated virulence genes related to biofilm formation and invasion through real-time polymerase chain reaction (RT-PCR). These findings demonstrate the potential application of SCFAs in the mitigation of Salmonella biofilm in food systems, but future research mimicking food environments encountered during the food chain is necessitated.

Antibiotic susceptibility and biofilm-forming ability of Veillonella strains

IntroductionVeillonella, known as early colonizers in oral biofilms, take part in some infections in human. Biofilm refers to complex, sessile communities of microbes, which function as strong barriers for bacteria to survive. Biofilm matrixes surrounding bacteria enable them to withstand harsh conditions, protect against immune cells, etc., and also make them resistant to antimicrobial treatments. Thus, the knowledge of antibiotic susceptibility and biofilm formation of Veillonella will shed light on their resistance mechanism. Materials and methodTheir morphology was observed by scanning electron microscopy (SEM). According to the performance standards for antibiotic susceptibility testing of the Clinical & Laboratory Standards Institute, the Agar dilution method was used to study the susceptibility of Veillonella strains to eight antibiotics (ampicillin, piperacillin-tazobactam, cefoxitin, tetracycline, moxifloxacin, clindamycin, metronidazole, and vancomycin). In addition, we applied the crystal violet staining method to reveal the processes of biofilm formation of these Veillonella strains. ResultsV. rogosae, V. nakazawae, and V. parvula were isolated from oral cavities of healthy adults and V. ratti was isolated from dairy goat droppings. Observations by scanning electron microscopy showed that Veillonella were spherical and arranged in single or short chains. The diameter of a single cell was about 0.3–0.5 μm. The Minimum Inhibitory Concentrations (MICs) of the antibiotics were determined and the results showed that these four strains were all sensitive to cefoxitin, tetracycline, moxifloxacin, clindamycin and metronidazole. Among the four strains, V. ratti was resistant to piperacillin-tazobactam, and V. rogosae and V. nakazawae were resistant to ampicillin. The vancomycin susceptibility of the four Veillonella strains varied greatly. The MICs of vancomycin against V. rogosae and V. ratti were greater than 256 μg/mL but the MICs of vancomycin against V. nakazawae and V. parvula were less than 2 μg/mL. V. parvula had significantly higher biofilm-forming ability than the other three strains (p < 0.05) and V. nakazawae had the weakest biofilm-forming ability. ConclusionIn this study, V. rogosae, V. nakazawae, V. parvula and V. ratti were isolated and identified. The four strains of Veillonella showed differences in MIC values for different antibiotics and biofilm-forming ability.

IN VITRO EVALUATION OF ANTI-BIOFILM POTENTIAL OF ALUM AND ITS BHASMA (SPHATIKA BHASMA)

Microbial biofilms may form on wide variety of surfaces and are problematic in medical, industrial and environmental settings. Natural antibiofilm agents are promising candidates for treatment of biofilm-associated infections. One such natural products used for therapeutic purposes is Alum (Sphatika). Besides, Ayurvedic Bhasmas are used for treatment of various ailments in India. In the present in vitro study, anti-Biofilm potential of Alum as well as its Bhasma- Sphatika Bhasma was evaluated against total 8 bacteria, viz., 4 gram negative and 4 gram positive using crystal violet staining method. Antibiofilm activity of Sphatika Bhasma was noted in the order of P. aeruginosa &gt; S. lutea &gt; E. coli &gt; K. pneumoniae &gt; S. typhi &gt; MRSA &gt; B. subtilis &gt; E. faecalis, whereas, Antibiofilm effect of Alum was found to be in the order of S. lutea &gt; P. aeruginosa &gt; E. coli &gt; MRSA &gt; S. typhi &gt; B. subtilis &gt; K. pneumoniae &gt; E. faecalis. In general, Sphatika Bhasma exhibited better Anti-Biofilm activity than Alum. Furthermore, four different combinations prepared from Alum (AL), Sphatika Bhasma (SB) and Ciprofloxacin (CP)- standard antibiotic were evaluated for their combinatorial effect against 8 bacterial biofilms. In general, the combination (AL + SB) displayed the highest Anti-biofilm activity against all the 8 bacterial biofilms. Antibiofilm effects of Alum and Sphatika Bhasma could be attributed to their Flavonoid content. Moreover, to best of our knowledge, this is the first study revealing the anti-biofilm potential of Sphatika Bhasma.

Effect of savirin in the prevention of biofilm-related Staphylococcus aureus prosthetic joint infection.

Background: Most of the arthroplasty surgery failure due to prosthetic joint infections (PJI) is caused by biofilm-associated Staphylococcus aureus. In a recent experimental study, savirin has been used to prevent and treat S. aureus skin infections in animal models. We explored the application of savirin in a PJI mouse model to determine its utility as an adjunct therapy to prevent PJI. Materials and methods: The in-vitro antibacterial and antibiofilm activity of savirin, with or without antibiotics (cefazolin, rifampicin, and vancomycin), against S. aureus were investigated using broth microdilution and crystal violet staining method, respectively. The effect of savirin treatment on the expression of the key biofilm-related genes (icaA, icaD, eno, fib, ebps, and agr) in S. aureus was studied using quantitative reverse transcriptase polymerase chain reaction (qRTPCR). The in-vivo efficacy of savirin alone and with cefazolin to prevent S. aureus PJI was determined using a clinically relevant PJI mouse model. Mice were randomized into five groups (n = 8/group): 1) infected K-wire savirin treated group, 2) infected K-wire cefazolin treated group, 3) infected K-wire savirin plus cefazolin treated group, 4) infected K-wire PBS treated group, 5) sterile K-wire group. Savirin was administered subcutaneously immediately post-surgery and intravenous cefazolin was given on day seven. Results: Savirin inhibited planktonic and biofilm in-vitro growth of S. aureus, showed enhanced inhibitory activity when combined with antibiotics, and down-regulated the expression of key S. aureus biofilm-related genes (icaA, icaD, eno, fib, ebps, and agr). Savirin significantly reduced bacterial counts on joint implants in comparison with the PBS treated control, while savirin plus cefazolin reduced bacterial counts on both implants and peri-prosthetic tissues. Conclusion: Savirin adjuvant therapy may prevent biofilm formation and S. aureus PJI. This study gives baseline data for using savirin for the prevention as well as treatment of S. aureus PJI in future animal studies.

Carbon dioxide can inhibit biofilms formation and cellular properties of Shewanella putrefaciens at both 30 °C and 4 °C

Shewanella putrefaciens (S. putrefaciens), which is a common specific spoilage organism (SSO) of marine fish, has strong spoilage ability even under low-temperature conditions. Carbon dioxide (CO2) was widely applied to control microorganisms in aquatic products package. To explore the regulation mechanism of CO2 on biofilm formation and cell properties of S. putrefaciens, the dynamic formation process of biofilms, cellular surface properties, and cellular metabolic characteristics of S. putrefaciens at both 30 °C and 4 °C in pure CO2 gas were evaluated. As evidenced by the crystal violet staining method, confocal laser scanning microscopy (CLSM) analysis, and field emission scanning electron microscopy (FESEM) observation, dynamic formation process of S. putrefaciens biofilms was apparently delayed by CO2 with integral cellular morphology. The number and viability of sessile cells in S. putrefaciens biofilms was significantly lower than those in normal air composition. The changes in cellular surface properties, such as decreased auto-aggregation and hydrophobicity, might be one of the reasons why biofilms were inhibited by CO2. Inhibition of swimming and swarming motility ability by CO2 could also be observed with significantly shorter bacterial halo diameter. What’s more, cellular metabolism was significantly decreased by CO2 according to the results of ATP content, ATPase activity and extracellular proteolytic activity. The influence of CO2 could be both observed whether combined with 30 °C or 4 °C. However, the inhibition produced by CO2 was more pronounced at the incubation temperature of 4 °C. In summary, it could be concluded that the dynamic formation process of S. putrefaciens biofilms and cellular metabolic properties could be inhibited by CO2. This research provided a theoretical basis for better application of CO2 to regulate spoilage microorganisms.