Quantification Of Biofilm Formation Research Articles

Evaluation of antibiofilm activity of Thymus syriacus essential oil against clinically isolated MDR bacteria

Finding alternative strategies to confront bacterial resistance is an urgent need. Biofilm-forming bacteria have become a serious problem in medicine and industry. Bacteria can use biofilm as a mechanism of resistance against antibacterial drugs and avoid the immune system. The aim of this study was to evaluate the antibacterial activity of Thymus syriacus (T. syriacus) essential oil in a solid and liquid medium and to study its antibiofilm formation activity. The T. syriacus essential oil was extracted from the aerial parts of the plants. The minimum inhibitory concentrations (MIC) measurements were used to identify the antibacterial activity of the essential oil against multidrug-resistant strains Pseudomonas aeruginosa (P. aeruginosa), Klebsiella pneumonia (K. pneumonia), Streptococcus pneumonia (S. pneumonia) isolated clinically from blood infections. The microtiter plate was used in order to quantify biofilm formation by bacteria. The minimum inhibitory concentration (MIC) against the three clinically isolated strains (P. aeruginosa, K. pneumonia, S. pneumonia) were (3.12, 1.56, 3.12 µL/mL) respectively. The formation of biofilm by (P. aeruginosa, K. pneumonia, S. pneumonia) was reduced up to (43%, 50%, 60%) respectively, when the essential oil was applied at MIC concentrations for each strain. The observed antibacterial activity of T. syriacus essential oil was significant against antibacterial-resistant strains and antibiofilm formation activity was identified. The novelty of this study is we confirmed that the essential oil of T. syriacus exhibited not just antibacterial properties but also antibiofilm formation effect. More studies are needed in order to continue studying this oil and evaluate its other medicinal properties and toxicity.

Salmonella Derby from pig production chain over a 10-year period: antimicrobial resistance, biofilm formation, and genetic relatedness.

The aim of this study was to evaluate 140 Salmonella Derby isolates collected over a 10-year period from porcine origins (environment, pig carcass, lymph nodes, intestinal content, and pork) for their phenotypic and genotypic antimicrobial resistance, their ability to produce biofilm, and their genetic relatedness. The minimum inhibitory concentration (MIC) was determined using microdilution broth method and antimicrobial resistance genes were investigated by PCR. The quantification of biofilm formation was performed in sterile polystyrene microtiter plates. Genetic relatedness was determined by Xba-I macrorestriction analysis. The highest frequencies of non-wildtype (nWT) populations were observed against tetracycline (75.7%), streptomycin (70%), and colistin (11.4%), whereas wildtype populations were observed against ciprofloxacin, ceftazidime, and gentamicin. The resistance genes found were blaTEM (ampicillin), aadA variant (streptomycin/spectinomycin), tetA (tetracycline), and floR (florfenicol). On 96-well polystyrene microtiter plate, 68.6% of the isolates proved to be biofilm producers. Among 36 S. Derby isolates selected to PFGE analysis, 22 were clustered with 83.6% of similarity. Additionally, 27 isolates were clustered in 11 pulsotypes, which presented more than one strain with 100% of similarity. Most of S. Derby isolates were able to form biofilm and were classified as nWT or resistant to tetracycline, streptomycin, and colistin. PFGE allowed the identification of closely related S. Derby isolates that circulated in pig slaughterhouses and pork derived products along a decade.

Biofilm formation, siderophore production, virulence-associated genes and genetic diversity of Yersinia enterocolitica from food

Yersinia enterocolitica is widely found in nature and foods including those implicated in foodborne infections. The ability of this organism to multiply at refrigeration temperatures in food products with a prolonged shelf-life renders it a potential health risk for consumers. Y. enterocolitica isolates from food were investigated for biofilm formation and siderophore production at different temperatures (12, 25 and 37 °C) using the microtiter plate and CAS agar plate methods, respectively. The isolates were also evaluated with PCR for the presence of virulence genes and rep-PCR for genetic diversity. Most of the isolates showed high capability to form biofilm at all temperatures after 24 and 48 h of incubation. The results at 12 and 25 °C (P < 0.05) as well as at 25 and 37 °C (P < 0.05) showed a statistically significant difference in quantification of biofilm formation after 24 h; however, no difference was observed between findings at 12 and 37 °C (P > 0.05). There was a significant difference at 12 and 37 °C (P < 0.05) as well as at 25 and 37 °C (P < 0.05) after 48 h of incubation; conversely, no significant difference was observed between 12 and 25 °C (P > 0.05). Strong biofilm-producing isolates at 12 °C were found to be more after 48 h than after 24 h of incubation. Siderophore production was observed in 83.3% and 100% of the isolates at 25 and 37 ºC, respectively. However, no isolate produced siderophores at 12 °C. Genes myfA, fepA, fepD, fes, hreP, tccC and ymoA were detected in 16.7%, 33.3%, 100%, 100%, 44.4%, 5.6%, and 33.3% of the isolates, respectively. The rep-PCR revealed a high level of genetic differentiation among the isolates. The results indicated that Y. enterocolitica from food may act as a potential pathogen producing biofilm and siderophore while carrying virulence-associated genes.

Screening for Biofilm-Stimulating Factors in the Freshwater Planctomycete Planctopirus limnophila to Improve Sessile Growth in a Chemically Defined Medium.

Planctomycetes such as Planctopirus limnophila offer a promising source of bioactive molecules, particularly when they switch from planktonic to sessile growth, but little is known about the corresponding biosynthetic gene clusters and how they are activated. We therefore screened for factors that promote sessile growth and biofilm formation to enable the cultivation of P. limnophila in a fixed-bed reactor. We carried out screening in microtiter plates focusing on biofilm formation and changes in optical density in response to various C:N ratios, metal ions, and oxidative stress. We used MTT assays and crystal violet staining to quantify biofilm formation. Positive factors were then validated in a fixed-bed bioreactor. The initial screen showed that D1ASO medium supplemented with NH4Cl to achieve a C:N ratio of 5.7:1, as well as 50 µM FeSO4 or CuSO4, increased the biofilm formation relative to the control medium. Exposure to H2O2 did not affect cell viability but stimulated biofilm formation. However, the same results were not replicated in the fixed-bed bioreactor, probably reflecting conditions that are unique to this environment such as the controlled pH and more vigorous aeration. Although we were able to cultivate P. limnophila in a fixed-bed bioreactor using a chemically defined medium, the factors that stimulate biofilm formation and inhibit planktonic growth were only identified in microtiter plates and further evaluation is required to establish optimal growth conditions in the bioreactor system.

Antimicrobial resistance, virulence genes, and biofilm formation in Staphylococcus aureus strains isolated from meat and meat products

ABSTRACTStaphylococcus aureus is an important food pathogen that has the ability to form biofilms. This pathogen has been implicated in foodborne outbreaks associated with the consumption of meat and meat products. Since the role of meat products as reservoirs of antimicrobial resistant S. aureus and biofilm formers has not been studied, this research was conducted to assess the incidence, antimicrobial resistance, and presence of biofilm‐forming genes in S. aureus bacteria from meat and meat products. For this purpose, 440 samples of meat and meat products were randomly collected from supermarkets of Shahrekord city, Iran. S. aureus isolates were highlighted and identified through biochemical tests. All S. aureus isolates were profiled for susceptibility to 14 antibiotics and the corresponding resistance genes were searched. The quantification of biofilm formation followed by a screening of biofilm formation encoding genes was done. Of the 440 samples collected, 61 were found to contain S. aureus. The incidence of S. aureus among the beef meat (16/61) was the highest followed by its incidence in sausage (10/61) and lunch meat (9/61). At least one isolate of S. aureus carries one of the following resistance gene blaZ, mecA, tet K, linA, tetM, erm A, ermB, and aac A‐D. High distribution of icaA, icaB, icaC, icaD, clfB, and clfA, biofilm formation genes and Enterotoxin encoding genes sea, seb, sec, and sed. The ability of biofilm production and the presence of virulence determinants in the genome of S. aureus can contribute to their pathogenicity. This research demonstrated the roles of meat and meat products as reservoirs of biofilm‐forming and antimicrobial‐resistant strains of S. aureus. This poses a food safety and public health problem. However, further work needs to be done to better understand the implication of icaABCD biofilm forming genes present in S. aureus from meat and meat products in their resistance.

New Adapted In Vitro Technology to Evaluate Biofilm Formation and Antibiotic Activity Using Live Imaging under Flow Conditions.

The polymicrobial nature of biofilms and bacterial interactions inside chronic wounds are keys for the understanding of bacterial cooperation. The aim of this present study was to develop a technique to study and visualize biofilm in live imaging under flow conditions (Bioflux™ 200, Fluxion Biosciences). The BiofluxTM system was adapted using an in vitro chronic wound-like medium (CWM) that mimics the environment encountered in ulcers. Two reference strains of Staphylococcus aureus (Newman) and Pseudomonas aeruginosa (PAO1) were injected in the BiofluxTM during 24 h to 72 h in mono and coculture (ratio 1:1, bacteria added simultaneously) in the CWM vs. a control medium (BHI). The quantification of biofilm formation at each time was evaluated by inverted microscopy. After 72 h, different antibiotics (ceftazidime, imipenem, linezolid, oxacillin and vancomycin) at 1x MIC, 10x MIC and 100x MIC were administrated to the system after an automatic increase of the flow that mimicked a debridement of the wound surface. Biofilm studies highlighted that the two species, alone or associated, constituted a faster and thicker biofilm in the CWM compared to the BHI medium. The effect of antibiotics on mature or “debrided” biofilm indicated that some of the most clinically used antibiotic such as vancomycin or imipenem were not able to disrupt and reduce the biofilm biomass. The use of a life cell imaging with an in vitro CWM represents a promising tool to study bacterial biofilm and investigate microbial cooperation in a chronic wound context.

Inhibition of biofilm formation on polystyrene substrates by atmospheric pressure plasma polymerization of siloxane‐based coatings

AbstractBiofilms pose important economic and health risks in biomedical applications and in food industries. In this study, coatings that reduce the biofilm formation of Pseudomonas aeruginosa on polystyrene cell culture plates are deposited by plasma polymerization of (3‐aminopropyl)triethoxysilane using an atmospheric pressure plasma jet system at three different power levels. Surface characterizations and quantification of biofilm formation during 1 week after deposition suggest that the higher concentration of oxygenated carbon groups on the coated samples than on uncoated ones can induce higher levels of oxidative stress in the bacteria in contact with the coatings. This causes an initial overproduction of extracellular polymeric substances that can avoid further bacterial attachment and biofilm formation at later cycles of biofilm development.

Use of Quorum Sensing Inhibition Strategies to Control Microfouling.

Interfering with the quorum sensing bacterial communication systems has been proposed as a promising strategy to control bacterial biofilm formation, a key process in biofouling development. Appropriate in vitro biofilm-forming bacteria models are needed to establish screening methods for innovative anti-biofilm and anti-microfouling compounds. Four marine strains, two Pseudoalteromonas spp. and two Vibrio spp., were selected and studied with regard to their biofilm-forming capacity and sensitivity to quorum sensing (QS) inhibitors. Biofilm experiments were performed using two biofilm cultivation and quantification methods: the xCELLigence® system, which allows online monitoring of biofilm formation, and the active attachment model, which allows refreshment of the culture medium to obtain a strong biofilm that can be quantified with standard staining methods. Although all selected strains produced acyl-homoserine-lactone (AHL) QS signals, only the P. flavipulchra biofilm, measured with both quantification systems, was significantly reduced with the addition of the AHL-lactonase Aii20J without a significant effect on planktonic growth. Two-species biofilms containing P. flavipulchra were also affected by the addition of Aii20J, indicating an influence on the target bacterial strain as well as an indirect effect on the co-cultured bacterium. The use of xCELLigence® is proposed as a time-saving method to quantify biofilm formation and search for eco-friendly anti-microfouling compounds based on quorum sensing inhibition (QSI) strategies. The results obtained from these two in vitro biofilm formation methods revealed important differences in the response of biosensor bacteria to culture medium and conditions, indicating that several strains should be used simultaneously for screening purposes and the cultivation conditions should be carefully optimized for each specific purpose.

Biofilm Forming Ability of Broiler Chicken Meat Associated <em>Salmonella</em> spp. on Food Contact Surfaces

Biofilms in the meat industry pose a significant impact on public health as many foodborne pathogens can form biofilms on equipment and utensils in meat processing plants. Biofilms formed on contact surfaces could be a continuous source of contaminants to food that come in contact with them. In the present study biofilm-forming ability of different Salmonella spp. isolated from broiler chicken meat from Sri Lanka was quantified, the presence of biofilm-related genes was investigated, and comparative evaluation on the biofilm formation on plastic, stainless steel, and glass surfaces was performed. It was revealed that the biofilm formation increased as a response to the nutrient stress. Quantification of biofilm formation by the microtitre plate method showed 7 strong (30.4%), 11 moderate (47.8%), and 5 weak (21.7%) biofilm formers at low nutrient media (TSB diluted 1:100), whereas there were only 4 moderate biofilm formers and 19 weak biofilm formers found in the undiluted TSB medium. All biofilm formers exhibited the presence of both adrA and gcpA regulatory genes. Investigation on the ability of biofilm formation on glass, stainless steel, and plastic at time points of 48, 96, 144, 192, and 240 hrs post incubation with the contamination of 106 CFU/cm2 revealed a significant (p≤0.05) variation among Salmonella isolates. All the isolates illustrated the highest ability to form biofilms on plastic, whereas that was minimum on stainless steel.

Transcriptional Profiling of Three Pseudomonas syringae pv. actinidiae Biovars Reveals Different Responses to Apoplast-Like Conditions Related to Strain Virulence on the Host.

Pseudomonas syringae pv. actinidiae is a phytopathogen that causes devastating bacterial canker in kiwifruit. Among five biovars defined by genetic, biochemical, and virulence traits, P. syringae pv. actinidiae biovar 3 (Psa3) is the most aggressive and is responsible for the most recent reported outbreaks; however, the molecular basis of its heightened virulence is unclear. Therefore, we designed the first P. syringae multistrain whole-genome microarray, encompassing biovars Psa1, Psa2, and Psa3 and the well-established model P. syringae pv. tomato, and analyzed early bacterial responses to an apoplast-like minimal medium. Transcriptomic profiling revealed i) the strong activation in Psa3 of all hypersensitive reaction and pathogenicity (hrp) and hrp conserved (hrc) cluster genes, encoding components of the type III secretion system required for bacterial pathogenicity and involved in responses to environmental signals; ii) potential repression of the hrp/hrc cluster in Psa2; and iii) activation of flagellum-dependent cell motility and chemotaxis genes in Psa1. The detailed investigation of three gene families encoding upstream regulatory proteins (histidine kinases, their cognate response regulators, and proteins with diguanylate cyclase or phosphodiesterase domains) indicated that cyclic di-GMP may be a key regulator of virulence in P. syringae pv. actinidiae biovars. The gene expression data were supported by the quantification of biofilm formation. Our findings suggest that diverse early responses to the host apoplast, even among bacteria belonging to the same pathovar, can lead to different virulence strategies and may explain the differing outcomes of infections. Based on our detailed structural analysis of hrp operons, we also propose a revision of hrp cluster organization and operon regulation in P. syringae.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Characterization of Staphylococcus aureus Isolates From Cases of Clinical Bovine Mastitis on Large-Scale Chinese Dairy Farms.

Bovine mastitis is a prevalent disease that causes serious economic problems globally in the dairy industry. Staphylococcus aureus is an important pathogen of bovine mastitis. This study was conducted to characterize S. aureus isolates from clinical bovine mastitis cases in large-scale dairy herds in China. S. aureus was isolated from 624 clinical mastitis cases and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In total, 62 S. aureus isolates were obtained. Cluster analysis, genetic diversity, quantification of biofilm formation, antimicrobial resistance, and detection of virulence genes were performed on these isolates of S. aureus. Eight isolates harbored the mecA gene and were sensitive to oxacillin. MALDI-TOF MS cluster analysis revealed that the 62 isolates were divided into three major clusters (I, II, III) and eight main groups (A–H) at the distance level of 700. The agr II was the most prevalent (56.5%). The 62 S. aureus isolates were assigned to seven spa types. The most common spa type was t529(58.1%), followed by t2196 (14.5%), t518 (14.5%), t571(6.5%), t034 (3.2%), t2734 (1.6%), and t730 (1.6%). Five STs were identified from seven representative isolates as follows: ST630/CC8, ST97/CC97, ST50, ST398, and ST705. All isolates had the ability to form biofilm. Antimicrobial resistance was most frequently observed to ciprofloxacin (29%), followed by penicillin (24.2%), and streptomycin (9.6%). All isolates harbored the fnbA, clfB (100%), icaA, and icaD genes. This study provides the basis for the development of bovine mastitis prevention program on large-scale dairy farms.

Anty-microbial, and anty-biofilm-forming properties of Origanum vulgare L. essential oils on Staphylococcus aureus and its antioxidant action

Essential oils (EOs) have been widely used in folk and traditional medicine, cooking, cosmetology and other sectors of the economy. The antimicrobial properties of essential oils determine the relevance of the study of their effectiveness against antibiotic-resistant biofuel clinical isolates. Our research was aimed to determine the antimicrobial and antibiofilm properties of essential oil of Origanum vulgare L. on clinical isolates of bacteria of the genus Staphylococcus and its antioxidant effect. The raw materials of Origanum vulgare L. for large-scale essential oil isolation were donated by established growers in Slovakia (Agrokarpaty, Ltd.). EOs were obtained by the large-scale distillation apparatus specifically designed for the aromatic and medicinal plants. Calendula Co., in Nova Lubovna, Slovakia. The analysis of EOs was carried out using a gas chromatograph Varian 3090. Antimicrobial activity of EOs was determined using well agar diffusion method. Quantification of biofilm formation was carried out using a microtiter plate assay and the spectrophotometric method. Biofilm formation was quantified by measuring optical density at 550 nm. Biochemical analysis of O. vulgare essential oil showed a high content of carvacrol and thymol (72%). All strains of Staphylococcus aureus were sensitive to this essential oil. At the same time, the lowest indicators of growth retardation zones were characteristic of S. aureus MRSA clinic biofilm creation. We have determined the antibio­film effect of O. vulgare essential oil on S. aureus isolates. The study showed the degradation of the biofilm under the influence of essential oil decreased with decreasing concentration. At the same time, even with the action of essential oil, the concentration of 0.01% showed a 42% decrease in the process of biofilm formation. At the highest concentration of 0.10%, a destruction of the biofilm by 78% was found. The study of the influence of essential oils on the formed biofilm showed that 0.1% solution of oil causes degradation of the biofilm by 73.2%; 0.05% – by 58% and 0.01% – by 51.5%. O. vulgare essential oil also showed high antioxidant properties. The neutralization rate of free radicals was 83%. The revealed antimicrobial properties of O. vulgare essential oil make it promising to be used as an antistatic agent, including as a component that contributes to the disintegration of biofilm in the treatment of boils, carbuncles, panari­tiums, phlegmon, wound suppuration, inhalation for respiratory diseases, as a component of rinses in inflammatory periodontal diseases.

Co-cultivation of cyanobacteria for raising nursery of chrysanthemum using a hydroponic system

The present investigation aimed to utilize plant growth–promoting diazotrophic cyanobacteria as an option to raise the chrysanthemum varieties Pusa Aditya and Jaya in a nursery by co-culturing hydroponically. Fresh stem cuttings of chrysanthemum were planted in BG 11 medium (−N) which was inoculated to log-phase cultures of Anabaena torulosa (BF1), Anabaena doliolum (BF4), and Anabaena laxa (RPAN8) individually. Analyses of chrysanthemum growth and biometric/biochemical parameters after 30 days of co-cultivation revealed that co-culturing treatments performed significantly better, as compared with BG11 medium alone. Anabaena laxa brought about an increment of 27–40% in IAA production in the root tissues of both varieties grown in hydroponics. Quantification of biofilm formation on roots (measured as OD550) illustrated a two- to four-fold increment in the co-culture treatments. PEP carboxylase activity was significantly enhanced in root and shoot tissues of cuttings in Jaya, and the medium chlorophyll enhanced by several folds in both varieties. Significant increases in root dry biomass were recorded, which positively correlated with root protein (r = 0.992) in Pusa Aditya, illustrating the superiority of co-culturing as a promising option for nursery propagation. The economic benefits of BG 11 medium (without combined N) as a novel growth medium for growing the cyanobacterium and raising chrysanthemum nursery, in co-culturing mode, were also highlighted. Anabaena torulosa (BF1) performed well in both Pusa Aditya and Jaya, while Anabaena laxa (RPAN8) was significantly superior in Jaya. Future research is focused towards integration of such novel and cheap organic inputs in maintaining disease-free and nutrient-enriched plants in long-term experiments up to flowering stage.

Biofilm-formation by drug-resistant Staphylococcus aureus from cow milk

The present study aims to quantify biofilm formation and determine the drug resistance profile of Staphylococcus aureus isolated from raw, pasteurized and ultra-high temperature (UHT) milk. Detection of S. aureus was made in 90 cow milk samples (30 raw, 30 pasteurized and 30 UHT). Biofilm biomass quantification was performed in 96-well polystyrene plates using the crystal violet technique. The resistance profile was determined by the disc-diffusion test. S. aureus was detected in 24.4% (22/90) of milk samples, with the highest prevalence in pasteurized milk. Biofilm production was observed in 100% of the S. aureus isolates from pasteurized and UHT milk, and in 81.8% of the strains from raw milk. Biofilm production by S. aureus drug-resistant strains was detected in 46.7% (14/30) of the isolates. In order to mitigate this problem, it is recommended to apply appropriate hygiene practices along the milk production chain.

Molecular Detection, Virulence Genes, Biofilm Formation, and Antibiotic Resistance of Salmonella enterica Serotype enteritidis Isolated from Poultry and Clinical Samples

Background: Salmonella spp. is one of the most important zoonotic pathogens transmitting among human and animals. Due to the similarity of antibiotic classes used to treat animals and humans, there is a high risk for emerging the multi-drug resistant (MDR) strains. Objectives: The current study aimed at evaluating molecular detection, virulence genes, biofilm formation, and antibiotic resistance of Salmonella enterica serotype enteritidis recovered from poultry and clinical isolates. Methods: A total of 282 isolates were recovered from chicken meat, live poultry feces, eggs, and human feces in Iran. The presence of virulent factors in the isolates was confirmed using biochemical and microbiological tests. The presence of Salmonella genus was determined using antiserum. Triplex polymerase chain reaction (PCR) was performed to detect Salmonella spp., serogroup D and the discriminate S. enteritidis from other species. Kirby-Bauer disk diffusion method was applied to perform the susceptibility testing. Quantification of biofilm formation was determined in 96-well microtiter plates as recommended by the defined protocol. The data were then analyzed with SPSS using consensus tables and Chi-square test. Results: Based on the results, all the isolates were positive for invA, sdiA, hilA, and ratA. Moreover, spvC had the lowest prevalence (37.6). Of all strains, 67 were MDR, 51.7 of which were recovered from humans. Furthermore, 34.5 of isolates were strong biofilm producers. There was a significant correlation between the strong biofilm formation and the antibiotic resistance to colistin, ceftazidime, chloramphenicol, gentamicin, trimethoprim, penicillin, and trimethoprim-sulfamethoxazole. Conclusions: The results of the current study showed a significant correlation between the strong biofilm formation and the antibiotic resistance to some antibiotics. © 2018, Author(s).

Biofilm formation by phytopathogenic bacteria Acidovorax citrulli subsp. citrulli and Ralstonia solanacearum

Phytopathogenic bacteria are responsible for causing several losses in agriculture economic world. Biofilm formation presents itself as an important structure related to bacterial virulence. The objective of this study was to investigate the biofilm formation by Ralstonia solanacearum and Acidovorax citrulli isolates. Quantification of biofilm formation was performed by the crystal violet method, using NYD as the standard medium for both bacteria, TZC as a specific medium for R. solanacearum and YDC for A. citrulli. The biofilm was observed in SEM and CLSM. Under the tested conditions, B5-5, CGH8, CGM10 and CGH26 R. solanacearum isolates and Aac1.43 and Aac1.73 A. citrulli isolate formed moderately or strongly biofilm on both media tested. However, the amount of biofilm produced by R. solanacearum was higher than those produced by A. citrulli. The SEM and CLSM revealed structurally distinct biofilms among isolates of R. solanacearum, which did not occur for A. citrulli isolates. We conclude that R. solanacearum is a strong biofilm producer, while A. citrulli not seem to be well suited to this condition by not adhere well to the surface. This species depicts potentials to become natural models to study plant biofilm infections due to the high number of host species.

Biofilm Formation by Coagulase-Positive Staphylococcus aureus Isolated from Mozzarella Cheese Elaborated with Buffalo Milk and its Effect on Sensitivity to Sanitizers

Background: The buffalo milk mozzarella cheese is a new product in the market, with high consumer acceptance and excellent prospects for trade. The cheese is rich in nutrients, which favors the proliferation of microorganisms that can cause food-borne diseases in the consumer. Staphylococcus aureus can cause gastro-enteritis in humans by the production of enterotoxins in food. One problem that may hinder the elimination of undesirable microorganisms in the food industry is the formation of biofilms. The objective of this study was to determine the effect of biofilm formation by Staphylococcus aureus isolated from buffalo mozzarella cheese on sensitivity to sanitizers.Materials, Methods & Results: Fifty samples of buffalo mozzarella cheese were analyzed to investigate the presence of S. aureus. The isolates were obtained through microbiological analysis and identified by PCR. The similarity of the strains was compared through rep-PCR. The distinct strains were tested for biofilm formation in microtiter plates. Soy Tripticase Broth (TSB) was placed in each well of the microtiter plate and overnight cultures of each strain was added. Wells without bacterial culture were used as controls. A villous cap was then placed on the plate and incubated for 48 h at 37°C. During incubation, the biofilms formed on the surface of the villi of the caps. For quantification of biofilm formation, material that remained attached to the cap was stained with crystal violet, the stained biofilm was extracted and the OD570 of each well was measured. Each strain was classified as non-biofilm forming, weak forming, moderately formed or formative strong. Strong forming and non-biofilm forming strains were tested on high density polyethylene, stainless steel and glass surfaces. Plates of 4 cm² of the different materials were placed in TSB where the culture of each isolate was inoculated separately. At each 48 h incubation the plates were washed to remove unbound cells and re-inserted into TSB without the inoculum. After five replicates of the procedure, sterile swabs were passed over the entire surface of each plate for counting in Baird-Parker agar. They were also tested for sensitivity to sodium hypochlorite and iodine after biofilm formation. The biofilm plates were immersed in flasks containing sanitizers, where they remained for 10 min. At the established contact time, the plates were immersed in neutralizing solution for 30 s. After washing with PBS, a sterile swab was passed on the surface of each plate and counts on Baird-Parker agar were performed. The bands profiles obtained on rep-PCR were identical when compared to isolates from the same sample, indicating that each sample was contaminated with only one S. aureus strain. From the twenty S. aureus strain identified, two isolates were classified as strong biofilm formers, seven as moderate formers, ten weak formers and one as non-biofilm builder. The two strong forming strains produced biofilm on the three surfaces tested. The application of sodium hypochlorite and iodine sanitizers promoted a reduction of approximately 2 log bacterial populations on all surfaces of both the biofilm and non-forming strains.Discussion: Most strains of S. aureus isolated from buffalo milk mozzarella cheese have the ability to form biofilm on the surfaces of equipment and utensils that have stainless steel, glass or high density polyethylene components. Although biofilm forming strains are no longer resistant to sanitizers sodium hypochlorite and iodine than non-forming sanitizers, they reach higher concentrations in the biofilm, resulting in larger bacterial populations remaining after application of the sanitizers. These results support the recommendation that the good hygienic practices adopted by industries processing buffalo milk mozzarella cheese should include specific measures to control the Staphylococcus aureus contamination.

English

The objective of this study was to assess the impact of subminimum inhibitory concentrations (sub-MICs) of chosen antimicrobial agents (Amikacin, imipenem, benzalkonium chloride, and chlorhexidine) and natural product (garlic) on biofilm formation ability, bacterial adherence and invasion. Susceptibility profiles of 50 non-repetitive Acinetobacter baumannii clinical isolates to eight antibiotics were investigated. MIC of various antibiotics, antiseptics and garlic were measured by the broth microdilution method. Quantification of biofilm formation was carried out using a microtiter plate assay. The ability of test compounds to affect the bacterial adherence and invasion was investigated using Type II pneumocyte cell line (A549) and the bacterial cells count was determined using flow cytometer. Screening for the presence of antiseptic resistant gene qacA/B was done using PCR. Ten isolates exhibited variable susceptibilities toward both amikacin and imipenem. The sub-MICs of benzalkonium chloride (BZC) markedly increased the biofilm formation. Additionally, amikacin, applied at sub minimal inhibitory concentrations, showed the highest induction in the bacterial adherence post treatment. Significant increase in bacterial invasion 3 h post treatment was detected upon applying BZC (616%) and imipenem (324%). BZC showed the greatest effect on the bacterial invasion. The highest impact on the bacterial invasion in case of 5 h post treatment was evident with the use of both BZC and Garlic. Results demonstrated that antibiotics, antiseptics and natural product at sub-MICs increased significantly the biofilm formation ability, bacterial adherence and invasion of A. baumanni clinical isolates, therefore careful consideration of sub-MIC effects is mandatory before their use. Key words: A. baumannii, Sub-MICs, biofilm formation, bacterial adherence and invasion, qac A/B

A dual energy micro-CT methodology for visualization and quantification of biofilm formation and dentin demineralization

ObjectiveThe aim of this study was to induce artificial caries in human sound dentin by means of a microcosm model using human saliva as source of bacteria and to apply a novel dual-energy micro-CT technique to quantify biofilm formation and evaluate its demineralization potential. DesignEight sound third molars had the occlusal enamel removed by cutting with a diamond disk and five cylindrical cavities (±2mm diameter; ±1.5mm depth) were prepared over the dentin surface in each specimen (n=40 cavities). After sterilization, each specimen received the bacterial salivary inoculum obtained from individuals without any systemic diseases presenting dentin caries lesions and were incubated in BHI added of with 5% sucrose for 96h to allow biofilm formation. After that, two consecutive micro-CT scans were acquired from each specimen (40kv and 70kv). Reconstruction of the images was performed using standardized parameters. After alignment, registration, filtering and image calculations, a final stack of images containing the biofilm volume was obtained from each prepared cavity. Dentin demineralization degree was quantified by comparison with sound dentin areas. All data were analyzed using Shapiro-Wilk test and Spearman correlation using α=5%. ResultsDual-energy micro-CT technique disclosed biofilm formation in all cavities. Biofilm volume inside each cavity varied from 0.30 to 1.57mm3. A positive correlation between cavity volume and volume of formed biofilm was obtained (0.77, p<0.01). The mineral decrease obtained in dentin was high (±90%) for all cavities and all demineralized areas showed mineral density values lower than a defined threshold for dentin caries (1.2g/cm3). ConclusionDual-energy micro-CT technique was successful in the quantification of a microcosm human bacterial biofilm formation and to quantify its demineralization potential in vitro.

Phosphoenolpyruvate phosphotransferase system components positively regulate Klebsiella biofilm formation.

Klebsiella pneumoniae is one of the leading causes of device-related infections (DRIs), which are associated with attachment of bacteria to these devices to form a biofilm. The latter is composed of not only bacteria but also extracellular polymeric substances (EPSes) consisting of extracellular DNAs, polysaccharides, and other macromolecules. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) regulates diverse processes of bacterial physiology. In the genome of K. pneumoniae MGH 78578, we found an uncharacterized enzyme II complex homolog of PTS: KPN00353 (EIIA homolog), KPN00352 (EIIB homolog), and KPN00351 (EIIC homolog). The aim of this study was to characterize the potential physiological role of KPN00353, KPN00352, and KPN00351 in biofilm formation by K. pneumoniae. We constructed the PTS mutants and recombinant strains carrying the gene(s) of PTS. The recombinant K. pneumoniae strain overexpressing KPN00353-KPN00352-KPN00351 produced more extracellular matrix than did the vector control according to transmission and scanning electron microscopy. Judging by quantification of biofilm formation, of extracellular DNA (eDNA), and of capsular polysaccharide, the recombinant strain overexpressing KPN00353-KPN00352-KPN00351 produced more biofilm and capsular polysaccharide after overnight culture and more eDNA in the log phase as compared to the vector control. The genes, KPN00353-KPN00352-KPN00351, encode a putative enzyme II complex in PTS and positively regulate biofilm formation by enhancing production of eDNA and capsular polysaccharide in K. pneumoniae. Five proteins related to chaperones, to the citric acid cycle, and to quorum sensing are upregulated by the KPN00353-KPN00352-KPN00351 system.