scholarly journals Biofilm formation by Prototheca zopfii isolated from clinical and subclinical bovine mastitis in distinct growth conditions under different dyes

2019 ◽  
Vol 49 (2) ◽  
Author(s):  
Valessa Lunkes Ely ◽  
Letícia Trevisan Gressler ◽  
Fernando Jonas Sutili ◽  
Márcio Garcia Ribeiro ◽  
Mateus Matiuzzi da Costa ◽  
...  
Keyword(s):  
Crystal Violet ◽  
Biofilm Formation ◽  
Bovine Mastitis ◽  
Growth Conditions ◽  
Blue Dye ◽  
Prototheca Zopfii ◽  
Biofilm Production ◽  
Cotton Blue ◽  
Dynamic Growth

ABSTRACT: Prototheca spp. have been reported as an emergent environmental mastitis pathogen in several countries. Biofilm formation is a significant factor associated with different degrees of virulence developed by many microorganisms, including Prototheca spp. The present study aimed to compare two growth conditions and two staining dyes to determine which combination was more appropriate to evaluate qualitatively and quantitatively the production of biofilm by P. zopfii. Biofilm formation was evaluated in polystyrene microplates under static and dynamic growth conditions and staining with crystal violet or cotton blue dye. All P. zopfii isolates from cows with mastitis were classified as biofilm-producers in all growth conditions and staining. The cotton blue dye proved to be more appropriate method to classify the intensity of P. zopfii biofilm production.

scholarly journals Motility activity, slime production, biofilm formation and genetic typing by ERIC-PCR for Pseudomonas aeruginosa strains isolated from bovine and other sources (human and environment)

2014 ◽  
Vol 17 (2) ◽  
pp. 321-329 ◽  
Author(s):  
K. Wolska ◽  
P. Szweda ◽  
K. Lada ◽  
E. Rytel ◽  
K. Gucwa ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Bovine Mastitis ◽  
Twitching Motility ◽  
Phenotypic Properties ◽  
Biofilm Production ◽  
Abiotic Surfaces ◽  
Hospital Patients ◽  
Relationship Of ◽  
Initial Biofilm ◽  
Different Sources

AbstractThe molecular-typing strategy, ERIC-PCR was used in an attempt to determine the genomic relationship of 28 P. aeruginosa strains isolated from faeces of healthy bovine, bovine mastitis and from faeces of hospital patients as well as from environment. ERIC-PCR fingerprinting revealed large molecular differentiation within this group of isolates. Twenty two out of 28 strains tested generated unique patterns of DNA bands and only three genotypes consisted of two isolates each were identified. We also tested the P. aeruginosa isolates for their ability to form a biofilm on abiotic surfaces including polyvinylchloride and polystyrene. Different biofilm-forming abilities were demonstrated among strains; however, most of them (64.3%) showed moderate-biofilm forming ability. The strains with increased swimming and twitching motility displayed elevated biofilm formation. However, a negative correlation was found between slime and initial biofilm production. On the basis of the results obtained, we suggest that there are no major differences in phenotypic properties between P. aeruginosa strains isolated from different sources

scholarly journals Phenotypic and Genotypic Characteristics and Resistance Profile of Staphylococcus spp. from Bovine Mastitis

2020 ◽  
Vol 48 ◽  
Author(s):  
Érica Chaves Lucio ◽  
Gisele Veneroni Gouveia ◽  
Mateus Matiuzzi Da Costa ◽  
Mário Baltazar De Oliveira ◽  
Rinaldo Aparecido Mota ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Milk Production ◽  
Biofilm Formation ◽  
Negative Impact ◽  
Meca Gene ◽  
Bovine Mastitis ◽  
Resistant Bacteria ◽  
Production Chain ◽  
Biofilm Production ◽  
Staphylococcus Spp

Background: Bovine mastitis remains one of the health problems that cause the most damage to milk producers. The negative impact of mastitis is due to reduced milk production, early slaughter of females, reduced commercial value of the affected animals, losses in the genetic potential of the herd, expenses with medicines and veterinary medical assistance. Staphylococcus spp. stands out as the cause of this disease and has been able to remain in the mammary gland, becoming resistant to several antimicrobials. The aims of the present study were to characterize the phenotypes, genotypes and resistance profiles of Staphylococcus spp. isolates from bovine mastitis cases in the state of Pernambuco, Brazil.Materials, Methods & Results: These isolates were classified according to biochemical tests and the presence of the nuc gene.  The polymerase chain reaction (PCR) for amplification of the mecA and blaZ genes was used to analyze the genetic potentials of antimicrobial resistance. Isolates were also phenotypically tested for resistance to nine antimicrobials (ampicillin, doxicillin, erythromycin, gentamicin, rifampicin, cephalothin, amoxicillin, nalidixic acid and oxacillin). The genetic potentials for biofilm production were evaluated by the amplifications of the icaD, icaA and bap genes. The phenotypic test of gentian violet was used for biofilm formation analyzes. Ninety-three (93.0%) of the isolates among the Staphylococcus spp. samples were classified as Staphylococcus aureus. The lowest percentage of sensitivity observed was for amoxicillin (28.0%). All of the isolates were sensitive to erythromycin and gentamicin, and 15 (15%) exhibited sensitivity to all of the drugs tested. All of the isolates were negative for the mecA gene, and 36 (36%) were positive for blaZ. In the adhesion microplate tests, 44 (44%) of the isolates were capable of biofilm formation. Of these, seven (15.9%) were strong formers, whereas 16 (36.3%) and 21 (47.8%) were moderate and weak formers, respectively. The icaD gene was confirmed in 89 (89%) of the isolates. The icaA gene was confirmed in 61 (61%) samples, and the bap gene in 52 (52%) samples. One of the samples did not possess icaA, icaD or bap and exhibited moderate biofilm formation according to the microplate adherence test. Sixteen isolates simultaneously exhibited the three genes tested for biofilm production (icaA, icaD and bap) and were negative according to the microplate adherence test.Discussion:  The indiscriminate use of antibiotics to treat mastitis is a common practice in the study area, which may have contributed to the high proportion of herds (88.23%; 15/17) with multi-resistant isolates, constituting a selection factor for the dissemination of resistant bacteria among herds.  The absence of the mecA gene in the present study may be associated with the development of resistant bacteria through another mechanism, such as the overproduction of beta-lactamases. The results demonstrate that antimicrobial resistance occurs in Staphylococcus spp. that cause bovine mastitis in herds of Pernambuco and that these isolates have the a great capacity for biofilm formation. It is necessary to sensitize the professionals involved in the milk production chain of Brazil regarding the importance of the adequate use of antimicrobials for the treatment and control of mastitis, since studies in the country indicate the dissemination of resistant bacterial strains.

Candidemia Candida albicans clusters have higher tendency to form biofilms than singleton genotypes†

2020 ◽  
Vol 58 (7) ◽  
pp. 887-895 ◽  
Author(s):  
Judith Díaz-García ◽  
Maiken C Arendrup ◽  
Rafael Cantón ◽  
Julio García-Rodríguez ◽  
Ana Gómez ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Candida Spp ◽  
Biofilm Production ◽  
Hospital Environments ◽  
Marked Variability ◽  
Inert Surfaces ◽  
Biofilm Development

Abstract The capacity of Candida spp. to form biofilms allows them to attach either to living or inert surfaces, promoting their persistence in hospital environments. In a previous study, we reported strain-to-strain variations in Candida spp. biofilm development, suggesting that some genotypes may be greater biofilm formers than others. In this study, we hypothesize that isolates pertaining to clusters may be found more frequently in the environment due to their ability to form biofilms compared to singleton genotypes. Two hundred and thirty-nine Candida spp. isolates (78 clusters) from candidemia patients admitted to 16 hospitals located in different cities and countries—and the same number of singleton genotypes used as controls—were tested in terms of biofilm formation using the crystal violet and the XTT reduction assays. Candida albicans clusters showed higher biofilm formation in comparison to singleton genotypes (P < .01). The biofilms formed by intra-hospital C. albicans clusters showed higher metabolic activity (P < .05). Furthermore, marked variability was found among species and type of cluster. We observed that the higher the number of isolates, the higher the variability of biofilm production by isolates within the cluster, suggesting that the production of biofilm by isolates of the same genotype is quite diverse and does not depend on the type of cluster studied. In conclusion, candidemia Candida spp. clusters—particularly in the case of C. albicans—show significantly more biomass production and metabolic activity than singleton genotypes.

scholarly journals Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

2002 ◽  
Vol 68 (6) ◽  
pp. 2950-2958 ◽  
Author(s):  
D. Djordjevic ◽  
M. Wiedmann ◽  
L. A. McLandsborough
Keyword(s):  
Listeria Monocytogenes ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Microtiter Plate ◽  
Cellular Growth ◽  
Epifluorescence Microscopy ◽  
Simple Method ◽  
Microtiter Plate Assay ◽  
Plate Assay ◽  
Biofilm Production

ABSTRACT Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32°C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.

scholarly journals Biofilm production by Listeria monocytogenes strains: detection with colorimetric analysis

2020 ◽  
Vol 30 (Supplement_5) ◽  
Author(s):  
P Centorame ◽  
L Iacone ◽  
R Salini ◽  
A Ciarulli ◽  
F Guidi ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Crystal Violet ◽  
Biofilm Formation ◽  
Colorimetric Analysis ◽  
Solution Versus ◽  
Crystal Violet Solution ◽  
Biofilm Production ◽  
Staining Methods

Abstract Background In literature, there are no standardized laboratory methods to detect formed biomass by colorimetric analysis. The purpose of this study was to compare three staining methods and two different wavelengths for determination of biofilm formation of Listeria monocytogenes (Lm) strains. Methods Three strains of Lm isolated from different origin were tested using 96 well polistirene plates at 12 °C and 30 °C, after incubation the wells were subjected to washing, detaching and staining with crystal violet (CV) at 0.2% and 2% (Panreac EU) in 95% ethanol and with Gram's crystal violet solution (Merck KGaA, Germany). The absorbance at 492nm and 540nm wavelengths was read using a spectrophotometer (SIRIO S, Seac, Firenze, Italia). Results The strains incubated at 12 °C displayed production of biofilm when stained with CV 2% and with Gram's crystal violet solution, both at 492 and 540 nm (with better evidence at 540 nm). If CV 0.2% was used to stain and reading at both optical densities there was evidence of weak or no biofilm production. At 30 °C, the biofilm production was displayed at both temperature and with all the stains. For all the strains and for all the conditions tested, the absorbance was greater but not proportional using the Gram's crystal violet solution, versus the CV 0,2% and CV 2%, and absorbance was higher at 540nm versus at 492nm. Conclusions Results confirmed the lack of reproducibility of each of the method used to detect and quantify the biomass produced during a biofilm formation test in vitro and the absence of ratio between the different results obtained using different CV concentration and wavelengths for reading. Key messages Biofilm production at 12 °C could not be adequately detected staining the wells with CV 0,2%. Absorbance could be influenced by the solvent in the stain used (ethanol, methanol or phenol or mixtures). To obtain data for assessment of biomass formation, being the method characterized by poor reproducibility, the laboratory should use at least the same stain and wavelength.

scholarly journals Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions

Antibiotics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1349
Author(s):  
Nina Patel ◽  
Jeremy C. Curtis ◽  
Balbina J. Plotkin
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Fold Increase ◽  
Growth Conditions ◽  
Yeast Nitrogen Base ◽  
Nitrogen Base ◽  
E Coli ◽  
Biofilm Production ◽  
Mueller Hinton ◽  
Static Conditions

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 μU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

scholarly journals Antibiotic resistance, biofilm formation, and biofilm-associated genes among Stenotrophomonas maltophilia clinical isolates

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Narjess Bostanghadiri ◽  
Abdollah Ardebili ◽  
Zohreh Ghalavand ◽  
Samane Teymouri ◽  
Mahsa Mirzarazi ◽  
...  
Keyword(s):  
Crystal Violet ◽  
Biofilm Formation ◽  
Staining Method ◽  
Clinical Isolates ◽  
Diffusion Method ◽  
Crystal Violet Staining ◽  
Disc Diffusion Method ◽  
Antimicrobial Susceptibility Pattern ◽  
Combination Strategies ◽  
Biofilm Production

Abstract Objective The purpose of the present study was to investigate the antimicrobial susceptibility pattern, biofilm production, and the presence of biofilm genes among the S. maltophilia clinical isolates. A total of 85 clinical isolates of S. maltophilia were collected from patients referred to several hospitals. Susceptibility to antibiotics was investigated by disc diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). By the crystal violet staining method, the capability of biofilm formation was examined. The genes associated with biofilm production were investigated by the PCR-sequencing techniques. Results All isolates were resistant to doripenem, imipenem, and meropenem. Minocycline, trimethoprim/sulfamethoxazole and levofloxacin exhibited the highest susceptibility of 100%, 97.65%, and 95.29%, respectively. The results of crystal violet staining assay showed that all isolates (100%) form biofilm. Moreover, 24 (28.23%), 32 (37.65%), and 29 (34.12%) of isolates were categorized as weak, moderate, and strong biofilm producers, respectively. Biofilm genes including rpfF, spgM and rmlA had an overall prevalence of 89.41% (76/85), 100% (85/85) and 84.71% (72/85), respectively. Rational prescribing of antibiotics and implementation of infection control protocols are necessary to prevent further infection and development of antimicrobial resistance. Combination strategies based on the appropriate antibiotics along with anti-biofilm agents can also be selected to eliminate biofilm-associated infections.

scholarly journals Increase in biofilm formation by Escherichia coli under conditions that mimic the mastitic mammary gland

2014 ◽  
Vol 44 (4) ◽  
pp. 666-671 ◽  
Author(s):  
João Carlos Miguel Costa ◽  
Isis de Freitas Espeschit ◽  
Fábio Alessandro Pieri ◽  
Laércio Anjos Benjamin ◽  
Maria Aparecida Scatamburlo Moreira
Keyword(s):  
Escherichia Coli ◽  
Mammary Gland ◽  
Biofilm Formation ◽  
Culture Media ◽  
Control Strategies ◽  
Bovine Mastitis ◽  
Microtiter Plate ◽  
Spectrophotometric Analysis ◽  
Biofilm Production ◽  
Glucose Availability

Bacterial biofilms are involved in the aggravation and recurrence of clinical mastitis in dairy herds. Several factors such as pH, temperature, concentration of O2 and glucose can affect their induction and growth rates. In this study, biofilm production was demonstrated by 27 Escherichia coli strains isolated from bovine mastitis at different pH values depending on the availability of glucose, mimicking conditions found in mammary glands affected by the disease. Biofilm formation was analyzed by spectrophotometric analysis in microtiter plate with 16 different culture media and by scanning electron microscopy. Biofilm formation was greater in isolates cultured under conditions associated with low glucose availability (0.5% or 1.5%) and with either an acidic (5.5) or alkaline (8.5) pH, compared to conditions associated with high glucose availability (2.5% or 3.5%) and near-neutral pH (6.5 or 7.5). Results indicate possible favoring of biofilm production in the later stages of the infectious process caused by E. coli, when the gland environment is less propitious to bacterial growth due to the stress conditions mentioned above; contrasting with the environment of the healthy mammary gland, in which there is no limitation on nutrients or conditions of particular alkalinity or acidity. Thus, knowledge of the stage in which is the infection and environmental conditions of the mammary gland that cause increased production of biofilms is of paramount importance to guide the most appropriate control strategies to prevent relapse after treatment of bovine mastitis, an economically important disease in dairy cattle worldwide.

Biofilm-formation by Staphylococcus aureus and Staphylococcus epidermidis isolates from subclinical mastitis in conditions mimicking the udder environment

2015 ◽  
Vol 18 (4) ◽  
pp. 787-792 ◽  
Author(s):  
R. Seixas ◽  
D. Varanda ◽  
R. Bexiga ◽  
L. Tavares ◽  
M. Oliveira
Keyword(s):  
Biofilm Formation ◽  
Antimicrobial Agents ◽  
Bovine Mastitis ◽  
Subclinical Mastitis ◽  
Protective Role ◽  
Dynamic Conditions ◽  
Biofilm Production ◽  
Static Conditions

AbstractStaphylococcusis the genus most commonly isolated from bovine mastitis in many countries. It may express several virulence factors including biofilm formation, which may protect the bacterial community from antimicrobials’ action, preventing these compounds from reaching its interior, where they reach subinhibitory concentrations (subMIC).Most biofilm production assays are performed in static conditions, while studies regarding antimicrobial resistance usually do not resemble the udder environment because they are performed at high concentrations. In this study we evaluated the influence of dynamic conditions and media, including Mueller Hinton Broth (MHB) and UHT whole milk (WM), as well as the effect of subMIC concentrations of five different antimicrobial agents on biofilm formation by staphylococci isolated from subclinical mastitis. Results suggest that dynamic conditions and media may influence biofilm formation and revealed that milking simulation may significantly increase biofilm production. Sub-MIC concentrations decrease biofilm formation in MHB but increase in WM, suggesting a protective role of milk against antimicrobial compounds’ action. Therefore,in vitroconditions that simulate the udder environment andin vivoconditions should be included as one of the parameters in evaluation of biofilm producing strains, in order to provide more reliable results.

scholarly journals luxS Gene and Biofilm Formation in Streptococcus uberis Isolated from Bovine Mastitis Cases

2021 ◽  
Author(s):  
A.J. Greeshma ◽  
R.N. Ramani Pushpa ◽  
K. Lakshmi Kavitha ◽  
T. Srinivasa Rao
Keyword(s):  
Biofilm Formation ◽  
Bovine Mastitis ◽  
Economic Loss ◽  
Microtiter Plate ◽  
Good Choice ◽  
Luxs Gene ◽  
Biochemical Tests ◽  
Streptococcus Uberis ◽  
Biofilm Production

Background: Streptococcus uberis (S. uberis) is an environmental pathogen causing mastitis in Dairy cattle. It causes recurrent mastitis and reduction in milk production in livestock causing economic loss. The prevalence of S. uberis intramammary infections is due to ability of the organism to form biofilm in udder tissue. The present study is on in-vitro biofilm production, the correlation of luxS gene and the biofilm formation in S. uberis. Methods: A total of 91 mastitic milk samples were collected from cattle and buffaloes brought at Veterinary Hospitals and farms in Krishna, Guntur and West Godavari districts, Andhra Pradesh. The identification of the culture isolates was based on cultural and biochemical characteristics and confirmed by Polymerase Chain Reaction (PCR). The Streptococcus species cultures showing greyish, pinpointed colonies and/or aesculin hydrolysis on Edwards medium were further identified by various biochemical tests viz., catalase test, ninhydrin test, sodium hippurate hydrolysis test and type of haemolysis on 7% sheep blood agar. Confirmation of the isolates by PCR was followed by detection of biofilm formation using qualitative Congo red agar (CRA) method, quantitative microtiter plate (MTP) assay and biofilm gene (luxS) was detected using PCR.Conclusion: From this study it is suggestable that for biofilm study both phenotypic and genotypic methods should be taken together which can be influenced by various other factors also. MTP assay was a good choice for quantitative biofilm determination, which was giving a more accurate and understandable results. The results express that any of the isolates without luxS didn’t produce a strong biofilm and it is concluded that there may be other genes for regulation of biofilm production and/or luxS gene has a regulatory role for one or more genes related to biofilm formation in S. uberis.

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