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.

scholarly journals Analysis of the bacterial biofilm formation in different models of the in vitro culture

2021 ◽  
Vol 19 (1) ◽  
pp. 40-45
Author(s):  
Agnieszka Bogut ◽  
◽  
Agnieszka Magryś ◽  
Keyword(s):  
Biofilm Formation ◽  
Culture Media ◽  
Microtiter Plate ◽  
Bacterial Biofilm ◽  
Atmospheric Conditions ◽  
Experimental Conditions ◽  
Microtiter Plate Assay ◽  
Biofilm Production ◽  
Strain Type

Introduction. Microtiter plate assay (MPA) remains one of workhorses of in vitro biofilm research but it requires optimization of experimental conditions to fulfill the biofilm formation requirements of different bacterial pathogens. Aim. The aim was to determine the effect of TSB and RPMI1640 culture media and selected culture variables (O2 vs. 5% CO2, extended incubation time) on the biofilm production by bacteria commonly involved in biofilm-related infections: Enterococcus faecalis (EF), Escherichia coli (EC), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Klebsiella pneumoniae (KP). Material and methods. The investigation was performed using the MPA with crystal violet. Results. Statistically significant (p<0.05) increase in biofilm production between 24h and 72h time points was observed for EF (TSB o2, RPMIo2 and RPMIco2), EC (TSBo2), SA (TSBo2, TSBco2), KP (TSBo2, TSBco2), PA (RPMIco2, TSBco2). The TSB caused a significantly greater stimulation of biofilm production compared to RPM1640. It outcompeted RPMI1640 irrespective of the atmospheric conditions for SA and KP and under aerobic conditions for EF. Conclusion. Although the TSB provided the most optimal conditions for biofilm production, the process was influenced by the strain type, atmospheric conditions and period of cultivation which limits the ability to design a single universal model of the in vitro biofilm investigation.

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.

scholarly journals Biofilm Formation on Biotic and Abiotic Surfaces in the Presence of Antimicrobials by Escherichia coli Isolates from Cases of Bovine Mastitis

2014 ◽  
Vol 80 (19) ◽  
pp. 6136-6145 ◽  
Author(s):  
Vitor O. Silva ◽  
Larissa O. Soares ◽  
Abelardo Silva Júnior ◽  
Hilário C. Mantovani ◽  
Yung-Fu Chang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Expression Profiles ◽  
Bovine Mastitis ◽  
Pcr Analysis ◽  
Content Type ◽  
E Coli ◽  
Biofilm Production ◽  
Abiotic Surfaces

ABSTRACTEscherichia coliis a highly adaptive microorganism, and its ability to form biofilms under certain conditions can be critical for antimicrobial resistance. The adhesion of fourE. coliisolates from bovine mastitis to bovine mammary alveolar (MAC-T) cells, biofilm production on a polystyrene surface, and the expression profiles of the genesfliC,csgA,fimA, andluxSin the presence of enrofloxacin, gentamicin, co-trimoxazole, and ampicillin at half of the MIC were investigated. Increased adhesion ofE. coliisolates in the presence of antimicrobials was not observed; however, increased internalization of some isolates was observed by confocal microscopy. All of the antimicrobials induced the formation of biofilms by at least one isolate, whereas enrofloxacin and co-trimoxazole decreased biofilm formation by at least one isolate. Quantitative PCR analysis revealed that all four genes were differentially expressed when bacteria were exposed to subinhibitory concentrations of antimicrobials, with expression altered on the order of 1.5- to 22-fold. However, it was not possible to associate gene expression with induction or reduction of biofilm formation in the presence of the antimicrobials. Taken together, the results demonstrate that antimicrobials could induce biofilm formation by some isolates, in addition to inducing MAC-T cell invasion, a situation that might occurin vivo, potentially resulting in a bacterial reservoir in the udder, which might explain some cases of persistent mastitis in herds.

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.

scholarly journals Association between Biofilm-Production and Antibiotic Resistance in Uropathogenic Escherichia coli (UPEC): An In Vitro Study

Diseases ◽  
2020 ◽  
Vol 8 (2) ◽  
pp. 17 ◽  
Author(s):  
Payam Behzadi ◽  
Edit Urbán ◽  
Márió Gajdács
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
In Vitro Study ◽  
Point Of View ◽  
Diffusion Method ◽  
Medical Attention ◽  
Disk Diffusion Method ◽  
Biofilm Production ◽  
Tract Infections

Urinary tract infections (UTIs) are among the most common infections requiring medical attention worldwide. The production of biofilms is an important step in UTIs, not only from a mechanistic point of view, but this may also confer additional resistance, distinct from other aspects of multidrug resistance (MDR). A total of two hundred and fifty (n = 250) Escherichia coli isolates, originating from clean-catch urine samples, were included in this study. The isolates were classified into five groups: wild-type, ciprofloxacin-resistant, fosfomycin-resistant, trimethoprim-sulfamethoxazole-resistant and extended spectrum β-lactamase (ESBL)-producing strains. The bacterial specimens were cultured using eosine methylene blue agar and the colony morphology of isolates were recorded. Antimicrobial susceptibility testing was performed using the Kirby–Bauer disk diffusion method and E-tests. Biofilm-formation of the isolates was carried out with the crystal violet tube-adherence method. n = 76 isolates (30.4%) produced large colonies (>3 mm), mucoid variant colonies were produced in n = 135 cases (54.0%), and n = 119 (47.6%) were positive for biofilm formation. The agreement (i.e., predictive value) of mucoid variant colonies in regard to biofilm production in the tube-adherence assay was 0.881 overall. Significant variation was seen in the case of the group of ESBL-producers in the ratio of biofilm-producing isolates. The relationship between biofilm-production and other resistance determinants has been extensively studied. However, no definite conclusion can be reached from the currently available data.

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.

Lactococcus lactis V7 inhibits the cell invasion of bovine mammary epithelial cells by Escherichia coli and Staphylococcus aureus

2015 ◽  
Vol 6 (6) ◽  
pp. 879-886 ◽  
Author(s):  
B. Seridan Assis ◽  
P. Germon ◽  
A.M. Silva ◽  
S. Even ◽  
J.R. Nicoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Mammary Gland ◽  
Lactococcus Lactis ◽  
Cell Invasion ◽  
Bovine Mastitis ◽  
Antibiotic Use ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
E Coli

Bovine mastitis, an inflammatory disease of the mammary gland often associated to bacterial infection, is the first cause of antibiotic use in dairy cattle. Because of the risk of antibioresistance emergence, alternative non-antibiotic strategies are needed to prevent or to cure bovine mastitis and reduce the antibiotic use in veterinary medicine. In this work, we investigated Lactococcus lactis V7, a strain isolated from the mammary gland, as a probiotic option against bovine mastitis. Using bovine mammary epithelial cell (bMEC) culture, and two representative strains for Escherichia coli and for Staphylococcus aureus, two major mastitis pathogens, we investigated L. lactis V7 ability to inhibit cell invasion (i.e. adhesion and internalization) of these pathogens into bMEC. L. lactis V7 ability to modulate the production of CXCL8, a key chemokine IL-8 responsible for neutrophil influx, in bMEC upon challenge with E. coli was investigated by an ELISA dosage of CXCL8 in bMEC culture supernatants. We showed that L. lactis V7 inhibited the internalisation of both E. coli and S. aureus strains into bMEC, whereas it inhibited the adhesion of only one out of the two S. aureus strains and of none of the E. coli strains tested. Investigation of the bMEC immune response showed that L. lactis V7 alone induced a slight increase in CXCL8 production in bMEC and that it increased the inflammatory response in bMEC challenged with the E. coli strains. Altogether these features of L. lactis V7 make it a potential promising candidate for a probiotic prevention strategy against bovine mastitis.

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.

CHARACTERIZATION OF BACTERIAL ADHESION AND BIOFILM FORMATION ABILITY OF LIVESTOCK-ASSOCIATED METHICILLIN-RESISTANTSTAPHYLOCOCCUS AUREUSISOLATED FROM SWINE AND SLAUGHTERHOUSE WASTEWATERS

2014 ◽  
Vol 40 (02) ◽  
pp. 101-107
Author(s):  
Min-Tao Wan ◽  
Chin-Cheng Chou
Keyword(s):  
Bacterial Adhesion ◽  
Biofilm Formation ◽  
Microtiter Plate ◽  
Ecological Niches ◽  
Microtiter Plate Assay ◽  
Environmental Group ◽  
Biofilm Production ◽  
The Difference ◽  
Wastewater Samples

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST9 has emerged as a potential zoonotic pathogen for humans and animals. Bacterial adhesion factors and biofilms mediate host colonization and infection of MRSA. This study investigated the dynamics of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), biofilm formation gene (intercellular adhesion [ica]), and biofilm expression in MRSA from the nasal samples of asymptomatic pigs (the nasal group, n = 147) and swine slaughterhouse wastewater samples (the environmental group, n = 86). Biofilm formation was quantified by microtiter plate assay. The most prevalent MSCRAMM profile was clfA-clfB-spa-eno-ebps-fib and more than 70% of the LA-MRSA ST9 isolates harbored the biofilm formation gene. Environmental MRSA harbored lower levels of the ica locus and MSCRAMMs (clfA and fib) than did the nasal group, suggesting possible gene loss. Biofilm production in the nasal group was higher than in the environmental group, indicating the difference in biofilm formation in MRSA isolates from different ecological niches. The higher prevalence of MSCRAMMs, biofilm formation gene, and biofilm production in LA-MRSA ST9 may enhance the persistence and infectivity of MRSA in the swine population and present a threat to the health of livestock as well as farm workers.

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