Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation

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 degrees 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.     

Microtiter Plate Assay for Assessment of Listeria monocytogenes Biofilm Formation

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.     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Investigation of alkane biodegradation using the microtiter plate method and correlation between biofilm formation, biosurfactant production and crude oil biodegradation

Among 25 crude oil-degrading bacteria isolated from a marine environment, four strains, which grew well on crude oil, were selected for more study. All the four isolated had maximum growth on 2.5% of crude oil and strain BC (Pseudomonas) could remove crude oil by 83%. The drop collapse method and microtiter assay show that this strain produces more biosurfactant, and its biofilm formation is higher compared to other strains. Bacterial adhesions to crude oil for strains CS-2 (Pseudomonas), BC, PG-5 (Rhodococcus) and H (Bacillus) were 30%, 46%, 10% and 1%, respectively. Therefore, strain H with a low production of biosurfactant and biofilm formation had showed the least growth on these compounds. PCR analysis of these four strains showed that all isolates had alk-B genes from group (III) alkane hydroxylase. All isolate strains could utilize cyclohexan, octane, hexadecane, octadecan and diesel fuel oil; however, the microtiter plate assay showed that strain BC had more growth, respiration and biofilm formation on octadecan.     

A Widely Used In Vitro Biofilm Assay Has Questionable Clinical Significance for Enterococcal Endocarditis

Biofilm formation may play an important role in the pathogenesis of infections caused by Enterococcus faecalis, including endocarditis. Most biofilm studies use a polystyrene dish assay to quantify biofilm biomass. However, recent studies of E. faecalis strains in tissue and animal models suggest that polystyrene dish results need to be interpreted with caution. We evaluated 158 clinical E. faecalis isolates using a polystyrene dish assay and found variation in biofilm formation, with many isolates forming little biofilm even when different types of media were used. However, all tested clinical isolates were able to form biofilms on porcine heart valve explants. Dextrose-enhanced biofilm formation in the polystyrene dish assay was found in 6/12 (50%) of clinical isolates tested and may explain some, but not all of the differences between the polystyrene dish assay and the heart valve assay. These findings suggest that in studies assessing the clinical relevance of enterococcal biofilm-forming ability, ex vivo biofilm formation on a relevant tissue surface may be warranted to validate results of in vitro assays.     

Phenotypic and Genotypic Characterizations of Campylobacter jejuni Isolated from the Broiler Meat Production Process

A set of C. jejuni isolates of different origins and flaA-genotypes obtained throughout the broiler meat production chain was tested in this study for a possible correlation of their origin, phylogenetic relationship, and phenotypic properties. Interestingly, the results showed a correlation of the origin and the phylogenetic relationship between the C. jejuni isolates and their ability to form biofilm, but not in their ability to survive at -18, 5, 20, and 48?°C. Two strains, a broiler cloacae isolate and a broiler fillet isolate, were unable to develop biofilm, while most of the C. jejuni isolates originating from meat and surfaces of the slaughterhouse readily formed biofilms after both 24, 48, and 72?h. Interestingly, these biofilm-forming strains were closely related. Furthermore, two strains that were isolated after disinfection developed significantly more biofilms after 24?h of incubation than the remaining strains. A comparative genomic analysis using DNA microarrays showed that the gene contents of strains that efficiently formed biofilms were different from those that did not. The study suggests that biofilm formation might be a lineage specific property, allowing C. jejuni to both survive environmental stress at the slaughterhouse and to attach to the surface of meat.     

Molecular characterization of clinical and environmental Pseudomonas aeruginosa isolated in a burn center

In burn centers, Pseudomonas aeruginosa acts as a major cause of nosocomial infections. Therefore, this study aimed to characterize molecularly P. aeruginosa isolates collected from environmental samples and burn patients. A total of 78 strains (including 58 clinical and 20 environmental isolates) of the P. aeruginosa were collected from Beasat hospital of Hamadan, west of Iran, and was identified using API 20NE. The disk diffusion method according to the CLSI was applied for determination of the antimicrobial resistance. Moreover, the microtiter plate test was used for the quantification of Biofilm formation. The genomic features of the isolated strains was evaluated using Pulsed Field Gel Electrophoresis (PFGE). We found that 94.8% of clinical and 80% environmental isolates were capable of forming biofilm. The rate of MDR in clinical and environmental isolates was 51.7% and 40%, respectively. A significant relationship was observed between biofilm formation capability and multiple drug resistance (p < 0.05). PFGE typing showed 11 different clusters with two major clusters A with 30 (38.5%) and B with 14 (17.9%) members, containing up to 56.4% of all isolates. There was no relationship between biofilm formation ability and antibiotic resistance patterns with PFGE patterns. According to the results, the clonal spread of environmental P. aeruginosa isolates is associated with clinical isolates, and both environmental and clinical isolates are attributed to a high prevalence of the antibiotic resistance and biofilm formation ability. This study highlighted that the prevention programs should be implemented in the hospital environment to control the spread of P. aeruginosa in burn units.     

Establishment and Early Succession of a Multispecies Biofilm Composed of Soil Bacteria

Most soil bacteria are likely to be organized in biofilms on roots, litter, or soil particles. Studies of such biofilms are complicated by the many nonculturable species present in soil, as well as the interspecific bacterial interactions affecting biofilm biology. We in this study describe the development of a biofilm flow model and use this system to establish an early (days 1–7) flow biofilm of soil bacteria from agricultural soil. It was possible to follow the succession in the early flow biofilm by denaturing gradient gel electrophoresis (DGGE) analysis, and it was demonstrated that the majority of strains present in the biofilm were culturable. We isolated and identified nine strains, all associated with unique DGGE profiles, and related their intrinsic phenotypes regarding monospecies biofilm formation in microtiter plates and planktonic growth characteristics to the appearance of the strains in the flow biofilm. The ability of the strains to attach to and establish biofilm in microtiter plates was reflected in their flow biofilm appearance, whereas no such reflection of the planktonic growth characteristics in the flow biofilm appearance was observed. One strain-specific synergistic interaction, strongly promoting biofilm formation of two strains when cultured together in a dual-species biofilm, was observed, indicating that some strains promote biofilm formation of others. Thus, the biofilm flow model proved useful for investigations of how intrinsic phenotypic traits of individual species affect the succession in an early soil biofilm consortium.     

Biofilm Formation by Salmonella Enterica Serovar 1,4,[5],12:i:- Portuguese Isolates: A Phenotypic,Genotypic, and Socio-geographic Analysis

Biofilm-forming ability is well established as an important virulence factor. However, there are no studies available regarding biofilm formation of Salmonella Typhimurium 1,4,[5],12:i:-, the new pandemic serovar in Europe. To address this problem, biofilm expression by Salmonella 1,4,[5],12:i:- was evaluated using 133 isolates from clinical, environmental and animal origins, collected in Portugal from 2006 to 2011. Biofilm detection was performed by phenotypic and genotypic methods, such growth characterization in agar and broth medium, optical density determination by microtiter assays and direct observation by fluorescent in situ hybridization. Biofilm-related genes adrA, csgD and gcpA were detected by PCR. A socio-geographic characterization of strains as biofilm producers was also performed. Results showed that biofilm formation in monophasic Salmonella is widely distributed in Portuguese isolates and could be one of the reasons for its dissemination in this country. Biofilm expression varies between locations, showing that isolates from some regions like Lisboa or Ponta Delgada have an increased ability to persist in the environment due to an enhanced biofilm production. Biofilm formation also varies between risk groups, with a higher prevalence in isolates from salmonellosis infections in women. Therefore, the analysis of the socio-geographic distribution of biofilm-forming bacteria should be considered for the establishment of more adequate regulatory measures or therapeutics regimens, especially important due to the continuous increase of infections caused by antimicrobial resistant microorganisms.     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

     

Esp-independent biofilm formation by Enterococcus faecalis

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.     

Evaluation of the biofilm-forming ability and genetic typing for clinical isolates of Pseudomonas aeruginosa by enterobacterial repetitive intergenic consensus-based PCR

Biofilm formation is an important phenotype associated with chronic Pseudomonas aeruginosa infections. In the present study, a total of 48 P. aeruginosa strains isolated from clinical specimens were examined for their biofilm-forming ability using a microtiter plate method. The different biofilm-forming abilities were demonstrated among the strains; however, most strains formed a larger biofilm than strain PAO1, a reference strain. The genetic typing was also carried out by enterobacterial repetitive intergenic consensus-based polymerase chain reaction. Although they were divided into five groups (A to E), most of the strains showing the higher biofilm-forming ability were found to be in groups D and E, suggesting a significant relationship between the biofilm-forming ability and the genetic group.     

Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci

Biofilm is one of the known virulence factors of staphylococci, a human and animal pathogen and commensal. Some of the strains become invasive under favorable conditions while others do not cause disease. Early detection and management of potentially pathogenic staphylococci is the essential step to prevent device-associated infections. There is also a need to evaluate one simple method for the detection of potential pathogens. Hence this study was planned to study the difference in potential of commensal, colonizing and invasive strains of staphylococci to produce biofilm. We used one qualitative (Congo red agar) and one quantitative (microtiter plate) method for detection of biofilm production and evaluated the sensitivity and specificity of Congo red agar method by using microtiter plate method as a gold standard. We consecutively enrolled staphylococcal strains isolated from peripheral intravenous device (IVD), venous blood, site of IVD insertion and nasal mucosa of patients admitted to pediatric ward with peripheral intravenous devices in place for more than 48 h. Total 100 invasive, 50 colonizing and 50 commensal isolates were studied. Of 100 invasive isolates 74% (74/100) were biofilm positive while only 68% (34/50) colonizing and 32% (16/50) commensal isolates were biofilm positive. The difference in biofilm production by commensal, colonizing and invasive strains was statistically significant (p<0.0001). Sensitivity and specificity of Congo red agar test for detection of biofilm producers were 90.63% and 90.79% for Staphylococcus aureus and 75.86% and 96.88% respectively for coagulase negative staphylococci. CRA is a method that could be used to determine whether an isolate has the potential for biofilm production or not.     

Evaluation of Adherence,Hydrophobicity, Aggregation,and Biofilm Development of <Emphasis Type="Italic">Flavobacterium johnsoniae</Emphasis>-Like Isolates

Flavobacterium spp. isolates have been identified in diverse biofilm structures, but the mechanism of adherence has not been elucidated. The absence of conventional biofilm-associated structures such as fimbriae, pili, and flagella suggest that surface hydrophobicity, and/or autoaggregation and coaggregation may play an important role in adherence and biofilm formation. The biofilm-forming capacity of 29 Flavobacterium johnsoniae-like isolates obtained from South African aquaculture systems was assessed using microtiter plate assays. The role of hydrophobicity [salting aggregation test (SAT) and bacterial adherence to hydrocarbons (BATH) assays], autoaggregation, and coaggregation on biofilm formation by Flavobacterium spp. was also investigated, while biofilm structure was examined using flow cells and microscopy. All isolates displayed a hydrophilic nature, but showed varying levels of adherence in microtiter assays. Significant negative correlations were observed between adherence and biofilm-forming capacity in nutrient-poor medium at 26°C and BATH hydrophobicity and motility, respectively. Isolates displayed strain-to-strain variation in their autoaggregation indices and their abilities to coaggregate with various Gram-negative and Gram-positive organisms. Microcolony and/or biofilm development were observed microscopically, and flavobacterial isolates displayed stronger biofilm structures and interaction with a Vibrio spp. isolate than with an Aeromonas hydrophila isolate. The role of extracellular polysaccharides and specific outer membrane proteins will have to be examined to reveal mechanisms of adherence and coaggregation employed by biofilm-forming F. johnsoniae-like strains.     

The gene <Emphasis Type="Italic">bap</Emphasis>, involved in biofilm production,is present in <Emphasis Type="Italic">Staphylococcus</Emphasis> spp. strains from nosocomial infections

This study analyzed ten strains of coagulase-negative staphylococci (CNS) involved in nosocomial infections in three Brazilian hospitals. Their antibiotic susceptibility profile showed that most strains exhibited multiple antibiotic resistance and possessed the mecA gene. The ability of these strains to adhere to polystyrene microtiter plates was also tested and nine of them proved to be biofilm producers at least in one of the three conditions tested: growth in TSB, in TSB supplemented with NaCl, or in TSB supplemented with glucose. The presence of the bap gene, which codes for the biofilm-associated protein (Bap), was investigated in all ten strains by PCR. AU strains were bop-positive and DNA sequencing experiments confirmed that the fragments amplified were indeed part of a bap gene. The presence of the icaA gene, one of the genes involved in polysaccharide intercellular adhesin (PIA) formation, was also detected by PCR in eight of the ten strains tested. The two icaA-negative strains were either weak biofilm producer or no biofilm producer, although they were bop-positive. To our knowledge, this is the first report demonstrating the presence of the bap gene in nosocomial isolates of CNS, being also the first report on the presence of this gene in Staphylococcus haemolyticus and S. cohnii. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Evaluation of biofilm production and prevalence of the icaD gene in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains isolated from patients with nosocomial infections and carriers

Staphylococcus aureus is one of the most important etiological agents responsible for healthcare-associated infections and is capable of producing many virulence factors including biofilm. The aim of the present study was to analyze the correlation between the presence of the icaD and icaA genes and the ability to produce biofilm in vitro in 302 methicillin-resistant (MRSA) and 268 methicillin-sensitive S. aureus (MSSA) strains isolated in the Provincial Hospital in Gdansk. Presence of the icaD and icaA genes was detected by PCR and the ability to produce biofilm in vitro was measured both spectrophotometrically and via Congo Red Agar plate culture methods. We found that 91% of MRSA strains harbored the icaD gene. Moreover, all icaD-negative strains were icaA-positive. Of MRSA and MSSA strains, 47% and 69%, respectively, produced biofilm in vitro. The level of consistency between the two applied phenotypic methods was 96%. Additionally, we found that strains with the same biofilm status may be present in asymptomatic carriers and cause infections.     

Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus,Pseudomonas aeruginosa,and Proteus mirabilis

The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80–220 nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO₂ was also evaluated.     

Quantitative analysis of biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) strains from patients with orthopaedic device-related infections

Biofilms play a pivotal role in medical device-related infections. However, epidemiological analysis of biofilm formation and genotyping among clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients with orthopaedic infections has rarely been reported. A total of 168 MRSA strains were examined: 23 strains from patients with device-related infection (the device group); 55 from patients with device-non-related infection (the nondevice group); and 90 from asymptomatic nasal carriers (the colonization group). Pulsed-field gel electrophoresis analysis and five genotyping methods including agr typing were performed. Biofilm formation was quantified using a microtitre plate assay. The device group had a significantly higher incidence of agr-2 than the colonization group (78.3% vs. 34.4%, P=0.001). The biofilm index of the agr-2 (0.523 ± 0.572) strains was significantly higher than those of agr-1 (0.260 ± 0.418, P<0.0001) and agr-3 (0.379 ± 0.557, P=0.045). The prevalence of strong biofilm formers in the device group (43.5%) was significantly higher than that in the nondevice group (12.7%, P=0.003) and the colonization group (20.0%, P=0.020). agr-2 MRSA strains may be more likely to cause orthopaedic device infection because of their strong biofilm formation ability.