Biofilm-derived Cells Research Articles

Fate of Planktonic and Biofilm-Derived Listeria monocytogenes on Unwaxed Apples during Air and Controlled Atmosphere Storage.

Multiple recalls and outbreaks involving Listeria monocytogenes-contaminated apples have been linked to the post-harvest packing environment where this pathogen can persist in biofilms. Therefore, this study assessed L. monocytogenes survival on apples as affected by harvest year, apple cultivar, storage atmosphere, and growth conditions. Unwaxed Gala, Granny Smith, and Honeycrisp apples were dip-inoculated in an 8-strain L. monocytogenes cocktail of planktonic- or biofilm-grown cells (~6.5 log CFU/mL), dried, and then examined for numbers of L. monocytogenes during air or controlled atmosphere (CA) (1.5% O2, 1.5% CO2) storage at 2 °C. After 90 days, air or CA storage yielded similar L. monocytogenes survival (p > 0.05), regardless of harvest year. Populations gradually decreased with L. monocytogenes quantifiable in most samples after 7 months. Apple cultivar significantly impacted L. monocytogenes survival (p < 0.05) during both harvest years with greater reductions (p < 0.05) seen on Gala compared to Granny Smith and Honeycrisp. Biofilm-derived cells survived longer (p < 0.05) on L. monocytogenes-inoculated Gala and Honeycrisp apples compared to cells grown planktonically. These findings should aid in the development of improved L. monocytogenes intervention strategies for apple growers and packers.

Ozonated oil is effective at killing Candida species and Streptococcus mutans biofilm-derived cells under aerobic and microaerobic conditions.

We demonstrated that ozonated sunflower oil is effective at killing the biofilms formed by Candida species, by the bacterium Streptococcus mutans, or by both micoorganisms that can interact in the oral cavity, making it a potential therapeutic option for the treatment of these infections.

Growth in a biofilm sensitizes Cutibacterium acnes to nanosecond pulsed electric fields

The Gram-positive anaerobic bacterium Cutibacterium acnes (C. acnes) is a commensal of the human skin, but also an opportunistic pathogen that contributes to the pathophysiology of the skin disease acne vulgaris. C. acnes can form biofilms; cells in biofilms are more resilient to antimicrobial stresses. Acne therapeutic options such as topical or systemic antimicrobial treatments often show incomplete responses. In this study we measured the efficacy of nanosecond pulsed electric fields (nsPEF), a new promising cell and tissue ablation technology, to inactivate C. acnes. Our results show that all tested nsPEF doses (250 to 2000 pulses, 280 ns pulses, 28 kV/cm, 5 Hz; 0.5 to 4 kJ/ml) failed to inactivate planktonic C. acnes and that pretreatment with lysozyme, a naturally occurring cell-wall-weakening enzyme, increased C. acnes vulnerability to nsPEF. Surprisingly, growth in a biofilm appears to sensitize C. acnes to nsPEF-induced stress, as C. acnes biofilm-derived cells showed increased cell death after nsPEF treatments that did not affect planktonic cells. Biofilm inactivation by nsPEF was confirmed by treating intact biofilms grown on glass coverslips with an indium oxide conductive layer. Altogether our results show that, contrary to other antimicrobial agents, nsPEF kill more efficiently bacteria in biofilms than planktonic cells.

Effect of sub-lethal chemical disinfection on the biofilm forming ability, resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.

Reduced phagocytosis and killing of Cryptococcus neoformans biofilm-derived cells by J774.16 macrophages is associated with fungal capsular production and surface modification

Cryptococcus neoformans is an opportunistic encapsulated pathogen that causes life-threatening meningoencephalitis in individuals with immunosuppression. We compared the interactions of C. neoformans planktonic and biofilm-derived cells with J774.16 macrophage-like cells. Planktonic cells are more phagocytized and killed by J774.16 cells than biofilm-derived fungal cells. Biofilm-derived cryptococci possess larger capsule size and release significantly more capsular polysaccharide than planktonic cells in culture. Biofilm-derived fungi exhibited upregulation of genes involved in capsular production. Capsular-specific monoclonal antibody 18B7 demonstrated differential binding to the surface of planktonic and biofilm-derived cryptococci providing a plausible strategy for fungal evasion of macrophages and persistence. Future studies are necessary to elucidate how C. neoformans biofilm-derived cells regulate their virulence factors when interacting with cells of the immune system.

Pharmacodynamics of ciprofloxacin against Pseudomonas aeruginosa planktonic and biofilm-derived cells.

The influence of growth phase and state on the survival and recovery of Pseudomonas aeruginosa exposed to ciprofloxacin was investigated using batch culture grown planktonic cells and disaggregated biofilm populations. Biofilms were either nonantibiotic exposed or previously exposed to ciprofloxacin before disaggregation and subsequent challenge with ciprofloxacin. Viable counts showed that late stationary phase cells were tolerant to ciprofloxacin over 24h exposure, while all other populations presented a biphasic killing pattern. In contrast, the metabolic activity of planktonic and biofilm-derived cells remained similar to controls during the initial 6h of ciprofloxacin exposure, despite a significant reduction in viable cell numbers. A similar effect was observed when assessing the postantibiotic effect of 1h ciprofloxacin exposure. Thus, although cell reduction occurred, the metabolic status of the cells remained unchanged. The recovery of disaggregated biofilm cells previously exposed to ciprofloxacin was significantly quicker than naïve biofilm cells, and this latter population's recovery was significantly slower than all planktonic populations. Results from this work have implications for our understanding of biofilm-related infections and their resilience to antimicrobial treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Removal of biofilms from surfaces and infection sites via disaggregation and induction of dispersion may reverse their antibiotic tolerant state. However, little is known of the recovery of the cells upon disaggregation from biofilms. Driven by this gap in knowledge we quantified the effect of ciprofloxacin on disaggregated biofilms of Pseudomonas aeruginosa, including those previously exposed to ciprofloxacin. Our results provide further insight into bacterial resilience, regrowth, and antimicrobial efficacy, as reduction in cell viability does not directly correlate with the metabolic activity of bacteria at the time of the exposure to antimicrobials. Thus, despite a perceived reduction in viability, the potential for cell persistence and regrowth remains and recovery is quicker upon subsequent exposure to antimicrobial, supporting the increase in resilience and recurrence of infections.

Miltefosine inhibits Candida albicans and non-albicans Candida spp. biofilms and impairs the dispersion of infectious cells

Candida spp. can adhere to and form biofilms over different surfaces, becoming less susceptible to antifungal treatment. Resistance of biofilms to antifungal agents is multifactorial and the extracellular matrix (ECM) appears to play an important role. Among the few available antifungals for treatment of candidaemia, only the lipid formulations of amphotericin B (AmB) and the echinocandins are effective against biofilms. Our group has previously demonstrated that miltefosine has an important effect against Candida albicans biofilms. Thus, the aim of this work was to expand the analyses of the in vitro antibiofilm activity of miltefosine to non-albicans Candida spp. Miltefosine had significant antifungal activity against planktonic cells and the development of biofilms of C. albicans, Candida parapsilosis, Candida tropicalis and Candida glabrata. The activity profile in biofilms was superior to fluconazole and was similar to that of AmB and caspofungin. Biofilm-derived cells with their ECM extracted became as susceptible to miltefosine as planktonic cells, confirming the importance of the ECM in the biofilm resistant behaviour. Miltefosine also inhibited biofilm dispersion of cells at the same concentration needed to inhibit planktonic cell growth. The data obtained in this work reinforce the potent inhibitory activity of miltefosine on biofilms of the four most pathogenic Candida spp. and encourage further studies for the utilisation of this drug and/or structural analogues on biofilm-related infections.

Heteroresistance to colistin in Klebsiella pneumoniae is triggered by small colony variants sub-populations within biofilms.

The emergence of Klebsiella pneumoniae multidrug-resistant strains paves the way to the re-introduction of colistin as a salvage therapy. However, recent planktonic studies have reported several cases of heteroresistance to this antimicrobial agent. The aim of this present work was to gain better understanding about the response of K. pneumoniae biofilms to colistin antibiotherapy and inspect the occurrence of heteroresistance in biofilm-derived cells. Biofilm formation and its susceptibility to colistin were evaluated through the determination of biofilm-cells viability. The profiling of planktonic and biofilm cell populations was conducted to assess the occurrence of heteroresistance. Colony morphology was further characterized in order to inspect the potential role of colistin in K. pneumoniae phenotypic differentiation. Results show that K. pneumoniae was susceptible to colistin in its planktonic form, but biofilms presented enhanced resistance. Population analysis profiles pointed out that K. pneumoniae manifest heteroresistance to colistin only when grown in biofilm arrangements, and it was possible to identify a resistant sub-population presenting a small colony morphology (diameter around 5 mm). To the best of our knowledge, this is the first report linking heteroresistance to biofilm formation and a morphological distinctive sub-population. Moreover, this is the first evidence that biofilm formation can trigger the emergence of heteroresistance in an apparently susceptible strain.

Dormant bacteria within Staphylococcus epidermidis biofilms have low inflammatory properties and maintain tolerance to vancomycin and penicillin after entering planktonic growth.

Staphylococcus epidermidis is the most commonly isolated aetiological agent of nosocomial infections, mainly due to its ability to establish biofilms on indwelling medical devices. Detachment of bacteria from S. epidermidis biofilms and subsequent growth in the planktonic form is a hallmark of the pathogenesis of these infections leading to dissemination. Here we showed that S. epidermidis cells collected from biofilms cultured in conditions that promote cell viability present marked changes in their physiological status upon initiating a planktonic mode of growth. When compared to cells growing in biofilms, they displayed an increased SYBR green I staining intensity, increased transcription of the rpiA gene, decreased transcription of the icaA gene, as well as higher susceptibility to vancomycin and penicillin. When bacteria collected from biofilms with high proportions of dormant cells were subsequently cultured in the planktonic mode, a large proportion of cells maintained a low SYBR green I staining intensity and increased resistance to vancomycin and penicillin, a profile typical of dormant cells. This phenotype further associated with a decreased ability of these biofilm-derived cells to induce the production of pro-inflammatory cytokines by bone marrow-derived dendritic cells in vitro. These results demonstrated that cells detached from the biofilm maintain a dormant cell-like phenotype, having a low pro-inflammatory effect and decreased susceptibility to antibiotics, suggesting these cells may contribute to the recalcitrant nature of biofilm infections.

The pga gene cluster in Aggregatibacter actinomycetemcomitans is necessary for the development of natural competence in Ca2+‐promoted biofilms

Natural competence is the ability of bacteria to incorporate extracellular DNA into their genomes. This competence is affected by a number of factors, including Ca(2+) utilization and biofilm formation. As bacteria can form thick biofilms in the presence of extracellular Ca(2+) , the additive effects of Ca(2+) -promoted biofilm formation on natural competence should be examined. We evaluated natural competence in Aggregatibacter actinomycetemcomitans, an important periodontal pathogen, in the context of Ca(2+) -promoted biofilms, and examined whether the pga gene cluster, required for bacterial cell aggregation, is necessary for competence development. The A.actinomycetemcomitans cells grown in the presence of 1mm CaCl2 exhibited enhanced cell aggregation and increased levels of cell-associated Ca(2+) . Biofilm-derived cells grown in the presence of Ca(2+) exhibited the highest levels of natural transformation frequency and enhanced expression of the competence regulator gene, tfoX. Natural competence was enhanced by the additive effects of Ca(2+) -promoted biofilms, in which high levels of pga gene expression were also detected. Mutation of the pga gene cluster disrupted biofilm formation and competence development, suggesting that these genes play a critical role in the ability of A.actinomycetemcomitans to adapt to its natural environment. The Ca(2+) -promoted biofilms may enhance the ability of bacteria to acquire extracellular DNA.

Role of Efflux Pump Inhibitors on the Antibiofilm Efficacy of Calcium Hydroxide, Chitosan Nanoparticles, and Light-activated Disinfection

Introduction The purpose of this study was to evaluate the role of efflux pumps in altering the susceptibility of Enterococcus faecalis biofilms to calcium hydroxide (Ca(OH) 2), chitosan nanoparticles, and light-activated disinfection (LAD). Methods E. faecalis as 4-day-old biofilms and biofilm-derived cells were tested with aqueous Ca(OH) 2 in concentrations of 25%, 50%, and 100%; chitosan nanoparticles in concentrations of 10 and 20 mg/mL (3, 12, and 24 hours); and methylene blue (MB) mediated LAD at an energy dose range of 2–40 J/cm 2. An efflux pump inhibitor (EPI) was incorporated into all 3 modalities of treatment. The antimicrobial activity was assessed by determining the colony-forming units. Results E. faecalis biofilms, in contrast to the biofilm-derived cells, were found to persist even after 24-hour treatment with different concentrations of Ca(OH) 2 and chitosan nanoparticles. LAD at an energy dose of 40 J/cm 2 completely inactivated 4-day-old E. faecalis biofilms. The addition of EPI improved the antibiofilm efficacy of Ca(OH) 2 at lower concentrations ( P < .001) and LAD ( P < .001). EPI did not influence the antibiofilm effect of chitosan nanoparticles and Ca(OH) 2 at higher concentrations. Conclusions E. faecalis biofilms were more susceptible to killing by LAD, when compared with the tested concentrations of Ca(OH) 2 and chitosan nanoparticles. The effect of EPI was more significant with LAD, when compared with Ca(OH) 2 and chitosan nanoparticles. This study highlighted the role of biofilm matrix in providing resistance to antimicrobials.

Evaluations of Shorter Exposures of Contact Lens Cleaning Solutions against Fusarium oxysporum Species Complex and Fusarium solani Species Complex To Simulate Inappropriate Usage

An outbreak of Fusarium keratitis in contact lens users resulted in withdrawal of ReNu with MoistureLoc solution, although the exact cause of the outbreak remains enigmatic. We evaluated current and discontinued multipurpose cleaning solutions (MPSs; MoistureLoc, Equate, MultiPlus, and OptiFree Express) against plankton- and biofilm-derived cells of Fusarium oxysporum species complex (FOSC) and F. solani species complex (FSSC). The methods included a traditional assay based on CFU counts and a novel flow cytometry (FC) assay based on percent cell subpopulation (PCS) stained with two fluorochromes (Sytox Red and 5-chloromethylfluorescein diacetate). The tests were done with the respective manufacturers' recommended cleaning regimens (240 to 360 min) and under shorter exposures (15 to 60 min) to simulate inappropriate usage by the customers. FC assay measured PCS, which was available rapidly, in 5 to 7 h, whereas 24 to 48 h was needed for CFU counts, and there was good correlation between the two methods (r2=0.97). FC assays allowed identification of injured fungal cells, which are likely to be missed with growth assays. In general, a time- and inoculum-dependent survival pattern was seen for both FOSC and FSSC cells, and biofilm-derived cells were more resistant than plankton-derived cells. MultiPlus and Equate produced 100% sterilization of fungi even under shorter exposures. However, biofilm FOSC and FSSC cells survived for up to 4 h in MoistureLoc solution and up to 6 h in OptiFree Express solution under shorter exposure times. This finding was enigmatic, as OptiFree Express is not associated with any outbreak of Fusarium keratitis. This study provides additional support for possible roles that improper lens cleaning regimens and fungal biofilms could play as predisposing factors for Fusarium keratitis.

Growth in a Biofilm Induces a Hyperinfectious Phenotype inVibrio cholerae

Biofilm formation plays a multifaceted role in the life cycles of a wide variety of microorganisms. In the case of pathogenic Vibrio cholerae, biofilm formation in its native aquatic habitats is thought to aid in persistence during interepidemic seasons and to enhance infectivity upon oral ingestion. The structure of V. cholerae biofilms has been hypothesized to protect the bacteria during passage through the stomach. Here, we directly test the role of biofilm architecture in the infectivity of V. cholerae by comparing the abilities of intact biofilms, dispersed biofilms, and planktonic cells to colonize the mouse small intestine. Not only were V. cholerae biofilms better able to colonize than planktonic cells, but the structure of the biofilm was also found to be dispensable: intact and dispersed biofilms colonized equally, and both vastly out-colonized planktonic cells. The infectious dose for biofilm-derived V. cholerae was orders of magnitude lower than that of planktonic cells. This biofilm-induced hyperinfectivity may be due in part to a higher growth rate of biofilm-derived cells during infection. These results suggest that the infectious dose of naturally occurring biofilms of V. cholerae may be much lower than previously estimated using cells grown planktonically in vitro. Furthermore, this work implies the existence of factors specifically induced during growth in a biofilm that augment infection by V. cholerae.

Expression of biofilm-associated genes of Streptococcus mutans in response to glucose and sucrose

Streptococcus mutans is known as a primary pathogen of dental caries, one of the most common human infectious diseases. Exopolysaccharide synthesis, adherence to tooth surface and biofilm formation are important physiological and virulence factors of S. mutans. In vitro comparative gene expression analysis was carried out to differentiate 10 selected genes known to be mostly involved in S. mutans biofilm formation by comparing the expression under biofilm and planktonic environments. Real-time RT-PCR analyses indicated that all of the genes tested were upregulated in the biofilm compared to cells grown in planktonic conditions. The influence of simple dietary carbohydrates on gene expression in S. mutans biofilm was tested also. Among the tested genes, in the biofilm phase, the greatest induction was observed for gtf and ftf, which are genes encoding the extracellular polysaccharide-producing enzymes. Biofilm formation was accompanied by a 22-fold induction in the abundance of mRNA encoding glucosyltransferase B (GTFB) and a 14.8 -fold increase in mRNA encoding GTFC. Levels of mRNA encoding fructosyltransferase were induced approximately 11.8-fold in biofilm-derived cells. Another notable finding of this study suggests that glucose affects the expression of S. mutans GS5 biofilm genes. In spite of a significant upregulation in biofilm-associated gene expression in the presence of sucrose, the presence of glucose with sucrose reduced expression of most tested genes. Differential analysis of the transcripts from S. mutans, grown in media with various nutrient contents, revealed significant shifts in the expression of the genes involved in biofilm formation. The results presented here provide new insights at the molecular level regarding gene expression in this bacterium when grown under biofilm conditions, allowing a better understanding of the mechanism of biofilm formation by S. mutans.

Effect of triclosan onSalmonella typhimuriumat different growth stages and in biofilms

Triclosan is a potent biocide that is included in a diverse range of products. This research was aimed to investigate the susceptibility of planktonic and biofilm-associated Salmonella enterica serovar Typhimurium to triclosan, and to identify potential mechanisms of adaptation. The effect of triclosan was studied on planktonic Salmonella (log and stationary phases), on biofilm-associated cells, on bacteria derived from disrupted biofilms and on a biofilm-deficient mutant. An eight-log reduction of exponentially growing cells was observed with 1000 micro g mL(-1) triclosan within 10 min, a 3.6-log reduction in stationary cells and a 6.3-log reduction in stationary cells of a biofilm-deficient mutant (P<0.05). Biofilm-associated cells were tolerant (1-log reduction). However, biofilm-derived cells showed sensitivity to triclosan similar to stationary-phase cells. Triclosan induced the transcription of fabI and micF. Within biofilms, triclosan also up-regulated the transcription of acrAB, encoding for an efflux pump, marA, and the cellulose-synthesis-coding genes bcsA and bcsE. Thus, Salmonella within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. Our results demonstrated that the tolerance of Salmonella towards triclosan in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials.

Role of anosXHomolog inStreptococcus gordoniiin Aerobic Growth and Biofilm Formation

Oral streptococci such as Streptococcus gordonii are facultative anaerobes that initiate biofilm formation on tooth surfaces. An isolated S. gordonii::Tn917-lac biofilm-defective mutant contained a transposon insertion in an open reading frame (ORF) encoding a homolog of NosX of Ralstonia eutropha, a putative maturation factor of nitrous oxide reductase. Located downstream are two genes, qor1 and qor2, predicted to encode two putative NADPH quinone oxidoreductases. These three genes are cotranscribed, forming a putative oxidative stress response (osr) operon in S. gordonii. Inactivation of nosX, qor1, or qor2 resulted in biofilm-defective phenotypes. Expression of nosX, measured by the beta-galactosidase activity of the nosX::Tn917-lac mutant, was growth-phase dependent and enhanced when grown under aerobic conditions or in the presence of paraquat. Real-time reverse transcription-PCR revealed that nosX-specific mRNA levels were increased approximately 8.4 and 3.5 fold in biofilm-derived cells grown on plastic and glass, respectively, when compared to planktonic cells. Expression of nosX increased 19.9 fold in cells grown under aerated aerobic conditions and 4.7 fold in cells grown under static aerobic conditions. Two ORFs immediately adjacent to the osr operon encode a putative NADH oxidase (Nox) and a putative thiol-specific antioxidant enzyme (AhpC, for alkyl hydroperoxide peroxidase C). Expression of nox and ahpC was also significantly increased in cells grown under aerated and static aerobic conditions when compared to anaerobic conditions. In addition, nox expression was increased in biofilm cells compared to planktonic cells. These genes may be part of an island that deals with oxidoreductive response, some of which may be important in S. gordonii biofilm formation.

Effect of growth-rate on resistance of Gram-negative biofilms to cetrimide

A method of cell culture, which allows control of growth rate for sessile Gram-negative populations, has been employed to assess the sensitivity of Escherichia coli biofilms to the antiseptic compound, cetrimide. Growth-rate-dependent changes in sensitivity were compared for chemostat-grown, planktonic cells, for cells resuspended from the biofilm and also for newly formed daughter cells shed from the biofilm during its growth and development. Susceptibility to cetrimide decreased in all instances with increases in growth-rate up to mu = 0.15 h-1. As growth rate was increased beyond this value then sensitivity increased in proportion to the rate of division. At rates of growth less than mu = 0.15 h-1 the susceptibility of the biofilm-derived cells and their offspring was significantly less than that of cells of planktonic origin.