Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes

Bacteria living as biofilm are frequently reported to exhibit inherent tolerance to antimicrobial compounds, and might therefore contribute to the persistence of infections. Antimicrobial peptides are attracting increasing interest as new potential antimicrobial therapeutics; however, little is known about potential mechanisms, which might contribute to resistance or tolerance development towards these compounds in biofilms. Here we provide evidence that a spatially distinct subpopulation of metabolically active cells in Pseudomonas aeruginosa biofilms is able to develop tolerance to the antimicrobial peptide colistin. On the contrary, biofilm cells exhibiting low metabolic activity were killed by colistin. We demonstrate that the subpopulation of metabolically active cells is able to adapt to colistin by inducing a specific adaptation mechanism mediated by the pmr operon, as well as an unspecific adaptation mechanism mediated by the mexAB-oprM genes. Mutants defective in either pmr -mediated lipopolysaccharide modification or in mexAB-oprM -mediated antimicrobial efflux were not able to develop a tolerant subpopulation in biofilms. In contrast to the observed pattern of colistin-mediated killing in biofilms, conventional antimicrobial compounds such as ciprofloxacin and tetracycline were found to specifically kill the subpopulation of metabolically active biofilm cells, whereas the subpopulation exhibiting low metabolic activity survived the treatment. Consequently, targeting the two physiologically distinct subpopulations by combined antimicrobial treatment with either ciprofloxacin and colistin or tetracycline and colistin almost completely eradicated all biofilm cells.     

Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms

During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane-targeting antimicrobial agents, such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulfate. The stalk-forming subpopulation, on the other hand, was sensitive to the membrane-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with lipopolysaccharide modification (pmr) eliminated the biofilm-associated colistin tolerance phenotype. Experiments with a PAO1 strain harboring a pmr-gfp fusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.     

Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae

Bacteria have evolved multiple mechanisms, such as biofilm formation, to thwart antibiotic action. Yet antibiotics remain the drug of choice against clinical infections. It has been documented that young biofilm of Klebsiella pneumoniae could be eradicated significantly by ciprofloxacin treatment alone. Since age of biofilm is a decisive factor in determining the outcome of antibiotic treatment, in the present study biofilm of K. pneumoniae, grown for extended periods was treated with ciprofloxacin and/or depolymerase producing lytic bacteriophage (KPO1K2). The reduction in bacterial numbers of older biofilm was greater after application of the two agents in combination as ciprofloxacin alone could not reduce bacterial biomass significantly in older biofilms (P > 0.05). Confocal microscopy suggested the induction of structural changes in the biofilm matrix and a decrease in micro-colony size after KPO1K2 treatment. The role of phage associated depolymerase was emphasized by the insignificant eradication of biofilm by a non-depolymerase producing bacteriophage that, however, eradicated the biofilm when applied concomitantly with purified depolymerase. These findings demonstrate that a lytic bacteriophage alone can eradicate older biofilms significantly and its action is primarily depolymerase mediated. However, application of phage and antibiotic in combination resulted in slightly increased biofilm eradication confirming the speculation that antibiotic efficacy can be augmented by bacteriophage.     

Planktonic aggregates of Staphylococcus aureus protect against common antibiotics

Bacterial cells are mostly studied during planktonic growth although in their natural habitats they are often found in communities such as biofilms with dramatically different physiological properties. We have examined another type of community namely cellular aggregates observed in strains of the human pathogen Staphylococcus aureus. By laser-diffraction particle-size analysis (LDA) we show, for strains forming visible aggregates, that the aggregation starts already in the early exponential growth phase and proceeds until post-exponential phase where more than 90% of the population is part of the aggregate community. Similar to some types of biofilm, the structural component of S. aureus aggregates is the polysaccharide intercellular adhesin (PIA). Importantly, PIA production correlates with the level of aggregation whether altered through mutations or exposure to sub-inhibitory concentrations of selected antibiotics. While some properties of aggregates resemble those of biofilms including increased mutation frequency and survival during antibiotic treatment, aggregated cells displayed higher metabolic activity than planktonic cells or cells in biofilm. Thus, our data indicate that the properties of cells in aggregates differ in some aspects from those in biofilms. It is generally accepted that the biofilm life style protects pathogens against antibiotics and the hostile environment of the host. We speculate that in aggregate communities S. aureus increases its tolerance to hazardous environments and that the combination of a biofilm-like environment with mobility has substantial practical and clinical importance.     

Combinatorial approaches with selected phytochemicals to increase antibiotic efficacy against Staphylococcus aureus biofilms

Combinations of selected phytochemicals (reserpine, pyrrolidine, quinine, morin and quercetin) with antibiotics (ciprofloxacin, tetracycline and erythromycin) were tested on the prevention and control of Staphylococcus aureus biofilms. The phytochemicals were also studied for their ability to avoid antibiotic adaptation and to inhibit antibiotic efflux pumps. Morin, pyrrolidine and quercetin at subinhibitory concentrations had significant effects in biofilm prevention and/or control when applied alone and combined with antibiotics. Synergism between antibiotics and phytochemicals was found especially against biofilms of NorA overexpressing strain S. aureus SA1199B. This strain when growing with subinhibitory concentrations of ciprofloxacin developed increased tolerance to this antibiotic. However, this was successfully reversed by quinine and morin. In addition, reserpine and quercetin showed significant efflux pump inhibition. The overall results demonstrate the role of phytochemicals in co-therapies to promote more efficient treatments and decrease antimicrobial resistance to antibiotics, with substantial effects against S. aureus in both planktonic and biofilm states.     

Inhibition and destruction of Pseudomonas aeruginosa biofilms by antibiotics and antimicrobial peptides

Pseudomonas aeruginosa is one of the major nosocomial pathogen that can causes a wide variety of acute and chronic infections P. aeruginosa is a dreaded bacteria not just because of the high intrinsic and acquired antibiotic resistance rates but also the biofilm formation and production of multiple virulence factors. We investigated the in vitro activities of antibiotics (ceftazidime, tobramycin, ciprofloxacin, doripenem, piperacillin and colistin) and antimicrobial cationic peptides (AMPs; LL-37, CAMA: cecropin(1–7)-melittin A(2–9) amide, melittin, defensin and magainin-II) alone or in combination against biofilms of laboratory strain ATCC 27853 and 4 clinical strains of P. aeruginosa. The minimum inhibitory concentrations (MIC), minimum bactericidal concentration (MBC) and minimum biofilm eradication concentrations (MBEC) were determined by microbroth dilution technique. The MBEC values of antibiotics and AMPs were 80–>5120 and 640–>640 mg/L, respectively. When combined with the LL-37 or CAMA at 1/10× MBEC, the MBEC values of antibiotics that active against biofilms, were decreased up to 8-fold. All of the antibiotics, and AMPs were able to inhibit the attachment of bacteria at the 1/10× MIC and biofilm formation at 1× or 1/10× MIC concentrations. Time killing curve studies showed 3-log₁₀ killing against biofilms in 24 h with almost all studied antibiotics and AMPs. Synergism were seen in most of the studied combinations especially CAMA/LL-37 + ciprofloxacin against at least one or two strains’ biofilms. Since biofilms are not affected the antibiotics at therapeutic concentrations, using a combination of antimicrobial agents including AMPs, or inhibition of biofilm formation by blocking the attachment of bacteria to surfaces might be alternative methods to fight with biofilm associated infections.     

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.Subject terms: Microbiology, Diseases     

Starvation,Together with the SOS Response,Mediates High Biofilm-Specific Tolerance to the Fluoroquinolone Ofloxacin

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin–antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.     

Quorum-sensing mutations affect attachment and stability of Burkholderia cenocepacia biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.     

The effect of subminimal inhibitory concentrations of antibiotics on virulence factors expressed by Staphylococcus aureus biofilms

Aims: The effect of subminimal inhibitory concentrations (sub‐MICs) of cefalexin, ciprofloxacin and roxithromycin was investigated on some virulence factors [e.g. coagulase, Toxic Shock Syndrome Toxin 1 (TSST‐1) and biofilm formation] expressed by Staphylococcus aureus biofilms. Methods and Results: Biofilms were grown with and without the presence of 1/16 MIC of antibiotics on Sorbarod filters. Eluate supernatants were collected, and coagulase and TSST‐1 production were evaluated. Coagulase production was reduced in eluates exposed to roxithromycin when compared to control, while TSST‐1 production was reduced in biofilms exposed to cefalexin and to a lesser extent, ciprofloxacin. In addition, the ability of Staph. aureus to produce biofilm in microtitre plates in the presence of sub‐MIC antibiotics indicated that cefalexin induced biofilm formation at a wide range of sub‐MICs. TSST‐1 produced from the challenged and control biofilms was purified, and its proliferative activity was studied on single cell suspension of mouse splenocytes using MTS/PMS assay. No significant difference in the activity between the treated toxin and the control has been observed. Conclusions: Antibiotics at sub‐MIC levels interfere with bacterial biofilm virulence expression depending on the type and concentration of antibiotic used. Significance and Impact of the Study: The establishment of sub‐MICs of antibiotics in clinical situations may result in altered virulence states in pathogenic bacteria.     

Pattern formation in Pseudomonas aeruginosa biofilms

Bacteria are capable of forming elaborate multicellular communities called biofilms. Pattern formation in biofilms depends on cell proliferation and cellular migration in response to the available nutrients and other external cues, as well as on self-generated intercellular signal molecules and the production of an extracellular matrix that serves as a structural 'scaffolding' for the biofilm cells. Pattern formation in biofilms allows cells to position themselves favorably within nutrient gradients and enables buildup and maintenance of physiologically distinct subpopulations, which facilitates survival of one or more subpopulations upon environmental insult, and therefore plays an important role in the innate tolerance displayed by biofilms toward adverse conditions.     

The ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency

The penetration ability of 12 antimicrobial agents, including antibiotics and biocides, was determined against biofilms of B. cereus and P. fluorescens using a colony biofilm assay. The surfactants benzalkonium chloride (BAC) and cetyltrimethyl ammonium bromide (CTAB), and the antibiotics ciprofloxacin and streptomycin were of interest due to their distinct activities. Erythromycin and CTAB were retarded by the presence of biofilms, whereas ciprofloxacin and BAC were not. The removal and killing efficacies of these four agents was additionally evaluated against biofilms formed in microtiter plates. The most efficient biocide was CTAB for both bacterial biofilms. Ciprofloxacin was the best antibiotic although none of the selected antimicrobial agents led to total biofilm removal and/or killing. Comparative analysis of the results obtained with colony biofilms and microtiter plate biofilms show that although extracellular polymeric substances and the biofilm structure are considered a determining factor in biofilm resistance, the ability of an antimicrobial agent to penetrate a biofilm is not correlated with its killing or removal efficiency. Also, the results reinforce the role of an appropriate antimicrobial selection as a key step in the design of disinfection processes for biofilm control.     

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.     

Strategies for adaptation to antibiotics in wild type <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and in the strains with small colony phenotype

Emergence of ciprofloxacin stress-induced mutants in the cultures of a collection strain Pseudomonas aeruginosa ATCC 27853 and of two strains with a small colony phenotype, which were isolated from a swimming pool biofilm, was studied. In biofilm cultures of the small colony phenotype strains, which were already resistant to hypochlorite, prolonged incubation (up to 16 days) with sublethal ciprofloxacin concentrations was shown to result in emergence of the cells, which are resistant to the antibiotic and form colonies on media with rifampicin (100 μg/mL) and streptomycin (50 μg/mL). Under the same conditions, the mechanisms of temporary adaptation are switched on in the cells of strain ATCC 27853, which enabled its shortterm survival at an average level in liquid media and provided for colony formation on solid medium with ciprofloxacin (0.2 μg/mL). Only 20% of these colonies remained viable when transferred to a higher antibiotic concentration (2 μg/mL).     

Oxidative stress involved in the antibacterial action of different antibiotics

Staphylococcus aureus and Escherichia coli sensitive to chloramphenicol incubated with this antibiotic suffered oxidative stress with increase of anion superoxide (O2-). This reactive species of oxygen was detected by chemiluminescence with lucigenin. S. aureus, E. coli, and Enterococcus faecalis sensitive to ciprofloxacin exhibited oxidative stress when they were incubated with this antibiotic while resistant strains did not show stimuli of O2-. Other bacteria investigated was Pseudomonas aeruginosa, strains sensitive to ceftazidime and piperacillin presented oxidative stress in presence of these antibiotics while resistant strains were not stressed. Higher antibiotic concentration was necessary to augment O2- in P. aeruginosa biofilm than in suspension, moreover old biofilms were resistant to oxidative stress caused by antibiotics. A ceftazidime-sensitive mutant of P. aeruginosa, coming from a resistant strain, exhibited higher production of O2- than wild type in presence of this antibiotic. There was relation between antibiotic susceptibility and production of oxidative stress.     

Variation in biofilm formation among strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.     

Biofilm formation and the presence of the intercellular adhesion locus ica among staphylococci from food and food processing environments

In clinical staphylococci, the presence of the ica genes and biofilm formation are considered important for virulence. Biofilm formation may also be of importance for survival and virulence in food-related staphylococci. In the present work, staphylococci from the food industry were found to differ greatly in their abilities to form biofilms on polystyrene. A total of 7 and 21 of 144 food-related strains were found to be strong and weak biofilm formers, respectively. Glucose and sodium chloride stimulated biofilm formation. The biofilm-forming strains belonged to nine different coagulase-negative species of Staphylococcus. The icaA gene of the intercellular adhesion locus was detected by Southern blotting and hybridization in 38 of 67 food-related strains tested. The presence of icaA was positively correlated with strong biofilm formation. The icaA gene was partly sequenced for 22 food-related strains from nine different species of Staphylococcus, and their icaA genes were found to have DNA similarities to previously sequenced icaA genes of 69 to 100%. Northern blot analysis indicated that the expression of the ica genes was higher in strong biofilm formers than that seen with strains not forming biofilms. Biofilm formation on polystyrene was positively correlated with biofilm formation on stainless steel and with resistance to quaternary ammonium compounds, a group of disinfectants.