Bacteriocin-mediated competition in cystic fibrosis lung infections

Bacteriocins are toxins produced by bacteria to kill competitors of the same species. Theory and laboratory experiments suggest that bacteriocin production and immunity play a key role in the competitive dynamics of bacterial strains. The extent to which this is the case in natural populations, especially human pathogens, remains to be tested. We examined the role of bacteriocins in competition using Pseudomonas aeruginosa strains infecting lungs of humans with cystic fibrosis (CF). We assessed the ability of different strains to kill each other using phenotypic assays, and sequenced their genomes to determine what bacteriocins (pyocins) they carry. We found that (i) isolates from later infection stages inhibited earlier infecting strains less, but were more inhibited by pyocins produced by earlier infecting strains and carried fewer pyocin types; (ii) this difference between early and late infections appears to be caused by a difference in pyocin diversity between competing genotypes and not by loss of pyocin genes within a lineage over time; (iii) pyocin inhibition does not explain why certain strains outcompete others within lung infections; (iv) strains frequently carry the pyocin-killing gene, but not the immunity gene, suggesting resistance occurs via other unknown mechanisms. Our results show that, in contrast to patterns observed in experimental studies, pyocin production does not appear to have a major influence on strain competition during CF lung infections.     

Diverse LXG toxin and antitoxin systems specifically mediate intraspecies competition in Bacillus subtilis biofilms

Biofilms are multispecies communities, in which bacteria constantly compete with one another for resources and niches. Bacteria produce many antibiotics and toxins for competition. However, since biofilm cells exhibit increased tolerance to antimicrobials, their roles in biofilms remain controversial. Here, we showed that Bacillus subtilis produces multiple diverse polymorphic toxins, called LXG toxins, that contain N-terminal LXG delivery domains and diverse C-terminal toxin domains. Each B. subtilis strain possesses a distinct set of LXG toxin–antitoxin genes, the number and variation of which is sufficient to distinguish each strain. The B. subtilis strain NCIB3610 possesses six LXG toxin–antitoxin operons on its chromosome, and five of the toxins functioned as DNase. In competition assays, deletion mutants of any of the six LXG toxin–antitoxin operons were outcompeted by the wild-type strain. This phenotype was suppressed when the antitoxins were ectopically expressed in the deletion mutants. The fitness defect of the mutants was only observed in solid media that supported biofilm formation. Biofilm matrix polymers, exopolysaccharides and TasA protein polymers were required for LXG toxin function. These results indicate that LXG toxin-antitoxin systems specifically mediate intercellular competition between B. subtilis strains in biofilms. Mutual antagonism between some LXG toxin producers drove the spatial segregation of two strains in a biofilm, indicating that LXG toxins not only mediate competition in biofilms, but may also help to avoid warfare between strains in biofilms. LXG toxins from strain NCIB3610 were effective against some natural isolates, and thus LXG toxin–antitoxin systems have ecological impact. B. subtilis possesses another polymorphic toxin, WapA. WapA had toxic effects under planktonic growth conditions but not under biofilm conditions because exopolysaccharides and TasA protein polymers inhibited WapA function. These results indicate that B. subtilis uses two types of polymorphic toxins for competition, depending on the growth mode.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Effect of long-term starvation in salty microcosm on biofilm formation and motility in Pseudomonas aeruginosa

The development of antibiotic resistance in the opportunistic pathogen Pseudomonas aeruginosa is a major cause of the pathogen’s morbidity and is strongly correlated with the biofilm formation. Motility and adherence capacity in long-term stressed cells have not been extensively analyzed even though P. aeruginosa considered a model organism for the study of biofilm formation. In this investigation, P. aeruginosa ATCC 27853 strain has been stored for 12 months in LB broth with 0.5 M NaCl. Several experiments demonstrated that the strain recovery from the salty microcosm had the ability to increase the biofilm formation and to reduce motility comparing with that of the original strain. To identify genes involved in the regulation of biofilm and/or in stress response by the recovered P. aeruginosa, differential display “DDRT-PCR” technique was used. The genes speD and ccoN2, coding, respectively, for an S-adenosylmethionine decarboxylase and Cbb3-type cytochrome oxidase, were identified in recovered strain of P. aeruginosa ATCC 27853 as two differentially expressed gene fragments. A comparison of the biofilm produced by the wild-type strain PA14 and the transposon insertion mutant for speD gene suggested that spermidine has a potential role in the adaptive response in P. aeruginosa incubated in long-term stress conditions.     

Targeting Acyl Homoserine Lactones (AHLs) by the quorum quenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa

Navigating novel biological strategies to mitigate bacterial biofilms have great worth to combat bacterial infections. Bacterial infections caused by the biofilm forming bacteria are 1000 times more resistant to antibiotics than the planktonic bacteria. Among the known bacterial infections, more than 70% involve biofilms which severely complicates treatment options. Biofilm formation is mainly regulated by the Quorum sensing (QS) mechanism. Interference with the QS system by the quorum quenching (QQ) enzyme is a potent strategy to mitigate biofilm. In this study, bacterial strains with QQ activity were identified and their anti-biofilm potential was investigated against the Multidrug Resistant (MDR) Pseudomonas aeruginosa. A Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136-based bioassays were used to confirm the degradation of different Acyl Homoserine Lactones (AHLs) by QQ isolates. The 16S rRNA gene sequencing of the isolated strains identified them as Bacillus cereus strain QSP03, B. subtilis strain QSP10, Pseudomonas putida strain QQ3 and P. aeruginosa strain QSP01. Biofilm mitigation potential of QQ isolates was tested against MDR P. aeruginosa and the results suggested that 50% biofilm reduction was observed by QQ3 and QSP01 strains, and around 60% reduction by QSP10 and QSP03 bacterial isolates. The presence of AHL degrading enzymes, lactonases and acylases, was confirmed by PCR based screening and sequencing of the already annotated genes aiiA, pvdQ and quiP. Altogether, these results exhibit that QQ bacterial strains or their products could be useful to control biofilm formation in P.aeruginosa.     

Kind Discrimination and Competitive Exclusion Mediated by Contact-Dependent Growth Inhibition Systems Shape Biofilm Community Structure

Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein (called BcpA in Burkholderia species) to inhibit the growth of neighboring bacteria upon cell-cell contact. CDI systems are present in a wide range of Gram-negative proteobacteria and a hallmark feature is polymorphism amongst the exoprotein C-termini (BcpA-CT in Burkholderia) and amongst the small immunity proteins (BcpI) that protect against CDI in an allele-specific manner. In addition to CDI, the BcpAIOB proteins of Burkholderia thailandensis mediate biofilm formation, and they do so independent of BcpA-mediated interbacterial competition, suggesting a cooperative role for CDI system proteins in this process. CDI has previously only been demonstrated between CDI⁺ and CDI⁻ bacteria, leaving the roles of CDI system-mediated interbacterial competition and of CDI system diversity in nature unknown. We constructed B. thailandensis strains that differed only in the BcpA-CT and BcpI proteins they produced. When co-cultured on agar, these strains each participated in CDI and the outcome of the competition depended on both CDI system efficiency and relative bacterial numbers initially. Strains also participated in CDI during biofilm development, resulting in pillar structures that were composed of only a single BcpA-CT/BcpI type. Moreover, a strain producing BcpA-CT/BcpI proteins of one type was prevented from joining a pre-established biofilm community composed of bacteria producing BcpA-CT/BcpI proteins of a different type, unless it also produced the BcpI protein of the established strain. Bacteria can therefore use CDI systems for kind recognition and competitive exclusion of ‘non-self’ bacteria from a pre-established biofilm. Our data indicate that CDI systems function in both cooperative and competitive behaviors to build microbial communities that are composed of only bacteria that are related via their CDI system alleles.     

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.     

Capture and Retention of Cryptosporidium parvum Oocysts by Pseudomonas aeruginosa Biofilms

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.     

Role of Mutation in Pseudomonas aeruginosa Biofilm Development

The survival of bacteria in nature is greatly enhanced by their ability to grow within surface-associated communities called biofilms. Commonly, biofilms generate proliferations of bacterial cells, called microcolonies, which are highly recalcitrant, 3-dimensional foci of bacterial growth. Microcolony growth is initiated by only a subpopulation of bacteria within biofilms, but processes responsible for this differentiation remain poorly understood. Under conditions of crowding and intense competition between bacteria within biofilms, microevolutionary processes such as mutation selection may be important for growth; however their influence on microcolony-based biofilm growth and architecture have not previously been explored. To study mutation in-situ within biofilms, we transformed Pseudomonas aeruginosa cells with a green fluorescent protein gene containing a +1 frameshift mutation. Transformed P. aeruginosa cells were non-fluorescent until a mutation causing reversion to the wildtype sequence occurs. Fluorescence-inducing mutations were observed in microcolony structures, but not in other biofilm cells, or in planktonic cultures of P. aeruginosa cells. Thus microcolonies may represent important foci for mutation and evolution within biofilms. We calculated that microcolony-specific increases in mutation frequency were at least 100-fold compared with planktonically grown cultures. We also observed that mutator phenotypes can enhance microcolony-based growth of P. aeruginosa cells. For P. aeruginosa strains defective in DNA fidelity and error repair, we found that microcolony initiation and growth was enhanced with increased mutation frequency of the organism. We suggest that microcolony-based growth can involve mutation and subsequent selection of mutants better adapted to grow on surfaces within crowded-cell environments. This model for biofilm growth is analogous to mutation selection that occurs during neoplastic progression and tumor development, and may help to explain why structural and genetic heterogeneity are characteristic features of bacterial biofilm populations.     

Investigating the Link Between Imipenem Resistance and Biofilm Formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC?≤?2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC?≥?8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.     

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H₂O₂) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H₂O₂ and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H₂O₂ was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H₂O₂ mediated cell lysis.     

PslG,a self-produced glycosyl hydrolase,triggers biofilm disassembly by disrupting exopolysaccharide matrix

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.     

Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms

In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express ‘cooperative traits'', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.     

A Systems-Level Approach for Investigating Pseudomonas aeruginosa Biofilm Formation

Prevention of the initiation of biofilm formation is the most important step for combating biofilm-associated pathogens, as the ability of pathogens to resist antibiotics is enhanced 10 to 1000 times once biofilms are formed. Genes essential to bacterial growth in the planktonic state are potential targets to treat biofilm-associated pathogens. However, the biofilm formation capability of strains with mutations in these essential genes must be evaluated, since the pathogen might form a biofilm before it is eliminated. In order to address this issue, this work proposes a systems-level approach to quantifying the biofilm formation capability of mutants to determine target genes that are essential for bacterial metabolism in the planktonic state but do not induce biofilm formation in their mutants. The changes of fluxes through the reactions associated with the genes positively related to biofilm formation are used as soft sensors in the flux balance analysis to quantify the trend of biofilm formation upon the mutation of an essential gene. The essential genes whose mutants are predicted not to induce biofilm formation are regarded as gene targets. The proposed approach was applied to identify target genes to treat Pseudomonas aeruginosa infections. It is interesting to find that most essential gene mutants exhibit high potential to induce the biofilm formation while most non-essential gene mutants do not. Critically, we identified four essential genes, lysC, cysH, adk, and galU, that constitute gene targets to treat P. aeruginosa. They have been suggested by existing experimental data as potential drug targets for their crucial role in the survival or virulence of P. aeruginosa. It is also interesting to find that P. aeruginosa tends to survive the essential-gene mutation treatment by mainly enhancing fluxes through 8 metabolic reactions that regulate acetate metabolism, arginine metabolism, and glutamate metabolism.     

Monitoring of biofilm production by Pseudomonas aeruginosa strains under different conditions of UVC irradiation and phage infection

Pseudomonas aeruginosa is an opportunistic pathogen that commonly causes infection in immunocompromised individuals. This bacterium forms complex communities known as biofilms. Biofilms are also beneficial for bioremediation processes in terms of eliminating oil spills and sewage treatment; however, they can be harmful, particularly in the food industry sector and in water distribution systems. This paper focuses on the development of a concept that limits biofilm formation by P. aeruginosa strains. The results suggest that combined treatment with lytic phage and UVC radiation may provide an alternative control strategy. Indeed, the enhancement of physical disinfection step efficiency by the use of a biological agent instead of chemical one such as chlorine can be considered as a safety procedure to reduce and/or to prevent biofilm formation.     

Broad-Spectrum Anti-biofilm Peptide That Targets a Cellular Stress Response

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.