Increased tolerance of Staphylococcus aureus to vancomycin in viscous media

The increased viscosity observed in biofilms, adherent communities of bacterial cells embedded in a polymeric matrix, was hypothesized to induce increased tolerance of bacteria to antibiotics. To test this concept, planktonic Staphylococcus aureus cells were grown and exposed to vancomycin in media brought to specific viscosities in order to mimic the biofilm extracellular polymeric matrix. A viscous environment was observed to decrease the vancomycin susceptibility of planktonic S. aureus to levels seen for biofilms. Both planktonic S. aureus at a viscosity of 100 mPa s and staphylococcal biofilms were able to survive at >500 times the levels of the antibiotic effective against planktonic populations in standard medium. Time-dependent and dose-dependent viability curves revealed that more than one mechanism was involved in high S. aureus tolerance to vancomycin in viscous media. Increased viscosity affects antibiotic susceptibility by reducing diffusion and the mass transfer rate; this mechanism alone, however, cannot explain the increased tolerance demonstrated by S. aureus in viscous media, suggesting that viscosity may also alter the phenotype of the planktonic bacteria to one more resistant to antimicrobials, as seen in biofilms. However, these latter changes are not yet understood and will require further study.     

Shaping the Growth Behaviour of Biofilms Initiated from Bacterial Aggregates

Bacterial biofilms are usually assumed to originate from individual cells deposited on a surface. However, many biofilm-forming bacteria tend to aggregate in the planktonic phase so that it is possible that many natural and infectious biofilms originate wholly or partially from pre-formed cell aggregates. Here, we use agent-based computer simulations to investigate the role of pre-formed aggregates in biofilm development. Focusing on the initial shape the aggregate forms on the surface, we find that the degree of spreading of an aggregate on a surface can play an important role in determining its eventual fate during biofilm development. Specifically, initially spread aggregates perform better when competition with surrounding unaggregated bacterial cells is low, while initially rounded aggregates perform better when competition with surrounding unaggregated cells is high. These contrasting outcomes are governed by a trade-off between aggregate surface area and height. Our results provide new insight into biofilm formation and development, and reveal new factors that may be at play in the social evolution of biofilm communities.     

Erosion from Staphylococcus aureus biofilms grown under physiologically relevant fluid shear forces yields bacterial cells with reduced avidity to collagen

An estimated 65% of infective diseases are associated with the presence of bacterial biofilms. Biofilm-issued planktonic cells promote blood-borne, secondary sites of infection by the inoculation of the infected sites with bacteria from the intravascular space. To investigate the potential role of early detachment events in initiating secondary infections, we studied the phenotypic attributes of Staphylococcus aureus planktonic cells eroding from biofilms with respect to expression of the collagen adhesin, CNA. The collagen-binding abilities of S. aureus have been correlated to the development of osteomyelitis and septic arthritis. In this study, we focused on the impact of CNA expression on S. aureus adhesion to immobilized collagen in vitro under physiologically relevant shear forces. In contrast to the growth phase-dependent adhesion properties characteristic of S. aureus cells grown in suspension, eroding planktonic cells expressed invariant and lower effective adhesion rates regardless of the age of the biofilm from which they originated. These results correlated directly with the surface expression level of CNA. However, subsequent analysis revealed no qualitative differences between biofilms initiated with suspension cells and secondary biofilms initiated with biofilm-shed planktonic cells. Taken together, our findings suggest that, despite their low levels of CNA expression, S. aureus planktonic cells shed from biofilms retain the capacity for metastatic spread and the initiation of secondary infection. These findings demonstrate the need for a better understanding of the phenotypic properties of eroding planktonic cells, which could lead to new therapeutic strategies to target secondary infections.     

Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background

Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods

Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results

Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion

We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.     

Application of a bacteriophage lysin to disrupt biofilms formed by the animal pathogen Streptococcus suis

Bacterial biofilms are crucial to the pathogenesis of many important infections and are difficult to eradicate. Streptococcus suis is an important pathogen of pigs, and here the biofilm-forming ability of 32 strains of this species was determined. Significant biofilms were completely formed by 10 of the strains after 60 h of incubation, with exopolysaccharide production in the biofilm significantly higher than that in the corresponding planktonic cultures. S. suis strain SS2-4 formed a dense biofilm, as revealed by scanning electron microscopy, and in this state exhibited increased resistance to a number of antibiotics (ampicillin, amoxicillin, ciprofloxacin, kanamycin, and rifampin) compared to that of planktonic cultures. A bacteriophage lysin, designated LySMP, was used to attack biofilms alone and in combination with antibiotics and bacteriophage. The results demonstrated that the biofilms formed by S. suis, especially strains SS2-4 and SS2-H, could be dispersed by LySMP and with >80% removal compared to a biofilm reduction by treatment with either antibiotics or bacteriophage alone of less than 20%; in addition to disruption of the biofilm structure, the S. suis cells themselves were inactivated by LySMP. The efficacy of LySMP was not dose dependent, and in combination with antibiotics, it acted synergistically to maximize dispersal of the S. suis biofilm and inactivate the released cells. These data suggest that bacteriophage lysin could form part of an effective strategy to treat S. suis infections and represents a new class of antibiofilm agents.     

The Pseudomonas aeruginosa Type III Translocon Is Required for Biofilm Formation at the Epithelial Barrier

Clinical infections by Pseudomonas aeruginosa, a deadly Gram-negative, opportunistic pathogen of immunocompromised hosts, often involve the formation of antibiotic-resistant biofilms. Although biofilm formation has been extensively studied in vitro on glass or plastic surfaces, much less is known about biofilm formation at the epithelial barrier. We have previously shown that when added to the apical surface of polarized epithelial cells, P. aeruginosa rapidly forms cell-associated aggregates within 60 minutes of infection. By confocal microscopy we now show that cell-associated aggregates exhibit key characteristics of biofilms, including the presence of extracellular matrix and increased resistance to antibiotics compared to planktonic bacteria. Using isogenic mutants in the type III secretion system, we found that the translocon, but not the effectors themselves, were required for cell-associated aggregation on the surface of polarized epithelial cells and at early time points in a murine model of acute pneumonia. In contrast, the translocon was not required for aggregation on abiotic surfaces, suggesting a novel function for the type III secretion system during cell-associated aggregation. Supernatants from epithelial cells infected with wild-type bacteria or from cells treated with the pore-forming toxin streptolysin O could rescue aggregate formation in a type III secretion mutant, indicating that cell-associated aggregation requires one or more host cell factors. Our results suggest a previously unappreciated function for the type III translocon in the formation of P. aeruginosa biofilms at the epithelial barrier and demonstrate that biofilms may form at early time points of infection.     

Aloe-emodin inhibits Staphylococcus aureus biofilms and extracellular protein production at the initial adhesion stage of biofilm development

Staphylococcus aureus (S. aureus) biofilms are clinically serious and play a critical role in the persistence of chronic infections due to their ability to resist antibiotics. The inhibition of biofilm formation is viewed as a new strategy for the prevention of S. aureus infections. Here, we demonstrated that minimum inhibitory concentrations (MICs) of aloe-emodin exhibited no bactericidal activity against S. aureus but affected S. aureus biofilm development in a dose-dependent manner. Further studies indicated that aloe-emodin specifically inhibits the initial adhesion and proliferation stages of S. aureus biofilm development. Scanning electron microscopy (SEM) indicated that the S. aureus ATCC29213 biofilm extracellular matrix is mainly composed of protein. Laser scanning confocal microscope assays revealed that aloe-emodin treatment primarily inhibited extracellular protein production. Moreover, the Congo red assay showed that aloe-emodin also reduced the accumulation of polysaccharide intercellular adhesin (PIA) on the cell surface. These findings will provide new insights into the mode of action of aloe-emodin in the treatment of infections by S. aureus biofilms.     

Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biofilms

AIMS: The objective of this study was to investigate the antagonistic interactions between bacteriocin-producing enteric bacteria in dual species biofilms and the interspecies interactions correlated with sensitivity to biocides. METHODS AND RESULTS: When compared with their single species counterparts, the dual species biofilms formed by bacteriocin-producing strains exhibited a decrease in biofilm size and an increase in sensitivity to the antimicrobial agents hypochlorite, triclosan and benzalkonium chloride. The five dual species biofilms studied all resulted in biofilms containing a mixture of the two strains. This was attributed to the spatial distribution of cells within the biofilm, with each strain forming its own microcolonies. The production of a bacteriocin also gave a strain a competitive advantage when interacting with a bacteriocin-sensitive strain within a biofilm, both in gaining a foothold in a new environment and in preventing the colonization of a potential competitor into a pre-established biofilm. CONCLUSIONS: It was concluded that bacteriocins might be used specifically for interacting with competing strains within a biofilm, as opposed to a planktonic, environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike planktonically grown bacteriocin-producing populations, where one strain will always be out-competed, bacteriocin-producing and bacteriocin-sensitive strains can coexist in biofilm communities, clearly demonstrating major differences between biofilm and planktonic competition. This paper highlights the importance of bacteriocin production in the development of biofilm communities.     

Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations.     

Pseudomonas aeruginosa PAO1 Preferentially Grows as Aggregates in Liquid Batch Cultures and Disperses upon Starvation

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.     

Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3–10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.     

Antibiotic resistance of biofilms

Microbial biofilms are notably recalcitrant towards treatment with antibiotics, biocides or disinfectants that would adequately control the same organisms growing in planktonic mode. Much of this resistance has been attributed to an organisation of the biofilm cells within exopolymer matrices. Whilst such exopolymers are unlikely to hinder the diffusion and access of antimicrobial agents to the underlying cells, they will chemically quench reactive biocides such as chlorine and peroxygens, and bind highly charged antibiotics, such as tobramycin and gentamycin, thereby providing some protection to the more deep lying cells. Extracellular enzymes, bound within the glycocalyx and able to degrade the treatment agents, will further reduce the access of susceptible compounds. Diffusion limitation however, is unlikely to be the sole moderator of the resistance properties of microbial biofilms. In addition, gradients of oxygen and nutrients established across the biofilm community will cause growth rates to be much reduced at points remoted from the accessible nutrient. Slow growth rates, and the associated induction of stringent responses further contribute towards this resistance. Finally, there have been recent demonstrations that attachment of microorganisms to surfaces promotes the expression of genes that are not normally expressed in planktonic culture. Whether or not the expression of such genes alters the phenotype in a manner which alters the response of the cells to antimicrobial agents remains to be demonstrated.     

Phage release from biofilm and planktonic Staphylococcus aureus cells

The ability of pathogenic staphylococci to form biofilms facilitates colonization and the development of chronic infections. Therapy is hampered by the high tolerance of biofilms towards antibiotic treatment and the immune system. We found evidence that lysogenic Staphylococcus aureus cells in a biofilm and in planktonic cultures spontaneously release phages into their surroundings. Phages were detected over a much longer period in biofilm cultures than in planktonic supernatants because the latter were degraded by secreted proteases. Phage release in planktonic and biofilm cultures was artificially increased by adding mitomycin C. Two morphologically distinct phages in the S. aureus strain used in this work were observed by electron microscopy. We postulate that phage-release is a frequent event in biofilms. The resulting lysis of cells in a biofilm might promote the persistence and survival of the remaining cells, as they gain a nutrient reservoir from their dead and lysed neighboring cells. This might therefore be an early differentiation and apoptotic mechanism.     

Antibiotic resistance of biofilms

Microbial biofilms are notably recalcitrant towards treatment with antibiotics, biocides or disinfectants that would adequately control the same organisms growing in planktonic mode. Much of this resistance has been attributed to an organisation of the biofilm cells within exopolymer matrices. Whilst such exopolymers are unlikely to hinder the diffusion and access of antimicrobial agents to the underlying cells, they will chemically quench reactive biocides such as chlorine and peroxygens, and bind highly charged antibiotics, such as tobramycin and gentamycin, thereby providing some protection to the more deep lying cells. Extracellular enzymes, bound within the glycocalyx and able to degrade the treatment agents, will further reduce the access of susceptible compounds. Diffusion limitation however, is unlikely to be the sole moderator of the resistance properties of microbial biofilms. In addition, gradients of oxygen and nutrients established across the biofilm community will cause growth rates to be much reduced at points remoted from the accessible nutrient. Slow growth rates, and the associated induction of stringent responses further contribute towards this resistance. Finally, there have been recent demonstrations that attachment of microorganisms to surfaces promotes the expression of genes that are not normally expressed in planktonic culture. Whether or not the expression of such genes alters the phenotype in a manner which alters the response of the cells to antimicrobial agents remains to be demonstrated.     

Characterization and gene cloning of a novel serine protease with nematicidal activity from Trichoderma pseudokoningii SMF2

Pseudomonas aeruginosa is a pathogenic bacterium widely investigated for its high incidence in clinical environments and its ability to form strong biofilms. During biofilm development, sessile cells acquire physiological characteristics differentiating them from planktonic cells. But after treatment with disinfectants, or to ensure survival of the species in hostile environments, biofilm cells can detach. This complicates disinfection procedures. This study aimed to physiologically characterize cells detached from a P. aeruginosa biofilm and to compare them with their sessile and planktonic counterparts. We first tested planktonic growth kinetics and capacities to form new biofilms. Then we investigated cell-surface properties. And finally, we tested in vitro susceptibility to antibiotics. The results first indicated that sessile and detached cells have similar planktonic growth kinetics and cell-surface properties, distinguishable from those of planktonic cells. Interestingly, the three populations exhibited different biofilm-forming capacities, suggesting that there is a transitional phenotype between sessile and planktonic states, at least during the first hours following cell detachment. It is important to consider this observation when developing treatments to optimize disinfection processes. Surprisingly, the three populations showed the same antibiotic susceptibility profile.     

细菌生物膜研究技术

细菌生物膜是细菌生长过程中为适应生存环境而在固体表面上生长的一种与游走态细胞相对应的存在形式。只要条件允许,绝大多数细菌都可以形成生物膜。一旦形成了生物膜细菌就具有极强的耐药性,在医疗、食品、工业、军事等诸多领域给人类社会带来了严重的危害,造成巨大的经济损失。因此,细菌生物膜已成为全球关注的重大难题,也是目前科学界研究的前沿和热点。本文结合细菌生物膜研究技术的最新进展,重点介绍了几种常用生物膜发生装置及检测量化技术,并对其原理及优缺点进行了讨论。     

Study of the structures of biofilms formed by <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> bacteria on abiotic surfaces by the methods of light and transmission electron microscopy

Process of biofilm formation by different Salmonella strains on abiotic surfaces has been studied. Differences in the structural organization were revealed by the analysis of the fine cell structure in the planktonic and biofilm cultures. It was shown, by the methods of light and electron microscopy and also by cytochemistry, that the two strains share similarities in structure and have individual features. Differences in the density of the extracellular matrix and the sizes of cell aggregates were established. The stages in the processes of growth and death of biofilms were demonstrated by the investigation of the process dynamics. The signs of aging in biofilms were disorganization of extracellular matrix and appearance of the planktonic cells. Microscopic and cytochemical methods used in this work were recommended for the investigation of the effects of various biocide agents on biofilms.