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Biofilm and Chronic Typhoid Carriers with Special Reference to Bacteriophage Therapy
... The formation of biofilms by probiotic bacteria is considered advantageous as it promotes colonization and longer residence in the host's mucosa, preventing colonization by pathogenic bacteria (Terraf et al., 2012). Biofilms formed by various organisms typically consist of exopolysaccharides (EPS), extracellular proteins, DNA, and lipids (Yadav et al., 2021). Exopolysaccharides have been found to effectively biosorb heavy metals from water and food products (Singh, Saini, Srivastava, & Kushwaha, 2021). ...
Heavy metals represent significant environmental pollutants in today's world. The primary sources of human exposure to these heavy metals are through food and water, which can lead to various life‐threatening diseases. Therefore, it is imperative to focus on the removal of these contaminants to safeguard human health. The study describes the optimization of biosorption conditions of chromium [Cr(III)] and zinc [Zn(II)] through biofilm and exopolysaacharides (EPS) of Lactobacillus plantarum. The strain was tested for minimum inhibitory concentration and assessed at 100–350 ppm for Cr (III) and Zn (II) in terms of log cfu/mL. Metal adsorption capacity of biofilm was slightly higher as compared to EPS. Biosorption of Cr (III) at pH 2 showed 90% biosorption through biofilms and 88% through EPS. Zn (II) showed maximum binding of about 86% and 78% by biofilm and EPS, respectively, at pH 8 at 37°C for 24 h. The binding of metal is mostly dependent upon the functional group present, the surface morphology of biofilm, and the EPS of strain.
Microbial biofilms are communities of aggregated microbial cells embedded in a self-produced matrix of extracellular polymeric substances (EPS). Biofilms are recalcitrant to extreme environments, and can protect microorganisms from ultraviolet (UV) radiation, extreme temperature, extreme pH, high salinity, high pressure, poor nutrients, antibiotics, etc., by acting as “protective clothing”. In recent years, research works on biofilms have been mainly focused on biofilm-associated infections and strategies for combating microbial biofilms. In this review, we focus instead on the contemporary perspectives of biofilm formation in extreme environments, and describe the fundamental roles of biofilm in protecting microbial exposure to extreme environmental stresses and the regulatory factors involved in biofilm formation. Understanding the mechanisms of biofilm formation in extreme environments is essential for the employment of beneficial microorganisms and prevention of harmful microorganisms.
With the emerging threat of infections caused by multidrug resistant bacteria, phages have been reconsidered as an alternative for treating infections caused by tenacious pathogens. However, instead of replacing antibiotics, the combination of both types of antimicrobials can be superior over the use of single agents. Enhanced bacterial suppression, more efficient penetration into biofilms, and lowered chances for the emergence of phage resistance are the likely advantages of the combined strategy. While a number of studies have provided experimental evidence in support of this concept, negative interference between phages and antibiotics have been reported as well. Neutral effects have also been observed, but in those cases, combined approaches may still be important for at least hampering the development of resistance. In any case, the choice of phage type and antibiotic as well as their mixing ratios must be given careful consideration when deciding for a dual antibacterial approach. The most frequently tested bacterium for a combined antibacterial treatment has been Pseudomonas aeruginosa, but encouraging results have also been reported for Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, and Burkholderia cepacia. Given the immense play area of conceivable phage-antibiotic combinations and their potential excess value, it is time to recapitulate of what has been achieved so far. This review therefore gathers and compares the results from most relevant studies in order to help researchers and clinicians in their strategies to combat multidrug resistant bacteria. Special attention is given to the selected bacterial model organisms, the phage families and genera employed, and the experimental design and evaluation (e.g., in vitro vs. in vivo models, biofilm vs. planktonic culture experiments, order and frequency of administration etc.). The presented data may serve as a framework for directed further experimental approaches to ultimately achieve a resolute challenge of multidrug resistant bacteria based on traditional antibiotics and phages.
Antibiotic resistance is arguably the biggest current threat to global health. An increasing number of infections are becoming harder or almost impossible to treat, carrying high morbidity, mortality, and financial cost. The therapeutic use of bacteriophages, viruses that infect and kill bacteria, is well suited to be part of the multidimensional strategies to combat antibiotic resistance. Although phage therapy was first implemented almost a century ago, it was brought to a standstill after the successful introduction of antibiotics. Now, with the rise of antibiotic resistance, phage therapy is experiencing a well-deserved rebirth. Among the admittedly vast literature recently published on this topic, this review aims to provide a forward-looking perspective on phage therapy and its role in modern society. We cover the key points of the antibiotic resistance crisis and then explain the biological and evolutionary principles that support the use of phages, their interaction with the immune system, and a comparison with antibiotic therapy. By going through up-to-date reports and, whenever possible, human clinical trials, we examine the versatility of phage therapy. We discuss conventional approaches as well as novel strategies, including the use of phage-antibiotic combinations, phage-derived enzymes, exploitation of phage resistance mechanisms, and phage bioengineering. Finally, we discuss the benefits of phage therapy beyond the clinical perspective, including opportunities for scientific outreach and effective education, interdisciplinary collaboration, cultural and economic growth, and even innovative use of social media, making the case that phage therapy is more than just an alternative to antibiotics.
Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.
Bacteriophages (phages) are viruses that infect bacteria. The “predator–prey” interactions are recognized as a potentially effective way to treat infections. Phages, as well as phage-derived proteins, especially enzymes, are intensively studied to become future alternative or supportive antibacterials used alone or in combination with standard antibiotic regimens treatment. There are many publications presenting phage therapy aspects, and some papers focused separately on the application of phage-derived enzymes. In this review, we discuss advantages and limitations of both agents concerning their specificity, mode of action, structural issues, resistance development, pharmacokinetics, product preparation, and interactions with the immune system. Finally, we describe the current regulations for phage-based product application.
Typhoid fever is caused by the human-restricted pathogen Salmonella enterica sv. Typhi. Approximately 5% of people that resolve the disease become chronic carriers, with the gallbladder as the main reservoir of the bacteria. Of these, about 90% present with gallstones, on which Salmonella form biofilms. Because S. Typhi is a human-restricted pathogen, these carriers are the main source of dissemination of the disease; unfortunately, antibiotic treatment has shown to be an ineffective therapy. This is believed to be caused by the inherent antibiotic resistance conferred by Salmonella biofilms growing on gallstones. The gallstone mouse model with S. Typhimurium has proven to be an excellent surrogate for S. Typhi chronic infection. In this study, we test the hypothesis that the biofilm state confers Salmonella with the increased resistance to antibiotics observed in cases of chronic carriage. We found that, in the biofilm state, Salmonella is significantly more resistant to ciprofloxacin, a common antibiotic used for the treatment of Salmonella, both in vitro (p < 0.001 for both S. Typhi and S. Typhimurium with respect to planktonic cells) and in vivo (p = 0.0035 with respect to control mice).
Many bacteria are adapted for attaching to surfaces and for building complex communities, termed biofilms. The biofilm mode of life is predominant in bacterial ecology. So too is the exposure of bacteria to ubiquitous viral pathogens, termed bacteriophages. Although biofilm–phage encounters are likely to be common in nature, little is known about how phages might interact with biofilm-dwelling bacteria. It is also unclear how the ecological dynamics of phages and their hosts depend on the biological and physical properties of the biofilm environment. To make headway in this area, we develop a biofilm simulation framework that captures key mechanistic features of biofilm growth and phage infection. Using these simulations, we find that the equilibrium state of interaction between biofilms and phages is governed largely by nutrient availability to biofilms, infection likelihood per host encounter and the ability of phages to diffuse through biofilm populations. Interactions between the biofilm matrix and phage particles are thus likely to be of fundamental importance, controlling the extent to which bacteria and phages can coexist in natural contexts. Our results open avenues to new questions of host–parasite coevolution and horizontal gene transfer in spatially structured biofilm contexts.
Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.
With an increase in cases of multidrug-resistant Pseudomonas aeruginosa, alternative and adjunct treatments are needed, leading to renewed interest in bacteriophage therapy. There have been few clinically relevant studies of phage therapy against chronic lung infections. Using a novel murine model that uses a natural respiratory inhalation route of infection, we show that phage therapy is an effective treatment against chronic P. aeruginosa lung infections. We also show efficacy against P. aeruginosa in a biofilm-associated cystic fibrosis lung-like environment. These studies demonstrate the potential for phage therapy in the treatment of established and recalcitrant chronic respiratory tract infections.
Ulcerative colitis (UC) is characterized by presence of ulcer in colon and bloody diarrhea. The present study explores the possibility of association between
Salmonella
and ulcerative colitis. The present study comprised 59 cases of UC, 28 of colon cancer (CC), 127 of irritable bowel syndrome (IBS), and 190 of healthy control. The serological study was done by Widal and Indirect Haemagglutination Assay (IHA) for ViAb. Nested PCR was performed targeting
fliC
,
staA,
and
stkG
gene for Typhi and Paratyphi A, respectively. A total of 15.3% patients were positive for
Salmonella
“O” antigen among them 18.6% UC, 35.5% CC, 12.6% IBS, and 15.3% healthy control. A total of 36.9% patients were positive for “H” antigen including 39.0%, 57.1%, and 67.7% UC, CC, and IBS, respectively. About 1.73% show positive agglutination for AH antigen including 3.4%, 3.6%, and 1.6%, UC, CC, and IBS. A total of 10.89% were positive for ViAb. While 6.8% of UC, 10.7% of CC, 11.0% of IBS, and 12.1% of healthy subjects were positive for the antibody, the PCR positivity rates for
Salmonella
specific sequences were 79.7% in UC, 53.6% in CC, 66.1% in IBS, and 16.3% in healthy controls. The present study suggested that higher prevalence of
Salmonella
might play important role in etiopathogenesis of UC, IBS, and CC.
Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.
Bacteriophages are bacterial viruses, capable of killing even multi-drug resistant bacterial cells. For this reason, therapeutic use of phages is considered as a possible alternative to conventional antibiotics. However, phages are very host specific in comparison to wide-spectrum antibiotics and thus preparation of phage-cocktails beforehand against pathogens can be difficult. In this study, we evaluate whether it may be possible to isolate phages on-demand from environmental reservoir. We attempted to enrich infectious bacteriophages from sewage against nosocomial drug-resistant bacterial strains of different medically important species in order to evaluate the probability of discovering novel therapeutic phages. Stability and host-range were determined for the acquired phages. Our results suggest that on-demand isolation of phages is possible against Pseudomonas aeruginosa, Salmonella and extended spectrum beta-lactamase Escherichia coli and Klebsiella pneumoniae. The probability of finding suitable phages was less than 40% against vancomycin resistant Enterococcus and Acinetobacter baumannii strains. Furthermore, isolation of new phages against methicillin resistant Staphylococcus aureus strains was found to be very difficult.
Carcinoma of the gallbladder (CaGB) is the fifth commonest gastrointestinal tract cancer and is endemic in several countries. The interplay of genetic susceptibility, infections, and life style factors has been proposed to be responsible for carcinogenesis of gallbladder. Persistence of infection leading to chronic inflammation, and production of certain toxins and metabolites with carcinogenic potentials, by certain bacteria has been speculated to be involved in the transformation of the gallbladder epithelium. Therefore, any bacteria that have evolved to acquire both of the above carcinogenic mechanisms can cause cancer. Salmonella typhi has been found to be prominently associated with CaGB. Chronic typhoid carriage (persistence) and production of mediators of chronic infl ammation and a genotoxic toxin (cytotoxic distending toxin, CdtB) are also known for this bacterium. Furthermore, the natural concentrating function of the gallbladder might amplify the carcinogenic effect of the mediators of carcinogenesis. In addition to S. typhi, certain species of Helicobacter (H. bilis and H. hepaticus) and Escherichia coli have also been implicated in carcinogenesis. As the isolation rate is verypoor with the presently available culture techniques, the existence of bacteria in a viable but non-cultivable state is quite likely; therefore, sensitive and specif ic molecular techniques might reveal the etiological role of bacterial infection in gallbladder carcinogenesis. If bacteria are found to be causing cancers, then eradication of such infections might help in reducing the incidence of some cancers.
Studies of antagonistic coevolution between hosts and parasites typically focus on resistance and infectivity traits. However, coevolution could also have genome-wide effects on the hosts due to pleiotropy, epistasis or selection for evolvability. Here we investigate these effects in the bacterium Pseudomonas fluorescens SBW25 during ~400 generations of evolution in the presence or absence of bacteriophage (coevolution or evolution treatments respectively). Coevolution resulted in variable phage resistance, lower competitive fitness in the absence of phages, and greater genome-wide divergence both from the ancestor and between replicates, in part due to the evolution of increased mutation rates. Hosts from coevolution and evolution treatments had different suites of mutations. A high proportion of mutations observed in coevolved hosts were associated with a known phage target binding site, the Lipopolysaccharide (LPS), and correlated with altered LPS length and phage resistance. Mutations in evolved bacteria were correlated with higher fitness in the absence of phages. However, the benefits of these growth-promoting mutations were completely lost when these bacteria were subsequently coevolved with phages, indicating that they were not beneficial in the presence of resistance mutations (consistent with negative epistasis). Our results show that in addition to affecting genome-wide evolution in loci not obviously linked to parasite resistance, coevolution can also constrain the acquisition of mutations beneficial for growth in the abiotic environment.
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Bacteriophage infection and antibiotics used individually to reduce biofilm mass often result in the emergence of significant levels of phage and antibiotic resistant cells. In contrast, combination therapy in Escherichia coli biofilms employing T4 phage and tobramycin resulted in greater than 99% and 39% reduction in antibiotic and phage resistant cells, respectively. In P. aeruginosa biofilms, combination therapy resulted in a 60% and 99% reduction in antibiotic and PB-1 phage resistant cells, respectively. Although the combined treatment resulted in greater reduction of E. coli CFUs compared to the use of antibiotic alone, infection of P. aeruginosa biofilms with PB-1 in the presence of tobramycin was only as effective in the reduction of CFUs as the use of antibiotic alone. The study demonstrated phage infection in combination with tobramycin can significantly reduce the emergence of antibiotic and phage resistant cells in both E. coli and P. aeruginosa biofilms, however, a reduction in biomass was dependent on the phage-host system.
The global threat to public health posed by emerging multidrug-resistant bacteria in the past few years necessitates the development of novel approaches to combat bacterial infections. Endolysins encoded by bacterial viruses (or phages) represent one promising avenue of investigation. These enzyme-based antibacterials efficiently kill Gram-positive bacteria upon contact by specific cell wall hydrolysis. However, a major hurdle in their exploitation as antibacterials against Gram-negative pathogens is the impermeable lipopolysaccharide layer surrounding their cell wall. Therefore, we developed and optimized an approach to engineer these enzymes as outer membrane-penetrating endolysins (Artilysins), rendering them highly bactericidal against Gram-negative pathogens, including Pseudomonas aeruginosa and Acinetobacter baumannii. Artilysins combining a polycationic nonapeptide and a modular endolysin are able to kill these (multidrug-resistant) strains in vitro with a 4 to 5 log reduction within 30 min. We show that the activity of Artilysins can be further enhanced by the presence of a linker of increasing length between the peptide and endolysin or by a combination of both polycationic and hydrophobic/amphipathic peptides. Time-lapse microscopy confirmed the mode of action of polycationic Artilysins, showing that they pass the outer membrane to degrade the peptidoglycan with subsequent cell lysis. Artilysins are effective in vitro (human keratinocytes) and in vivo (Caenorhabditis elegans).
IMPORTANCE Bacterial resistance to most commonly used antibiotics is a major challenge of the 21st century. Infections that cannot be treated by first-line antibiotics lead to increasing morbidity and mortality, while millions of dollars are spent each year by health care systems in trying to control antibiotic-resistant bacteria and to prevent cross-transmission of resistance. Endolysins-enzymes derived from bacterial viruses-represent a completely novel, promising class of antibacterials based on cell wall hydrolysis. Specifically, they are active against Gram-positive species, which lack a protective outer membrane and which have a low probability of resistance development. We modified endolysins by protein engineering to create Artilysins that are able to pass the outer membrane and become active against Pseudomonas aeruginosa and Acinetobacter baumannii, two of the most hazardous drug-resistant Gram-negative pathogens.
Millions of indwelling devices are implanted in patients every year, and staphylococci (S. aureus, MRSA and vancomycin-resistant S. aureus (VRSA)) are responsible for a majority of infections associated with these devices, thereby leading to treatment failures. Once established, staphylococcal biofilms become resistant to antimicrobial treatment and host response, thereby serving as the etiological agent for recurrent infections. This study investigated the efficacy of octenidine hydrochloride (OH) for inhibiting biofilm synthesis and inactivating fully-formed staphylococcal biofilm on different matrices in the presence and absence of serum protein. Polystyrene plates and stainless steel coupons inoculated with S. aureus, MRSA or VRSA were treated with OH (zero, 0.5, one, 2 mM) at 37 °C for the prevention of biofilm formation. Additionally, the antibiofilm effect of OH (zero, 2.5, five, 10 mM) on fully-formed staphylococcal biofilms on polystyrene plates, stainless steel coupons and urinary catheters was investigated. OH was effective in rapidly inactivating planktonic and biofilm cells of S. aureus, MRSA and VRSA on polystyrene plates, stainless steel coupons and urinary catheters in the presence and absence of serum proteins. The use of two and 10 mM OH completely inactivated S. aureus planktonic cells and biofilm (>6.0 log reduction) on all matrices tested immediately upon exposure. Further, confocal imaging revealed the presence of dead cells and loss in biofilm architecture in the OH-treated samples when compared to intact live biofilm in the control. Results suggest that OH could be applied as an effective antimicrobial to control biofilms of S. aureus, MRSA and VRSA on appropriate hospital surfaces and indwelling devices.
Objectives:
Streptococcus pyogenes, or Group A streptococcus (GAS), has a propensity to colonize human tissues and form biofilms. Significantly, these biofilms are a contributing mechanism of antibiotic treatment failure in streptococcal disease. In this study, we evaluate a streptococcal-specific bacteriophage-encoded endolysin (PlyC), which is known to lyse planktonic streptococci, on both static and dynamic streptococcal biofilms.
Methods:
PlyC was benchmarked against antibiotics for MIC, MBC and minimum biofilm eradication concentration (MBEC). A biomass eradication assay based on crystal violet staining of the biofilm matrix was also used to quantify the anti-biofilm properties of PlyC. Finally, conventional fluorescence microscopy and laser scanning confocal microscopy were used to study the effects of PlyC on static and dynamic biofilms of GAS.
Results:
PlyC and antibiotics had similar MIC (range 0.02-0.08 mg/L) and MBC (range 0.02-1.25 mg/L) values on planktonic GAS. However, when GAS grew in biofilms, the MBEC values for antibiotics rose to clinically resistant values (≥400 mg/L) whereas PlyC had MBEC values two orders of magnitude lower by mass and four orders of magnitude lower by molarity than the conventional antibiotics. Laser scanning confocal microscopy revealed that PlyC destroys the biofilm as it diffuses through the matrix in a time-dependent fashion.
Conclusions:
Our findings indicate that while streptococcal cells within a biofilm rapidly become refractory to traditional antibiotics, the biofilm matrix is readily destroyed by the lytic actions of PlyC.
We report here the complete genome sequence of Salmonella enterica subsp. enterica serovar Typhi P-stx-12, a clinical isolate obtained from a typhoid carrier in India.
This study evaluates the occurrence of bacteriophages carrying antibiotic resistance genes in animal environments. blaTEM, blaCTX-M (clusters 1 and 9), and mecA were quantified by quantitative PCR in 71 phage DNA samples from pigs, poultry, and cattle fecal wastes. Densities of 3 to
4 log10 gene copies (GC) of blaTEM, 2 to 3 log10 GC of blaCTX-M, and 1 to 3 log10 GC of mecA per milliliter or gram of sample were detected, suggesting that bacteriophages can be environmental vectors for the horizontal
transfer of antibiotic resistance genes.
Aspergillus fumigatus is an increasingly prevalent opportunistic fungal pathogen of various immuno-compromised individuals. It has the ability to filament within the lungs forming dense intertwined mycelial balls. These morphological characteristics resemble those of microbial biofilms, which are matrix enclosed microbial populations, adherent to each other and/or to surfaces or interfaces. The purpose of this paper is to review some recent experiments that indicate the potential biofilm forming capacity of A. fumigatus in vitro. Initially it was established that conidial seeding density is important for stable biofilm development. In the optimized model conidial germination and filamentous growth characteristics were not observed until 8 h, after which a multi-cellular population expanded exponentially forming a thick structure (approx. 250 microm). Calcofluor white staining of this revealed the presence of extracellular polymeric matrix material, which increased as the biofilm matured. Subsequent antifungal sensitivity testing of this structure showed that azoles, polyenes and echinocandins were ineffective in reducing the cellular viability at therapeutically attainable concentrations. Microarray and real-time PCR analysis demonstrated the up-regulation of AfuMDR4 during multicellular growth and development, which may account the recalcitrance observed. Overall, A. fumigatus appears to possess the classical elements of biofilm growth, namely multicellularity, matrix production and sessile resistance. This controversial approach to understanding the biology of A. fumigatus infection may provide crucial information on how to treat this pathogenic fungus more effectively.
Formation of a protected biofilm environment is recognized as one of the major causes of the increasing antibiotic resistance development and emphasizes the need to develop alternative antibacterial strategies, like phage therapy. This study investigates the in vitro degradation of single-species Pseudomonas putida biofilms, PpG1 and RD5PR2, by the novel phage ϕ15, a 'T7-like virus' with a virion-associated exopolysaccharide (EPS) depolymerase. Phage ϕ15 forms plaques surrounded by growing opaque halo zones, indicative for EPS degradation, on seven out of 53 P. putida strains. The absence of haloes on infection resistant strains suggests that the EPS probably act as a primary bacterial receptor for phage infection. Independent of bacterial strain or biofilm age, a time and dose dependent response of ϕ15-mediated biofilm degradation was observed with generally a maximum biofilm degradation 8 h after addition of the higher phage doses (10(4) and 10(6) pfu) and resistance development after 24 h. Biofilm age, an in vivo very variable parameter, reduced markedly phage-mediated degradation of PpG1 biofilms, while degradation of RD5PR2 biofilms and ϕ15 amplification were unaffected. Killing of the planktonic culture occurred in parallel with but was always more pronounced than biofilm degradation, accentuating the need for evaluating phages for therapeutic purposes in biofilm conditions. EPS degrading activity of recombinantly expressed viral tail spike was confirmed by capsule staining. These data suggests that the addition of high initial titers of specifically selected phages with a proper EPS depolymerase are crucial criteria in the development of phage therapy.
Bacterial exopolysaccharides have always been suggested to play crucial roles in the bacterial initial adhesion and the development of complex architecture in the later stages of bacterial biofilm formation. However, Escherichia coli group II capsular polysaccharide was characterized to exert broad-spectrum biofilm inhibition activity. In this study, we firstly reported that a bacterial exopolysaccharide (A101) not only inhibits biofilm formation of many bacteria but also disrupts established biofilm of some strains. A101 with an average molecular weight of up to 546 KDa, was isolated and purified from the culture supernatant of the marine bacterium Vibrio sp. QY101 by ethanol precipitation, iron-exchange chromatography and gel filtration chromatography. High performance liquid chromatography traces of the hydrolyzed polysaccharides showed that A101 is primarily consisted of galacturonic acid, glucuronic acid, rhamnose and glucosamine. A101 was demonstrated to inhibit biofilm formation by a wide range of Gram-negative and Gram-positive bacteria without antibacterial activity. Furthermore, A101 displayed a significant disruption on the established biofilm produced by Pseudomonas aeruginosa, but not by Staphylococcus aureus. Importantly, A101 increased the aminoglycosides antibiotics' capability of killing P. aeruginosa biofilm. Cell primary attachment to surfaces and intercellular aggregates assays suggested that A101 inhibited cell aggregates of both P. aeruginosa and S. aureus, while the cell-surface interactions inhibition only occurred in S. aureus, and the pre-formed cell aggregates dispersion induced by A101 only occurred in P. aeruginosa. Taken together, these data identify the antibiofilm activity of A101, which may make it potential in the design of new therapeutic strategies for bacterial biofilm-associated infections and limiting biofilm formation on medical indwelling devices. The found of A101 antibiofilm activity may also promote a new recognition about the functions of bacterial exopolysaccharides.
The microorganisms in biofilms live in a self-produced matrix of hydrated extracellular polymeric substances (EPS) that form their immediate environment. EPS are mainly polysaccharides, proteins, nucleic acids and lipids; they provide the mechanical stability of biofilms, mediate their adhesion to surfaces and form a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells. In addition, the biofilm matrix acts as an external digestive system by keeping extracellular enzymes close to the cells, enabling them to metabolize dissolved, colloidal and solid biopolymers. Here we describe the functions, properties and constituents of the EPS matrix that make biofilms the most successful forms of life on earth.
The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter(-1) chlorine, 21 min for 50 mg liter(-1) chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.
Although well studied the association between chronic typhoid carrier state and carcinoma of the gallbladder (CaGB) remains unproven.
The study was performed at a tertiary care medical center in North India and involved 52 patients with CaGB, 223 patients with benign gallbladder diseases, 508 healthy individuals and, 424 corpses. For the detection of Salmonella enterica serovar Typhi, hepatobiliary specimens were subjected to DNA extraction for specific nested- PCR amplification of the S. Typhi flagellin gene. Anti-Vi S. Typhi antibodies were detected in serum samples from patients by indirect haemagglutination.
Thirty five of the 52 (67.3%) CaGB patients were PCR-positive for the S. Typhi flagellin gene; significantly higher than for patients with benign gallbladder diseases (95/223, 42.6%; p<0.01) and corpses (35/424, 8.2%; p<0.001). The numbers of individuals that had significant anti-Vi antibody titres (> or = 160) in their serum were 20/52 (38.5%) for CaGB patients, 31/223 (13.9%) for patients with benign gallbladder diseases, and 47/508 (9.2%) for healthy individuals.
Specific nested-PCR amplification of the S. Typhi flagellin gene in hepato-biliary specimens was more sensitive for detection of S. Typhi carriage than anti-Vi antibody titres in serum. The results demonstrate an association between typhoid carriage and gallbladder diseases, both CaGB and benign. S. Typhi specific immunosuppression is also suggested in patients with gallbladder diseases.
Staphylococcus aureus is a proficient biofilm former on host tissues and medical implants. We mutagenized S. aureus strain SH1000 to identify loci essential for ica-independent mechanisms of biofilm maturation and identified multiple insertions in the rsbUVW-sigB operon. Following construction and characterization of a sigB deletion, we determined that the biofilm phenotype was due to a lack of sigma factor B (SigB) activity. The phenotype was
conserved in a sigB mutant of USA300 strain LAC, a well-studied community-associated methicillin-resistant S. aureus isolate. We determined that agr RNAIII levels were elevated in the sigB mutants, and high levels of RNAIII expression are known to have antibiofilm effects. By introducing an agr mutation into the SH1000 or LAC sigB deletion strain, S. aureus regained biofilm capacity, indicating that the biofilm phenotype was agr dependent. Protease activity is linked to agr activity and ica-independent biofilm formation, and we observed that the protease inhibitors phenylmethylsulfonyl fluoride and α-macroglobulin
could reverse the sigB biofilm defect. Similarly, inactivating genes encoding both the aureolysin and Spl extracellular proteases in the sigB mutant restored biofilm capacity. Due to the growing link between murein hydrolase activity and biofilm maturation, autolysin
zymography was performed, which revealed an altered profile in the sigB mutant; again, the phenotype could be repaired through protease inactivation. These findings indicate that the lack of SigB
activity results in increased RNAIII expression, thus elevating extracellular protease levels and altering the murein hydrolase
activity profile. Altogether, our observations demonstrate that SigB is an essential regulator of S. aureus biofilm maturation.
Bacteria are sophisticated systems with high capacity and flexibility to adapt to various environmental conditions. Each prokaryote however possesses a defined metabolic network, which sets its overall metabolic capacity, and therefore the maximal growth rate that can be reached. To achieve optimal growth, bacteria adopt various molecular strategies to optimally adjust gene expression and optimize resource allocation according to the nutrient availability. The resulting physiological changes are often accompanied by changes in the growth rate, and by global regulation of gene expression. The growth-rate-dependent variation of the abundances in the cellular machineries, together with condition-specific regulatory mechanisms, affect RNA metabolism and fate and pose a challenge for rational gene expression reengineering of synthetic circuits.
This article is part of a Special Issue entitled: RNA and gene control in bacteria, edited by Dr. M. Guillier and F. Repoila.
Cell-free transcription-translation (TXTL) has become a highly versatile technology to construct, characterize and interrogate genetically programmed biomolecular systems implemented outside living organisms. By recapitulating gene expression in vitro, TXTL offers unparalleled flexibility to take apart, engineer and analyze quantitatively the effects of chemical, physical and genetic contexts on the function of biochemical systems, from simple regulatory elements to millimeter-scale pattern formation. Here, we review the capabilities of the current cell-free platforms for executing DNA programs in vitro. We describe the recent advances in programming using cell-free expression, a multidisciplinary playground that has enabled a myriad of novel applications in synthetic biology, biotechnology, and biological physics. Finally, we discuss the challenges and perspectives in the research area of TXTL-based constructive biology.
Nonhealing ulcers are a great challenge to surgeons as they may occasionally culminate in amputation of the affected part. Mostly nonhealing of wounds results due to infection by antibiotic-resistant bacteria and subsequent biofilm formation. However, customized bacteriophage therapy may take care of both of the above-mentioned hurdles. A total of 48 study subjects of age group 12 to 70 years, having minimum one eligible full-thickness wound and failed to heal in 6-week duration with conventional therapy, were included in this exploratory prospective study. Patients with systemic diseases, that is, burn, malignancy, dermatological disorders, and ulcers with leprosy or tuberculosis, were excluded. However, subjects having diabetes and hypertension were included in the study. The customized monophage for single bacterial infection and cocktail of phages specific to 2 or more infecting bacteria were applied on an alternate day over the wound surface. A total of 5 to 7 applications were made till the wound became free of infecting bacteria. The study period extended from August 2018 to May 2019. The study subjects were followed for 3 months since the start of therapy. A cure rate of 81.2% could be obtained, of which 90.5% (19/21) patients were nondiabetic and 74.1% (20/27) diabetic. The wounds infected with Klebsiella pneumoniae were observed with relatively delayed healing. Post phage therapy, the mean hemoglobin level and percentage of lymphocytes increased significantly. The customized local phage therapy is very promising in nonhealing ulcers.
The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.
Background:
A chronic wound usually results due to halt in the inflammatory phase of wound healing. Bacterial infections and biofilm formation are considered to be the basic cause of it. Chronic wounds significantly impair the quality of life. Antibiotics are now failing due to biofilm formation emergence of drug-resistant bacteria.
Objective:
This study aims to see the effect of bacteriophage therapy in chronic nonhealing wound infected with the following bacteria: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.
Subject:
Patients with chronic nonhealing wound not responding to conventional local debridement and antibiotic therapy were included in the study. The age of patients ranged between 12 and 60 years.
Method:
A total of 20 patients selected and tissue biopsies and wound swabs were taken for isolation of the bacteria. After confirmation of organism, a cocktail of customized bacteriophages was topically applied over the wound on alternate days till the wound surface became microbiologically sterile. Mean bacterial count and clinical assessment were done and compared at the time of presentation and after 3 and 5 doses of application.
Results:
A significant improvement was observed in the wound healing, and there were no signs of infection clinically and microbiologically after 3 to 5 doses of topical bacteriophage therapy. Seven patients achieved complete healing on day21 during follow up while in others healthy margins and healthy granulation tissue were observed.
Conclusion:
Topical bacteriophage application may be quite effective therapy for the treatment of chronic nonhealing wounds.
Bacteriophage therapy is a commonly used treatment for Staphylococcus aureus infections in countries of the former Soviet Union, using both single phages and phage cocktails. The scarce data available on Eastern phage cocktails prompted an investigation into commercially‐available Pyophage cocktails from two different manufacturers used to treat skin and wound infections. Comparison of the metagenomic composition of two Pyophage products from Georgia and Russia revealed substantial differences in phage‐types targeting Escherichia, Enterococcus, Salmonella, Pseudomonas aeruginosa, and Proteus, therefore indicating multiple strategies for composing phage cocktails against these bacterial pathogens. Closely‐related Kayvirus‐like Myoviruses were, however, a shared component against S. aureus within all products, except for the inclusion of a secondary S. aureus Podovirus in one Microgen cocktail. Metagenomic analysis also revealed the presence of several probable prophage sequences, but detected no genetic safety risks in terms of virulence factors or antibiotic resistance genes. The safety of broad‐spectrum cocktails was tested by comparing the effects of nasal and oral exposure to Eliava Pyophage, a monospecies counterpart, and placebo in healthy human carriers of S. aureus. The lack of adverse effects in any treatment groups supports the clinical safety of S. aureus phages administered as a single phage or as phage cocktail.
This article is protected by copyright. All rights reserved.
The complex heterogeneous structure of biofilms confers to bacteria an important survival strategy. Biofilms are frequently involved in many chronic infections in consequence of their low susceptibility to antibiotics as well as resistance to host defences. The increasing need of novel and effective treatments to target these complex structures has led to a growing interest on bacteriophages (phages) as a strategy for biofilm control and prevention. Phages can be used alone, as a cocktail to broaden the spectra of activity, or in combination with other antimicrobials to improve their efficacy. Here, we summarize the studies involving the use of phages for the treatment or prevention of bacterial biofilms, highlighting the biofilm features that can be tackled with phages or combined therapy approaches.
The observation of aggregated microbes surrounded by a self-produced matrix adhering to surfaces or located in tissues or secretions is old since both Leeuwenhoek and Pasteur have described the phenomenon. In environmental and technical microbiology, biofilms, 80–90 years ago, were already shown to be important for biofouling on submerged surfaces, for example, ships. The concept of biofilm infections and their importance in medicine was, however, initiated in the early 1970s by the observation of heaps of Pseudomonas aeruginosa cells in sputum and lung tissue from chronically infected cystic fibrosis patients. The term biofilm was introduced into medicine in 1985 by J. W. Costerton. During the following decades, the number of published biofilm articles and methods for studying biofilms increased rapidly and it was shown that adhering and nonadhering biofilm infections are widespread in medicine. The medical importance of biofilm infections is now generally accepted and guidelines for prophylaxis, diagnosis, and treatment have been published.
The human periodontium health is commonly compromised by chronic inflammatory conditions and has become a major public health concern. Dental plaque, the precursor of periodontal disease, is a complex biofilm consisting mainly of bacteria, but also archaea, protozoa, fungi and viruses. Viruses that specifically infect bacteria - bacteriophages - are most common in the oral cavity. Despite this, their role in the progression of periodontal disease remains poorly explored. This review aims to summarize how bacteriophages interact with the oral microbiota, their ability to increase bacterial virulence and mediate the transfer of resistance genes and suggests how bacteriophages can be used as an alternative to the current periodontal disease therapies.
Objective:
Enterococcus faecalis is a Gram-positive, facultative anaerobic bacterium that is associated with failed endodontic cases and nosocomial infections. E. faecalis can form biofilms, penetrate dentinal tubules and survive in root canals with scarce nutritional supplies. These properties can make E. faecalis resistant to conventional endodontic disinfection therapy. Furthermore, treatment may be complicated by the fact that many E. faecalis strains are resistant to antibiotics. A potential alternative to antibiotic therapy is phage therapy. ϕEf11 is a temperate phage that infects strains of E. faecalis. It was previously sequenced and genetically engineered to modify its properties in order to render it useful as a therapeutic agent in phage therapy. In the current study, we have further genetically modified the phage to create phage ϕEf11/ϕFL1C(Δ36)(PnisA). The aim of this study was to evaluate the efficacy of bacteriophage ϕEf11/ϕFL1C(Δ36)(PnisA), to disrupt biofilms of two Enterococcus faecalis strains: JH2-2 (vancomycin-sensitive) and V583 (vancomycin-resistant).
Methods:
24h static biofilms of E. faecalis strains JH2-2(pMSP3535 nisR/K) and V583 (pMSP3535nisR/K), formed on cover slips, were inoculated with bacteriophage ϕEf11/ϕFL1C(Δ36)(PnisA). After 24 and 48h incubation, the bacterial biomass was imaged by confocal microscopy and viable cells were quantified by colony forming unit measurement.
Results:
The results showed a 10-100-fold decrease in viable cells (CFU/biofilm) after phage treatment, which was consistent with comparisons of treated and untreated biofilm images visualized as max projections of the Z-series.
Conclusion:
The biomass of both vancomycin-sensitive and vancomycin-resistant E. faecalis biofilms is markedly reduced following infection by bacteriophage ϕEf11/ϕFL1C(Δ36)(PnisA).
Increasing development of antimicrobial resistance is driving a resurgence in interest in phage therapy: the use of bacteriophages to treat bacterial infections. As the lytic action of bacteriophages is unaffected by the antibiotic resistance status of their bacterial target it is thought that phage therapy may have considerable potential in the treatment of a wide range of topical and localized infections. As yet this interest has not extended to intravenous (IV) use, which is surprising given that the historical record shows that phages are likely to be safe and effective when delivered by this route. Starting almost 100 years ago, phages were administered intravenously in treatment of systemic infections including typhoid, and Staphylococcal bacteremia. There was extensive IV use of phages in the 1940s to treat typhoid, reportedly with outstanding efficacy and safety. The safety of IV phage administration is also underpinned by the detailed work of Ochs and colleagues in Seattle who have over 4 decades experience with IV injection into human subjects of large doses of highly purified coliphage PhiX174. Though these subjects included a large number of immune deficient children no serious side effects were observed over this extended time period. The large and continuing global health problems of typhoid and Staphylococcus aureus are exacerbated by the increasing antibiotic resistance of these pathogens. We contend that these infections are excellent candidates for use of intravenous phage therapy.
Significance
Antibiotic resistance of pathogens is a growing concern to human health, reviving interest in phage therapy. This therapy uses phages (natural bacterial enemies) to kill pathogens. However, it encounters many obstacles such as delivery barriers into the tissues and bacterial resistance to phages. Here, we use phages for delivering a programmable DNA nuclease, clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas), to reverse antibiotic resistance and eliminate the transfer of resistance between strains. This approach combines CRISPR-Cas delivery with lytic phage selection of antibiotic-sensitized bacteria. The strategy may reduce the prevalence of antibiotic-resistant bacteria in treated surfaces and on skin of medical personnel, as it uses phages in a unique way that overcomes many of the hurdles encountered by phage therapy.
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Cancer is fueled by deregulation of signaling pathways in control of cellular growth and proliferation. These pathways are also targeted by infectious pathogens en route to establishing infection. Gallbladder carcinoma (GBC) is frequent in the Indian subcontinent, with chronic Salmonella enterica serovar Typhi infection reported as a significant risk factor. However, direct association and causal mechanisms between Salmonella Typhi infection and GBC have not been established. Deconstructing the epidemiological association between GBC and Salmonella Typhi infection, we show that Salmonella enterica induces malignant transformation in predisposed mice, murine gallbladder organoids, and fibroblasts, with TP53 mutations and c-MYC amplification. Mechanistically, activation of MAPK and AKT pathways, mediated by Salmonella enterica effectors secreted during infection, is critical to both ignite and sustain transformation, consistent with observations in GBC patients from India. Collectively, our findings indicate that Salmonella enterica can promote transformation of genetically predisposed cells and is a causative agent of GBC.
Copyright © 2015 Elsevier Inc. All rights reserved.
Microbial biofilms are known to support a number of human infections, including those related to medical devices. This work is focused on the development of novel dual-function amphiphilic random copolymers to be employed as coatings for medical devices. Particularly, copolymers were obtained by polymerization of an antimicrobial cationic monomer (bearing tertiary amine) and an antioxidant and antimicrobial hydrophobic monomer (containing hydroxytyrosol, HTy), To obtain copolymers with a various amphiphilic balance, different molar ratios of the two monomers were used. (1)H-NMR and DSC analysis evidenced that HTy aromatic rings are able to interact each other leading to a supra-macromolecular re-arrangement and decreasing the copolymer size in water. All copolymers showed good antioxidant activity and Fe(2+) chelating ability. Cytotoxicity and hemolytic tests evidenced that the amphiphilic balance, cationic charge density and polymer size in solution are key determinants for polymer biocompatibility. As for the antimicrobial properties, the lowest minimal inhibitory concentration (MIC=40 μg/mL) against S. epidermidis was shown by the water-soluble copolymer having the highest HTy molar content (0.3). This copolymer layered onto catheter surfaces was also able to prevent staphylococcal adhesion. The approach here reported permits not only to prevent biofilm infections but also to reduce the risk of emergence of drug-resistant bacteria. Indeed, the combination of two active compounds in the same polymer can provide a synergistic action against biofilm and suppress reactive species oxygen (ROS), known to promote the occurrence of antibiotic resistance.
Copyright © 2015. Published by Elsevier Ltd.
Isothermal microcalorimetry (IMC) is particularly suited to the study of microbiological samples in complex or heterogeneous environments because it does not require optical clarity of the sample and can detect metabolic activity from as few as 10(4)CFU/mL cells. While the use of IMC for studying planktonic cultures is well established, in the clinical environment bacteria are most likely to be present as biofilms. Biofilm prevention and eradication present a number of challenges to designers and users of medical devices and implants, since bacteria in biofilm colonies are usually more resistant to antimicrobial agents. Analytical tools that facilitate investigation of biofilm formation are therefore extremely useful. While it is possible to study pre-prepared biofilms in closed ampoules, better correlation with in vivo behaviour can be achieved using a system in which the bacterial suspension is flowing. Here, we discuss the potential of flow microcalorimetry for studying biofilms and report the development of a simple flow system that can be housed in a microcalorimeter. The use of the flow system is demonstrated with biofilms of Staphylococcus aureus.
Copyright © 2014. Published by Elsevier Inc.
In hospital settings, biofilm-based medical device-related infections are considered a threat to patients, the sessile growing bacteria playing a key role in the spreading of healthcare-associated infections. In last decades, the design of antifouling coatings for medical devices able to prevent microbial adhesiveness has emerged as one of the most promising strategies to face this important issue. In order to obtain suitable antifouling materials, segmented polyurethanes characterized by a hard/soft domain structure, having the same hard domain but a variable soft domain, have been synthesized. The soft domain was constituted by one of the following macrodiols: polypropylenoxide (PPO), polycaprolactide (PCL) and poly-l-lactide (PLA). The effects of the polymer hydrophilicity and the degree of hard/soft domain separation on antifouling properties of the synthesized polyurethanes were investigated. Microbial adherence assays evidenced as the polymers containing PCL or PLA were able to significantly reduce the adhesion of Staphylococcus epidermidis with respect to the PPO-containing polymer. This article is protected by copyright. All rights reserved.
Infections with bacterial or fungal biofilms have emerged as a major public heath concern because biofilm-growing cells are highly resistant to both antibiotics and host immune defenses. This review focuses on the progress in understanding the mechanisms of biofilm-specific antimicrobial resistance and in developing innovative therapeutic measures based on novel antibiofilm agents.
Bacterial biofilms are the basis of many persistent diseases. The persistence of these infections is primarily attributed to the increased antibiotic resistance exhibited by the cells within the biofilms. This resistance is multifactorial; there are multiple mechanisms of resistance that act together in order to provide an increased overall level of resistance to the biofilm. These mechanisms are based on the function of wild-type genes and are not the result of mutations. This article reviews the known mechanisms of resistance, including the ability of the biofilm matrix to prevent antibiotics from reaching the cells and the function of individual genes that are preferentially expressed in biofilms. Evidence suggests that these mechanisms have been developed as a general stress response of biofilms that enables the cells in the biofilm to respond to all of the changes in the environment that they may encounter.
Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.
The increasing occurrence of antibiotic-resistant pathogens is of growing concern, and must be counteracted by alternative antimicrobial treatments. Bacteriophages represent the natural enemies of bacteria. However, the strong immune response following application of phages and rapid clearance from the blood stream are hurdles which need to be overcome. Towards our goal to render phages less immunogenic and prolong blood circulation time, we have chemically modified intact bacteriophages by conjugation of the non-immunogenic polymer monomethoxy-polyethylene glycol (mPEG) to virus proteins. As a proof of concept, we have used two different polyvalent and strictly virulent phages of the Myoviridae, representing typical candidates for therapeutical approaches: Felix-O1 (infects Salmonella) and A511 (infects Listeria). Loss of phage infectivity after PEGylation was found to be proportional to the degree of modification, and could be conveniently controlled by adjusting the PEG concentration. When injected into naïve mice, PEGylated phages showed a strong increase in circulation half-life, whereas challenge of immunized mice did not reveal a significant difference. Our results suggest that the prolonged half-life is due to decreased susceptibility to innate immunity as well as avoidance of cellular defence mechanisms. PEGylated viruses elicited significantly reduced levels of T-helper type 1-associated cytokine release (IFN-γ and IL-6), in both naïve and immunized mice. This is the first study demonstrating that PEGylation can increases survival of infective phage by delaying immune responses, and indicates that this approach can increase efficacy of bacteriophage therapy.
It has become evident that aggregation or biofilm formation is an important survival mechanism for bacteria in almost any environment. In this review, we summarize recent visualizations of bacterial aggregates in several chronic infections (chronic otitis media, cystic fibrosis, infection due to permanent tissue fillers and chronic wounds) both as to distribution (such as where in the wound bed) and organization (monospecies or multispecies microcolonies). We correlate these biofilm observations to observations of commensal biofilms (dental and intestine) and biofilms in natural ecosystems (soil). The observations of the chronic biofilm infections point toward a trend of low bacterial diversity and sovereign monospecies biofilm aggregates even though the infection in which they reside are multispecies. In contrast to this, commensal and natural biofilm aggregates contain multiple species that are believed to coexist, interact and form biofilms with high bacterial and niche diversity. We discuss these differences from both the diagnostic and the scientific point of view.
Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.