FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to the use of glycosaminoglycans degrading enzymes, such as, but not limited to, heparanases, connective tissue activating peptide III (CTAP), heparinases, hyaluronidases and chondroitinases, against surface protected bacteria, for reduction of bacterial alginate and for the disruption of bacterial biofilms. More particularly, the present invention relates to the use of glycosaminoglycans degrading enzymes for treating conditions resulting from infection by mucoid, alginate-producing and/or biofilm-producing bacteria.
Glycosaminoglycans degrading enzymes: Glycosaminoglycans (GAG) are unbranched polyanionic polysaccharides made up of repeating disaccharides. One component of which is always an amino sugar. Degradation of GAG is carried out by a battery of lysosomal hydrolases. These include certain endoglycosidases (heparanse and CTAP degrade heparan sulfate and to a lesser extent heparin, and hyaluronidase from sheep or bovine testes degrade hyaluronic acid and chondroitin sulfate), various exoglycosidases (β-glucoronidase), and sulfatases (iduronate sulfatase), generally acting in sequence to degrade the various GAG. Bacterial lyases such as heparinase I, II and III from Flavobacterium heparinum cleave heparin-like molecules, chondroitinase ABC from Proteus vulgaris, AC from Arthrobacter aurescens or Flavobacterium heparinum, B and C from Flavobacterium heparinum degrade chondroitin-sulfate.
Bergey's Manual of Determinative Bacteriology describes 149 species of the genus Pseudomonas. However as with species designations in other groups of organisms, many are based on minor points of difference, which may vary under different conditions of growth and nutrition. Most species are motile with polar flagella: straight rods or occasionally coccoid in shape. They grow well on conventional culture media, and many strains thereof produce characteristic pigmentation. All species except P. maltophilia are recognized as having a cytochrome C oxidase present when tested with tetramethyl-p-phenylenediamine, a characteristic that distinguishes them from the enterobacteroaceae. Although Pseudomonas are not particularly invasive, once they are established as infective agents, they are very difficult to eradicate (Handbook of Microbiology, Vol. 1 1974 pp. 239-242).
Pseudomonas aeruginosa is an opportunistic pathogen responsible for a wide range of infections, one of the most debilitating being chronic pulmonary infection in cystic fibrosis (CF) patients. The basic alteration in the bronchial/pulmonary environment of the CF lung causing increased secretion of hyperviscous mucus favors bacterial colonization by Staphylococcus aureus, Haemophilus influenzae and P. aeruginosa. Prolonged antibiotic therapy and the increasing life expectancy of CF patients may influence the prevalence of all of these organisms in the lung flora. P. aeruginosa is found in patients with moderate and severe pulmonary disease, being the sole pathogen found in sputum in the most advanced stages of the disease. P. aeruginosa is particularly resistant to even the most aggressive chemotherapy and has been found to colonize the lungs of 50-90% of all CF patients. It has been shown that the severity of lung infection in CF patients is directly correlated to the presence of mucoid strains. The mucoid P. aeruginosa isolates revert at a high frequency to a nonmucoid form upon serial transfers in the laboratory.
The pathogenicity of mucoid P. aeruginosa in the CF lung is attributed in part to the synthesis of the exopolysaccharide alginate by the bacterium. Nonmucoid strains of P. aeruginosa initially colonize the upper respiratory tract of CF patients. However, mucoid alginate-producing variants appear with prolonged infection and eventually predominate in the CF lung. The alginate produced by these mucoid strains of P. aeuriginosa compounds the problems related to the hyperviscous bronchial secretions of CF patients. Alginate-producing strains of P. aeruginosa are almost exclusively associated with respiratory tract infections that accompany CF. Although 80% of the P. aeruginosa isolates form CF patients are mucoid, only about 1% of clinical P. aeruginosa isolates from other types of infections are mucoid. Alginate appears to protect P. aeruginasa by shielding it from host immune defense and antibiotic therapy, and possibly enables it to adhere more effectively to respiratory tract tissues. Once established in the CF lung, these mucoid strains tend to persist and parallel the progressive clinical deterioration of the patient. Alginate is a linear acetylated copolymer consisting of β-1,4-linked D-mannuronic acid and variable amounts of its C-5 epimer L-guluronic acid. Alginate is produced by several bacterial species, the most widely known being Azotobacter vinelandii and P. aeruginosa. Bacterial alginates differ from algal alginate in that the former contain O-acetyl groups. The viscosity level of alginate may play a role in the pathogenesis of mucoid P. aeruginosa in the CF respiratory tract. Several enzymes are involved in the alginate biosynthetic pathway: Phosphomannose isomerase (PMI), GDP-mannose dehydrogenase (GMD), and GDP-mannose pyrophosphorylase (GMP) in mucoid, alginate-producing P. aeruginose. Activities of the enzymes are either absent or greatly reduced in nonmucoid strains.
Alginate synthesis by the highly mucoid P. aeruginosa 8821 M is growth-phase-dependent and the alginate produced per unit of biomass reaches maximum values in the deceleration phase of growth. However, the degree of polymerization increases as batch growth proceeds, reaching maximum values at the stationary phase of growth (Leitao J H, Sa-Correia I; Arch Microbiol March 1995; 163(3): 217-222).
Regulation of alginate synthesis: The regulation of alginate biosynthesis by P. aeruginosa appears to involve fine tuning of several factors. A pivotal step in alginate biosynthesis is the activation of the algD gene in mucoid, alginate-producing P. aeruginosa . algD is highly activated in response to increased concentrations of either KCl or NaCl. This is an interesting finding since the CF lung is rich in Na+, Cl− and K+ ions.
Alginate-producing strains of three other Pseudomonas species (P. fluorescens, P. putida, and P. mendocina) have been isolated in vitro by growth on subinhibitory concentrations of carbenicillin. Also, certain phytopathogen Pseudomonas species produce alginate both in planta and in vitro. These observations suggest that many species of Pseudomonas harbor genes involved in alginate biosynthesis, but that they are not normally expressed. Since many of the P. aeruginosa alginate genes had been cloned, it was possible to examine genomic DNA from various Pseudomonas species and phylogenetically related organisms for sequences homologous to the P. aeruginosa alg genes. Southern hybridization studies using algA, pmm, algD, and algR1 as probes showed some degree of homology with several Pseudomonas species belonging to Pseudomonas RNA homology group 1. Some probes also hybridized with Azotobacter, Azomonas, and Serpens species. In the laboratory, the alginate-producing (alg+) phenotype is somewhat unstable, and nonmucoid (alg−) revertants are commonly seen. Genetic mapping experiments have shown that the switching between alg+ and alg− is due to a genetic change in one region of the chromosome located at about 68 min on the 75-min chromosomal linkage map of Pseudomonas. This was originally referred to as the muc locus. Two additional recognized genes are involved in the regulation of alginate production. These are algR at 9 min and algB at 13 min, both of which are required for high-level alginate production. However, most of the alginate biosynthetic genes appear to be located in a larger gene cluster at 34 min.
Collectively, the regulation of the alginate biosynthetic pathway in P. aeruginosa is multignenic and appears to be relatively complex, which suggests that this system has a long evolutionary history. Alginate is secreted in copious amounts (e.g., 2 mg/ml in culture supernatants), thus channeling much of the available carbon and energy sources toward its production. It is not surprising that alginate biosynthesis would be tightly regulated. However, when production of alginate is advantageous to the organism (such as in the CF respiratory tract) the rare mucoid cells in the population become predominant because of their selective advantage. When alginate production is no longer advantageous, the instability of the Alg+ phenotype (controlled by the genetic switch algS) allows the nonmucoid population to quickly become predominant as a result of the ability to conserve carbon and energy resources. Environmental factors may also play a role in the frequency of alginate conversion. Because virtually all CF isolates of P. aeruginosa are mucoid, it was postulated that the lung environment of CF patients provides the trigger required to turn on the production of the alginate adhesin. To identify virulence genes of P. aeruginosa that are important in infection of CF patients, an in vivo selection system (IVST) was used to identify promoters that are specifically inducible by respiratory mucus derived from CF patients. Three genetic loci that are highly inducible by the mucus were identified (Wang J et al.; Mol Microbiol December 1996; 22(5): 1005-12).
It is unlikely that this complex regulatory scheme to activate alginate production evolved solely as a pathogenic mechanism specific for the infection of CF patients. P. aeruginosa normally dwells in the soil environment, and alginate conversion may have evolved to protect the bacterial population from destruction due to attack by bacteriophages or bactriocins or from desiccate during periods of dryness. However, P. aeruginosa is a remarkable opportunistic pathogen and has adapted the alginate conversion system to promote debilitating and life threatening pulmonary infections of CF patients. Understanding alginate gene regulation in P. aeruginosa may lead to treatments that could turn off alginate production by the organisms resident in the CF lung, thus improving the longevity and quality of life for these patients (Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. Edited by Simon Silver et al., 1990 Am. Soc. For Microbiol. Ch. 2 and 3, pp. 15-36)
Adhesion mechanisms of P. aeruginosa: Adherence through carbohydrate-binding adhesins is an early step in colonization of the lung by gram-negative organisms (Azghani AO et al.; Glycobiology February 1995; 5(1): 39-44). Pseusomonas aeruginosa is an opportunistic pathogen capable of causing serious localized infections of the cerebrospinal fluid (CSF), urinary tract, eye, ear, lung, skin, and other parts of the body. The organism is often isolated from peritoneal dialysis membranes. Generalized systemic infections tend to occur only in injured, immunodeficient, or otherwise compromised patients. It is not surprising, therefore, that this genus has developed a number of adhesion mechanisms, each being specific for a particular type of substratum. There are numerous articles reporting various mechanisms of adhesion for P. aeruginosa. These include the hydrophobic effect, adhesion to and by alginates, and lectin-dependent adhesion. Some reports maintain that the adhesion of P. aeruginosa is fimbriae-dependent. The exopolysaccharide alginate binds to buccal epithelial and tracheal cells, as well as to bronchotracheal mucin. Antipolysaccharide inhibits binding of the organisms to the tracheal cells. Also, mucoid strains of Pseudomonas adhere much better to tracheal cells compared to nonmucoid strains or compared to alginate-producing bacteria grown in an antibiotic medium to reduce alginate production. Alginate appears to play a role in the adhesion of Pseudomonas to contact lenses. Microscopic evidence suggests that mucoid strains may adhere to ciliated tracheal cells and to inert surfaces, including contact lenses, by polysaccharide-like materials. The presence of alginate in the reaction mixture causes an increase in the number of bacteria adherent to tracheal cells or immobilized mucin. It has been suggested that the alginate may act to trap and tether the organisms to the substrata, thereby allowing other adhesins, such as the fimbrial adhesin complex, to bind the specific receptors. If the target animal cell or substrata lacks the ability to bind alginate, then the presence of an exopolysaccharide coat on the bacterial surface may actually impede the ability of the organisms to attach to such animal cells. This may explain the rescued ability of the mucoid strains to bind to phagocytic cells or to primary cultures of cilliated epithelial cells. The phagocytic cells lack the ability to bind alginate.
There are several reports showing that adhesion of P. aeruginosa to various substrata may involve lectins specific for carbohydrates other than sialic acid. For example, adhesion of the bacteria to the tracheal epithelium is inhibited by D-galactose and N-acetyl-D-glucosamine. Moreover, putative nonfimbrial adhesins produced by both mucoid and nonmucoid strains of Pseudomonas, specific for Galβ1, 4GlcNAc or Galβ1, 3GlcNAc sequences, may be involved in mediating binding of the organisms to human bronchial mucins (Bacterial Adhesion to Cells and Tissues/Ofek I and Doyle R J, 1994 Chapman & Hall, Inc. pp. 114-116, 418-421).
P. aeruginosa biofilms: Bacteria in nature often exist as sessile communities called biofilms. These communities develop structures that are morphologically and physiologically differentiated from free living bacteria. A cell-to-cell signal is involved in the development of P. aeruginosa biofilms. The involvement of an intercellular signal molecule in the development of P. aeruginosa biofilms suggests possible targets to control biofilm growth on catheters, in CF and in other environments where P. aerugiosa biofilms are a persistent problem. Biofilms of P. aeruginosa develop on solid surfaces exposed to a continuous flow of nutrients. The biofilm structures consist primarily of an exopolysaccharide matrix or glycocalyx in which the bacteria are embedded. Cell to cell signal is required for the differentiation of individual cells of the common bacterium P. aeruginosa into complex multicellular structures. P. aeruginosa cells in biofilms secrete a particular homoserine lactone, 3-oxododecanoylhomoserine (OdDHL), that helps to control biofilm differentiation. OdDHL binds an R-protein inside the target cell, activating RNA polymerases that are involved in forming the biofilm. A mutation that blocks generation of the signal molecule hinders differentiation, and the resulting abnormal biofilm appears to be sensitive to the detergent biocide SDS. The control of biofilm differentiation and integrity by quorum sensing has important implications in medicine. Because of their innate resistance to antibiotics and other biocides, biofilms in these environments are difficult, if not impossible, to eradicate. Bacterial biofilms also present other problems of significant economic importance in both industry and medicine. The finding of a connection between biofilm differentiation into clusters of bacteria resistant to the detergent biocide SDS and a quorum-sensing signal suggests that inhibition of these cell-to-cell signals could aid in the treatment of biofilms (Davies D G et al.; Science vol 280 Apr. 10, 1998).
It was shown that in continuous flow biofilm cultures in medium resembling CF bronchial secretions, P. aeruginosa was not eradicated form biofilms by 1 week of treatment with high concentrations of ceftazidime and gentamicin, to which they are sensitive on conventional testing. The addition of rifampicin, which has little activity against the strains as measured by the minimum inhibitory concentration, led to the apparent elimination of the bacteria from the biofilms. The effect was not strain specific. Ghani M and Soothill J S; Can J microbial November 1997; 43(11): 999-1004.
Scanning electron microscopy (SEM) showed that the internal surfaces of catheters and drainage systems are commonly colonized by thick layers or biofilms of organisms embedded in a polysaccharide matrix. There is evidence from laboratory and clinical studies that the progressive spread of the biofilm along the luminal surfaces of the bag, tube and catheter leads to bladder infection (Stickler D J et al.; Br J Urol. 1996, 78; 579-88).
Biofilms are also an integral part of dental plaque, thereby contributing to tooth decay and periodontal disease. One target is Fusobacterium nucleatum, an oral bacteria found in biofilms coating the area between teeth and gums (Potera C; ASM News, 64(6), 1998).
The incidence of gram-negative bacteria in reviews of postoperative acute endophthalmitis ranges from 3-22% with P. aeruginosa being one of the more common etiological agents of fulminant infections. These infections usually develop within days of the surgery and have a poor prognosis. P. aeruginosa has been reported to adhere to damaged corneal cells as well as to contact lenses. Its adherence to silicone and latex materials is significantly greater that of several isolates of Staph. Epidermidis. The in vitro adherence of P. aeruginosa to a variety of hydrogel and rigid gas-permeable contact lenses correlates with clinical data. P. aeruginosa adheres to the optic material of intraoccular lenses: AcrySof-acrylic<PMMA<silicone 1<silicon 2, and form biofilms (Manal M et al.; J Cataract Refract Surg vol. 24, January 1998).
CF lung predilection for excessive inflammation and infection with P. aeruginosa: In CF, defective function of the CFTR in airway epithelial cells and submucosal glands, results in chronic pulmonary infection with P. aeruginosa. The pulmonary infection incites an intense host inflammatory response, causing progressive suppurative pulmonary disease. Several hypotheses have been proposed to explain CF lung predilection for excessive inflammation and infection with P. aeruginosa.
The role of alginate in pathogenesis is complex and appears to confer antiphagocytic properties and an adherence mechanism upon the organism. Autopsies show that mucoid P. aeruginosa forms adherent microcolonies in the lung. Alginate does not firmly adhere to the organisms but is released in large quantities into the respiratory environment. Because alginate is very viscous in aqueous solution, it probably contributes to the high viscosity of the bronchial secretions in the CF lung, resulting in obstruction of small airways, interference with mucociliary airway clearance, and impaired movement to phagocytes. The mucoid organisms may be more adapted to a chronic infection because they secrete lower levels of proteases, which would otherwise cause extensive lung damage and acute infection. Also P. aeuruginosa can utilize the respiratory secretions of the CF lung to support rapid growth and alginate biosynthesis; thus, the mucus-congested CF respiratory tract provides P. aeruginosa with a nutritionally rich environment favorable to clonization. The initial colonization of the CF upper respiratory tract appears to be with a nonmucoid strain and is often asympomatic. This usually precedes the emergence of mucoid variants of the original strain and is followed by chronic infection and a poor prognosis for the patient.
CF patients do produce opsonic antibodies to mucoid P. aeruginosa that are in a planktonic or suspended state, but these antibodies fail to kill P. aeruginosa growing in a biofilm (Pier G B; Behring Inst Mitt February 1997; 98: 350-60). Results showed that alginate and neutral polysaccharides are involved in phagocytic impairment of P. aeruginosa (Pasquier C et al.; FEMS Microbiol Lett Feb. 15, 1997; 147(2): 195-202). Alginate production inhibits opsonic and nonopsonic phagocytosis, protects cells form reactive oxygen intermediates and plays additional roles associated with biofilm phenomena (Hatano K et al.; Infect Immun. January 1995; 63(1): 21-26; Meluleni G J et al.; J. Immunol. Aug. 15, 1995; 155(4): 2029-38). The results support the findings of the previous extensive work carried out in vitro, suggesting that the phagocytic and other bactericidal systems in the lung are impaired: Some investigators suggest that the elevated salt content in the surface fluid of the CF airway renders human β-defensin-1 nonfunctional, eliminating the bactericidal activity of the respiratory epithelium. Another hypothesis suggests that failure of the respiratory epithelial cells in the CF lung to ingest bacteria and be sloughed allows for P. aeruginosa retention at the endobronchial surface. The airway epithelial cell ingestion of bacteria followed by cellular desquamation may protect the lung form infection, and epithelial cells expressing mutant forms of the CFTR may be defective in this function. It was found that transformed human airway epithelial cells homozygous for the delta F508 allele of CFTR were significantly defective in uptake of P. aeruginosa compared with the same cell line complemented with the wild-type allele of CFTR. Defective epithelial cell internalization of P. aeruginosa may be a critical factor in hyper susceptibility of CF patients to chronic lung infections (Pier G B et al.; Am J Respir Crit Care Med October 1996; 154(4 Pt 2): S175-82; Proc Natl Acad Sci U S A Oct. 28, 1997; 94(22): 12088-93). The majority of CF mucoid isolates carry mucA mutations which allow transcription of alginate biosynthetic genes, resulting in a mucoid phenotype. Mucoidy is caused by muc mutations that depress the alternative sigma factor encoded by algU, which in turn activates alginate biosynthetic and ancillary regulatory genes (Boucher J C et al.; Infec Immun September 1997; 65: 3838-46; Boucher J C et al.; J Bacteriol January 1996; 178(2): 511-23). Mucoid cells are cleared less efficiently and appears to linger in the lung longer that nonmucoid organisms. This finding suggests that mucoidy may confer an ability to resist innate clearance mechanisms in the lung and, along with other potentially contributing factors, could be the basis for selection of mucA mutants in CF (Yu H et al.; Infec and Immun. 1998, 66(1): 280-88). Another possibility is that Pseudomonas adheres to epithelial cells in the CF airway in greater numbers because of the abnormal surface properties of the cells, thus leading to infection.
Although details of the mechanism differ, all of these hypotheses predict that the basic defect in CF permits retention of bacteria at an otherwise sterile site, providing the stimulus for inflammation. Furthermore, it is suggested that the CF genotype is associated with excessive inflammatory response compared with the normal response, even if the initiating stimulus is similar. In order to understand the pathogenesis of pulmonary disease characteristic of CF, it is required to examine not only the impaired clearance of the bacteria, but also the excessive host response to P. aeruginosa (Van Heeckeren A et al.; J. Clin Inves. December 1997; 100(11): 2810-5).
Thus, P. aeruginosa is a remarkable opportunistic pathogen and has adapted the alginate conversion system to promote debilitating and life threatening pulmonary infections of CF patients. The environment of the CF lung is unique in its capacity to induce alginate production by P. aeruginosa. However, the factors which contribute to this unusual host-pathogen interaction have not yet been determined.
Understanding alginate gene regulation in P. aeruginosa may lead to treatments that could turn off alginate production by the organisms resident in the CF lung, thus improving the longevity and quality of life for these patients.
CF is the most common fatal genetic disease among the Caucasian population, affecting approximately 1:2500 newborns. The median age of survival of patients with CF has dramatically increased over the past 2 decades from less than 10 to more than 30 years. This progress has occurred primarily through improved nutritional support and aggressive management with antibiotic therapy of acute pulmonary infections. Currently there is no effective combination of therapies which completely eradicates alginate-producing P. aeruginosa from the CF lung environment. The development of new compounds effective in preventing alginate synthesis represents a major step towards reaching this goal. Such inhibitors of alginate synthesis have potential clinical applications in that elimination of the alginate capsule might render P. aeruginosa more susceptible to both antibiotic therapy and the host's immune system. Therefore, many laboratories are involved in an extensive study of the genetics and regulation of the alginate biosynthetic pathway in P. aeruginosa in an effort to identify factors unique to the CF lung environment that trigger expression of the genes involved in alginate biosynthesis and in an attempt to find nontoxic compounds that inhibit alginate synthesis by inhibiting the enzymes directly involved in the pathway.
Mrnsy R J et al. have investigated the use of an alginate lyase obtained from a bacterial source to disrupt P. aeruginosa alginate's polymeric nature and effect a change in the Theological properties of CF sputum in vitro. Their results suggested that bacterial alginate present within purulent CF sputum may be quite stable, that endogenous alginate lyase activities appear to be limited and that the in vitro addition of exogenous alginate lyase can lead to the disruption of alginate and a change in the viscoelastic properties of some purulent CF sputum samples (Mrsny R J et al.; Pulm Pharmacol December 1994; 7(6): 357-66).
A suspension of 2% P. aeruginosa alginate completely blocked the diffusion of gentamycin and tobramycin, but not that of carbenicillin, illustrating how alginate production can help protect P. aeruginosa growing within alginate microcolonies in patients with CF from the effects of aminoglycosides. This aminoglycoside diffusion barrier was degraded with a semipurified preparation of P. aeruginosa alginate lyase (Hatch R A, Schiller N L; Antimicrob Agents Chemother. April 1998; 42(4): 974-7).
A 41 kDa alginate lyase capable of degrading alginic acid of P. aeruginosa was prepared from the culture of Bacillus strain ATB-1015. The enzyme was found useful for the treatment of respiratory diseases caused by infection by P. aeruginosa (JP 95-181047, JP 09009962 A2 to Akira Nakagawa). Alginic acid lyase(s) which decompose alginic acid into sugar and the 4-deoxy-5-keto uronic acid is used for treatment of pulmonary cystic fibrosis (JP 06197760 A to Yakuhin Otsuka). However, these lyases fail to degrade glycosaminoglycans.
More therapeutic approaches: Phagocytosis of P. aeruginosa by macrophages is a unique two-step process; binding is glucose-independent but ingestion occurs only in the presence of D-glucose or D-mannose. Since glucose is present in only negligible quantities in the endobroncheal space, P. aeruginosa may be pathogenic by virtue of its capacity to exploit the opportunity presented in the lower airway to resist normal nonspecific phagocytic defenses. Because delivery of simple glucose by aerosol would not be effective, various approaches for targeting glucose to alvelolar macrophages by receptor-mediated endocytosis are under investigation (Speert D P et al.; Behring Inst Mitt February 1997; 98: 274-82).
The ongoing lung tissue damage in chronically P. aeruginosa infected CF patients has been shown to be caused by elastase liberated from polymorphonuclear leukocytes (PMN). Alginate alone appeared to be a weak inhibitor of the hydrolysis of long synthetic peptide substrates and [14C]elastin by elastase. Alginate also had effects on the antielastase function of naturally occurring protease inhibitors in the lung: It reduces the association rate of elastase and alpha 1-proteinase inhibitor, whereas it increases the association rate of elastase and secretory leukoprotease inhibitor.
Based on these finding, alginate may be an important factor in determining the local distribution of leukocyte elastase and perturbing the overall protease-antiprotease balance in the infected lungs of CF patients (Ying Q L et al.; Am j Respir Cell Mol Biol. August 1996; 15(2): 283-91).
Thus, prevention of the onset of the chronic infection or prevention of the dominance of the inflammation by PMNs would be important goals for a vaccine strategy against P. aeruginosa. Findings suggested that change from the Th2 like response seen in CF patients towards a Th1 response might improve their prognosis (Johansen H K et al.; Behring Inst Mitt February 1997; 98: 269-73).
Care should be taken when treating nonmucoid P. aeruginosa with gyrase inhibitors such as ciprofloxacin, norfloxacin and ofloxacin, which target the A subunit of topoisomerase II, since it resulted in 100% conversion to the mucoid phenotype. An increase in resistance was observed in populations that expressed the mucoid phenotype. Both mucoid conversion and antibiotic resistance were completely reversible when ciprofloxacin pressure was withdrawn, but only partially reversible by the removal of norfloxacin and ofloxacin. Thus, these experiments indicate that in the presence of some fluoroquinolones, a conditional response resulting in mucoid conversion and antibiotic resistance may occur (Pina S E, Mattingly S J; Curr Microbiol August 1997; 35(2): 103-8).
Other mucoid bacteria: Klebsiella Pneumoniae K1 synthesizes capsular polysaccharide. Non mucoid variants thereof are more susceptible to some bacteriophages, possibly due to the reduction or absence of capsular polysaccharide (Mengistu Y et al.; J Appl Bacteriol May 1994, 76(5): 424-30). There are two virulence factors of K. pneumoniae: aerobactin and the mucoid phenotype. Aerobactin is always associated with the mucoid phenotype (FEMS Microbiol Lett. Jul. 15, 1995; 130(1): 51-57).
Mucoid or highly encapsulated strains of group A Streptococci have been associated both with unusually sever infections and with acute rheumatic fever. The mucoid M-type 18 strain of a group A Streptococcus has a hyaluronic acid capsule which plays an important role in virulence. The region of the chromosome essential for capsular polysaccharide expression is conserved among diverse group A streptococcal strains. Wessels M R et al.; Infec Immun February 1994; 62(2): 433-41. In communities, where increases in cases of rheumatic fever had been reported, the serotypes M-1, 3, 5, and 18 were isolated which, on culture, produced characteristic mucoid colonies (Spencer R C; Eur J Clin Microbiol Infect Dis. 1995, 14 Suppl 1: S26-32). The mucoid serotype 3 of S. pneumoniae cause rapid fatal infections, despite adequate antibiotic therapy (Hsueh P R et al.; J Formaos Med Assoc May 1996; 95(5): 364-71).
The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A Streptococcus. The hyaluronic acid capsule also appeared to play an important role: studies of an acapsular mutant derived form the mucoid or highly encapsulated M protein type 18 group A strepococcal strain 282 indicated that loss of capsule expression was associated with decreased resistance to phagocytic killing and with reduced virulence in mice. The results provide further evidence that the hyaluronic acid capsule confers resistance to phagocytosis and enhances group A streptococcal virulence. Moses A E et al.; Infect Immun. January 1997; 65(1): 64-71.
Staphylococcus aureus arthritis is a rapidly progressive and highly erosive disease of the joints in which both host and bacterial factors are of pathogenic importance. One potential bacterial virulence factor is the ability to express a polysaccharide capsule (CP). Among 11 reported capsular serotypes, CP type 5 (CP5) and CP8 comprise 80-85% of all clinical blood isolates. The results clearly indicated that the expression of CP5 is a determinant of the virulence of S. aureus in arthritis and septicemia (Nilsson I M et al.; Infec Immun 1 October; 65(10): 4216-21).
Treponema denticola, which has been associated with periodontitis, synthesizes or acquires and extracellular polysaccharide layer (Scott D et al; Oral Microbiol Immunol April 1997, 12(2): 121-5).
The above described data implies that there is a widely recognized need for, and it would be highly advantageous to have agents effective in reducing mucus production by bacteria, by, for example, degradation or prevention of synthesis of the bacterial exopolysaccharide alginate. The benefits from employing such agents for the degradation of bacterial exopolysaccharide alginate include (i) viscosity reduction of alginate related hyperviscous bronchial secretions; (ii) disruption of bacterial biofilms which may render the bacteria more susceptible to host immune defense systems and antibiotic therapy; (iii) inhibition of alginate associated adhesion to host cells and enhancement of bacterial clearance, resulting in reduction of infection rate; (vi) reduction of host's inflammatory response to infection.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided a method of rendering a surface protected bacteria more susceptible to an anti-bacterial agent comprising the step of subjecting the bacteria to a glycosaminoglycans degrading enzyme.
According to another aspect of the present invention there is provided a method of rendering a surface protected bacteria less capable of adhering to a substratum comprising the step of subjecting the bacteria to a glycosaminoglycans degrading enzyme.
According to yet another aspect of the present invention there is provided a method of treating a disease for relieving disease associated symptoms comprising the step of administering a therapeutical composition including a glycosaminoglycans degrading enzyme.
According to still another aspect of the present invention there is provided a therapeutic composition for treating a surface protected bacteria associated disease or symptoms comprising a glycosaminoglycans degrading enzyme and an antibiotic.
According to yet another aspect of the present invention there is provided a bactericide composition effective in eliminating a surface protected bacteria comprising a glycosaminoglycans degrading enzyme and a bactericide.
According to further features in preferred embodiments of the invention described below, the surface protected bacteria is a mucoid bacteria.
According to still further features in the described preferred embodiments the surface protected bacteria is an alginate-producing bacteria.
According to still further features in the described preferred embodiments the surface protected bacteria is a biofilm-producing bacteria.
According to still further features in the described preferred embodiments the anti-bacterial agent is a bactericide.
According to still further features in the described preferred embodiments the anti-bacterial agent is an antibiotic.
According to still further features in the described preferred embodiments the anti-bacterial agent is an immune moiety.
According to still further features in the described preferred embodiments the glycosaminoglycans degrading enzyme is selected from the group consisting of a lysosomal hydrolase and a bacterial lyase.
According to still further features in the described preferred embodiments the glycosaminoglycans degrading enzyme is selected from the group consisting of an endoglycosidase, an exoglycosidase and a sulfatase.
According to still further features in the described preferred embodiments the glycosaminoglycans degrading enzyme is selected from the group consisting of heparanse, connective tissue activating peptide III (CTAP), hyaluronidase, glucoronidase, iduronate sulfatase, heparinase I, heparinase II heparinase III, chondroitinase ABC, chondroitinase AC, chondroitinase B and chondroitinase C.
According to still further features in the described preferred embodiments the bacteria is of a genus selected from the group consisting of Pseudomonas, Azotobacter, Azomonas, Serpens, Fusobacterium, Klebsiella, Streptococcus, Staphylococcus and Treponema
According to still further features in the described preferred embodiments the bacteria is of a genus Pseudomonas
According to still further features in the described preferred embodiments the bacteria is Pseudomonas aeruginosa
According to still further features in the described preferred embodiments the bacteria is in a lung of a patient suffering chronic pulmonary infection, the method being for relieving symptoms associated with the chronic pulmonary infection.
According to still further features in the described preferred embodiments the bacteria is in a lung of a cystic fibrosis patient suffering chronic pulmonary infection, the method being for relieving symptoms associated with the chronic pulmonary infection.
According to still further features in the described preferred embodiments the bacteria is growing on a non-living substratum.
According to still further features in the described preferred embodiments the non-living substratum forms a part of a medical device.
According to still further features in the described preferred embodiments the medical device is selected from the group consisting of an infusion device, a catheter device, a contact lens device, a dialysis device and a draining device.
The present invention successfully addresses the shortcomings of the presently known configurations by providing methods and compositions effective in combating surface protected bacteria (e.g., mucoid-, alginate-, biofilm-producing bacteria), by subjecting such bacteria to glycosaminoglycans degrading enzyme, rendering such bacteria surface non-protected and therefore more susceptible to anti-bacterial agents and less capable of adhering to various living and non-living substrata.