Neal L. Schiller

Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms

Once mucoid (alginate‐producing) strains of Pseudomonas aeruginosa have become established in the respiratory tracts of cystic fibrosis patients they can rarely be eliminated by antibiotic treatment alone; we have investigated, in an in vitro biofilm system, the putative role of co‐administration of alginate lyase with antibiotic. Biofilms were maintained in continuous flow culture in a medium resembling sputum from CF patients. Antibiotics and/or alginate lyase were added to some of the cultures. Biofilms of two mucoid CF strains of P. aeruginosa were, in most cases, not eradicated by a one‐week course of treatment with 64 μg/ml of gentamicin; the same concentration of gentamicin, under the same conditions, led to the apparent elimination of all biofilms of non‐mucoid derivatives of these strains. When alginate lyase and gentamicin were administered together the apparent elimination of mucoid bacteria from biofilms was achieved, whereas the mucoid bacteria in most control biofilms treated only with gentamicin persisted. Ceftazidime treatment of biofilms was more effective against those containing the non‐mucoid strains than those with mucoid strains. These studies support the view that co‐administration of antibiotics with alginate lyase, which degrades the exopolysaccharide produced by mucoid strains of P aeruginosa, might benefit CF patients by increasing the efficacy of antibiotic in the respiratory tract.

Characterization of an In Vitro-Selected Amoxicillin-Resistant Strain of Helicobacter pylori

ABSTRACT An amoxicillin-resistant (Amoxr) strain ofHelicobacter pylori was selected for by culturing an amoxicillin-sensitive (Amoxs) strain in increasingly higher concentrations of amoxicillin, resulting in a 133-fold increase in MIC, from 0.03 to 0.06 μg/ml to 4 to 8 μg/ml. This resistance was stable upon freezing for at least 6 months and conferred cross-resistance to seven other β-lactam antibiotics. β-Lactamase activity was not detected in this Amoxr strain; however, analysis of the penicillin-binding protein (PBP) profiles generated from isolated bacterial membranes of the Amoxs parental strain and the Amoxr strain revealed a significant decrease in labeling of PBP 1 by biotinylated amoxicillin (bio-Amox) in the Amoxrstrain. Comparative binding studies of PBP 1 for several β-lactams demonstrated that PBP 1 in the Amoxr strain had decreased affinity for mezlocillin but not significantly decreased affinity for penicillin G. In addition, PBP profiles prepared from whole bacterial cells showed decreased labeling of PBP 1 and PBP 2 in the Amoxr strain at all bio-Amox concentrations tested, suggesting a diffusional barrier to bio-Amox or a possible antibiotic efflux mechanism. Uptake analysis of 14C-labeled penicillin G showed a significant decrease in uptake of the labeled antibiotic by the Amoxr strain compared to the Amoxs strain, which was not affected by pretreatment with carbonyl cyanidem-chlorophenylhydrazone, eliminating the possibility of an efflux mechanism in the resistant strain. These results demonstrate that alterations in PBP 1 and in the uptake of β-lactam antibiotics in H. pylori can be selected for by prolonged exposure to amoxicillin, resulting in increased resistance to this antibiotic.

AlgX Is a Periplasmic Protein Required for Alginate Biosynthesis in Pseudomonas aeruginosa

Alginate, an exopolysaccharide produced by Pseudomonas aeruginosa, provides the bacterium with a selective advantage that makes it difficult to eradicate from the lungs of cystic fibrosis (CF) patients. Previous studies identified a gene, algX, within the alginate biosynthetic gene cluster on the P. aeruginosa chromosome. By probing cell fractions with anti-AlgX antibodies in a Western blot, AlgX was localized within the periplasm. Consistent with these results is the presence of a 26-amino-acid signal sequence. To examine the requirement for AlgX in alginate biosynthesis, part of algX in P. aeruginosa strain FRD1::pJLS3 was replaced with a nonpolar gentamicin resistance cassette. The resulting algXDelta::Gm mutant was verified by PCR and Western blot analysis and was phenotypically nonmucoid (non-alginate producing). The algXDelta::Gm mutant was restored to the mucoid phenotype with wild-type P. aeruginosa algX provided on a plasmid. The algXDelta::Gm mutant was found to secrete dialyzable oligouronic acids of various lengths. Mass spectroscopy and Dionex chromatography indicated that the dialyzable uronic acids are mainly mannuronic acid dimers resulting from alginate lyase (AlgL) degradation of polymannuronic acid. These studies suggest that AlgX is part of a protein scaffold that surrounds and protects newly formed polymers from AlgL degradation as they are transported within the periplasm for further modification and eventual transport out of the cell.

Inhibition of Macrophage Phagocytosis by Pseudomonas aeruginosa Rhamnolipids In Vitro and In Vivo

Abstract. Patients with cystic fibrosis often have chronic and ultimately lethal pulmonary infections with Pseudomonas aeruginosa. In order to understand why these bacteria resist pulmonary clearance, we have investigated the interaction of P. aeruginosa and phagocytic cells. In an earlier study we reported that sub-lytic concentrations of two glycolipids produced by P. aeruginosa (the mono- and dirhamnolipids) caused structural changes in human monocyte-derived macrophages, and at lower concentrations inhibited the phagocytosis of Staphylococcus epidermidis by these cells. In the present study we demonstrate that rhamnolipids also inhibit the in vitro phagocytosis of both P. aeruginosa and Saccharomyces cerevisiae by thioglycollate-elicited mouse peritoneal macrophages. Using lucifer yellow to label the lysosomal compartments of macrophages, we determined that rhamnolipids interfere with the internalization of attached particles and reduce the level of phagosome-lysosome fusion of internalized targets within macrophages. We also demonstrate that physiologically relevant concentrations of rhamnolipids injected intratracheally into rat lungs inhibited the response of alveolar macrophages to a challenge of zymosan particles in vivo. These studies further demonstrate the profound inhibitory effects of P. aeruginosa rhamnolipids on macrophage function and are consistent with our hypothesis that the in situ production of these rhamnolipids directly contributes to the persistence of this pathogen in cystic fibrosis patient lungs.

Effects of Pseudomonas aeruginosa rhamnolipids on human monocyte-derived macrophages.

Pseudomonas aeruginosa, a major opportunistic gram‐negative pathogen, produces and secretes two heat‐stable hemolytic glycolipids, a monorhamnolipid and a dirhamnolipid. In this paper a simplified method for the isolation of these rhamnolipids is described. The effect of these two rhamnolipids, both together and individually, on the viability and structural morphology of human monocyte‐derived macrophages (MDMs) was examined. These cells were found to be very susceptible to the cytolytic activity of the rhamnolipids, particularly the dirhamnolipid. The monorhamnolipid, although not as cytolytic as the dirhamnolipid, caused extensive blebbing of the MDM plasma membrane. Comparison studies with several detergents confirmed the different yet distinct detergent‐like activity of each rhamnolipid form. At sublethal doses, the rhamnolipids produced marked cellular distortions of the MDMs and inhibited the ability of these cells to bind and/or ingest preopsonized bacteria. The potential mechanism of action of these rhamnolipids on the MDM membranes is discussed, as well as the possible significance of these extracellular bacterial glycolipids as a virulence factor in the pathogenesis of P. aeruginosa.

Alginate Lyase (AlgL) Activity Is Required for Alginate Biosynthesis in Pseudomonas aeruginosa

To determine whether AlgLs lyase activity is required for alginate production in Pseudomonas aeruginosa, an algLdelta::Gm(r) mutant (FRD-MA7) was created. algL complementation of FRD-MA7 restored alginate production, but algL constructs containing mutations inactivating lyase activity did not, demonstrating that the enzymatic activity of AlgL is required for alginate production.

Bacteria and Bacterial rRNA Genes Associated with the Development of Colitis in IL-10-/-Mice

Background: Microorganisms appear to play important yet ill‐defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population‐based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL‐10−/− mice. Methods: Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue. Results: Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture‐independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4). Conclusions: The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease‐associated immunity in murine immune colitis and human Crohns disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host–microbe interactions underlying IBD.

A novel model of chronic wounds: importance of redox imbalance and biofilm-forming bacteria for establishment of chronicity.

Chronic wounds have a large impact on health, affecting ∼6.5 M people and costing ∼

Generating and Reversing Chronic Wounds in Diabetic Mice by Manipulating Wound Redox Parameters

25B/year in the US alone [1]. We previously discovered that a genetically modified mouse model displays impaired healing similar to problematic wounds in humans and that sometimes the wounds become chronic. Here we show how and why these impaired wounds become chronic, describe a way whereby we can drive impaired wounds to chronicity at will and propose that the same processes are involved in chronic wound development in humans. We hypothesize that exacerbated levels of oxidative stress are critical for initiation of chronicity. We show that, very early after injury, wounds with impaired healing contain elevated levels of reactive oxygen and nitrogen species and, much like in humans, these levels increase with age. Moreover, the activity of anti-oxidant enzymes is not elevated, leading to buildup of oxidative stress in the wound environment. To induce chronicity, we exacerbated the redox imbalance by further inhibiting the antioxidant enzymes and by infecting the wounds with biofilm-forming bacteria isolated from the chronic wounds that developed naturally in these mice. These wounds do not re-epithelialize, the granulation tissue lacks vascularization and interstitial collagen fibers, they contain an antibiotic-resistant mixed bioflora with biofilm-forming capacity, and they stay open for several weeks. These findings are highly significant because they show for the first time that chronic wounds can be generated in an animal model effectively and consistently. The availability of such a model will significantly propel the field forward because it can be used to develop strategies to regain redox balance that may result in inhibition of biofilm formation and result in restoration of healthy wound tissue. Furthermore, the model can lead to the understanding of other fundamental mechanisms of chronic wound development that can potentially lead to novel therapies.

Resistance Mechanisms in an In Vitro-Selected Amoxicillin-Resistant Strain of Helicobacter pylori

By 2025, more than 500 M people worldwide will suffer from diabetes; 125 M will develop foot ulcer(s) and 20 M will undergo an amputation, creating a major health problem. Understanding how these wounds become chronic will provide insights to reverse chronicity. We hypothesized that oxidative stress (OS) in wounds is a critical component for generation of chronicity. We used the db/db mouse model of impaired healing and inhibited, at time of injury, two major antioxidant enzymes, catalase and glutathione peroxidase, creating high OS in the wounds. This was necessary and sufficient to trigger wounds to become chronic. The wounds initially contained a polymicrobial community that with time selected for specific biofilm-forming bacteria. To reverse chronicity we treated the wounds with the antioxidants α-tocopherol and N-acetylcysteine and found that OS was highly reduced, biofilms had increased sensitivity to antibiotics, and granulation tissue was formed with proper collagen deposition and remodeling. We show for the first time generation of chronic wounds in which biofilm develops spontaneously, illustrating importance of early and continued redox imbalance coupled with the presence of biofilm in development of wound chronicity. This model will help decipher additional mechanisms and potentially better diagnosis of chronicity and treatment of human chronic wounds.