Polymicrobial interactions: impact on pathogenesis and human disease.

Microorganisms coexist in a complex milieu of bacteria, fungi, archaea, and viruses on or within the human body, often as multifaceted polymicrobial biofilm communities at mucosal sites and on abiotic surfaces. Only recently have we begun to appreciate the complicated biofilm phenotype during infection; moreover, even less is known about the interactions that occur between microorganisms during polymicrobial growth and their implications in human disease. Therefore, this review focuses on polymicrobial biofilm-mediated infections and examines the contribution of bacterial-bacterial, bacterial-fungal, and bacterial-viral interactions during human infection and potential strategies for protection against such diseases.

Resolution of Staphylococcus aureus biofilm infection using vaccination and antibiotic treatment.

Staphylococcus aureus infections, particularly those from methicillin-resistant strains (i.e., MRSA), are reaching epidemic proportions, with no effective vaccine available. The vast number and transient expression of virulence factors in the infectious course of this pathogen have made the discovery of protective antigens particularly difficult. In addition, the divergent planktonic and biofilm modes of growth with their accompanying proteomic changes also demonstrate significant hindrances to vaccine development. In this study, a multicomponent vaccine was evaluated for its ability to clear a staphylococcal biofilm infection. Antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) were chosen since they were found in previous studies to have upregulated and sustained expression in a biofilm, both in vitro and in vivo. Antibodies against these antigens were first used in microscopy studies to localize their expression in in vitro biofilms. Each of the four antigens showed heterogeneous production in various locations within the complex biofilm community in the biofilm. Based upon these studies, the four antigens were delivered simultaneously as a quadrivalent vaccine in order to compensate for this varied production. In addition, antibiotic treatment was also administered to clear the remaining nonattached planktonic cells since the vaccine antigens may have been biofilm specific. The results demonstrated that when vaccination was coupled with vancomycin treatment in a biofilm model of chronic osteomyelitis in rabbits, clinical and radiographic signs of infection significantly reduced by 67 and 82%, respectively, compared to infected animals that were either treated with vancomycin or left untreated. In contrast, vaccination alone resulted in a modest, and nonsignificant, decrease in clinical (34% reduction) and radiographic signs (9% reduction) of infection, compared to nonvaccinated animal groups untreated or treated with vancomycin. Lastly, MRSA biofilm infections were significantly cleared in 87.5% of vaccinated and antibiotic-treated animals, while antibiotics or vaccine alone could not significantly clear infection compared to controls (55.6, 22.2, and 33.3% clearance rates, respectively). This approach to vaccine development may lead to the generation of vaccines against other pathogenic biofilm bacteria.

Microbial biofilms and gastrointestinal diseases.

The majority of bacteria live not planktonically, but as residents of sessile biofilm communities. Such populations have been defined as 'matrix-enclosed microbial accretions, which adhere to both biological and nonbiological surfaces'. Bacterial formation of biofilm is implicated in many chronic disease states. Growth in this mode promotes survival by increasing community recalcitrance to clearance by host immune effectors and therapeutic antimicrobials. The human gastrointestinal (GI) tract encompasses a plethora of nutritional and physicochemical environments, many of which are ideal for biofilm formation and survival. However, little is known of the nature, function, and clinical relevance of these communities. This review summarizes current knowledge of the composition and association with health and disease of biofilm communities in the GI tract.

Effect of a synbiotic on microbial community structure in a continuous culture model of the gastric microbiota in enteral nutrition patients.

Patients with dysphagia require long-term nutritional support. This can be delivered by the enteral route via a percutaneous endoscopic gastrostomy (PEG) tube. Enteral nutrition (EN) bypasses the body's innate defences that prevent the microbial colonization of the proximal gut, which predisposes to microbial overgrowth. A continuous culture model simulating the upper gastrointestinal tract microbiota of EN patients was used to investigate the effects of a synbiotic (Lactobacillus acidophilus DUN-311, Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01, Synergy 1) on microbial community structure and metabolism. A PEG tube was inserted into the fermenters to study biofilm formation. The synbiotic delivered in sterile semi-skimmed milk (SSSM) was introduced either 48 h prior to or after PEG tube insertion. The synbiotic reduced biofilm formation on PEG tube surfaces, with suppression of Escherichia coli and Klebsiella pneumoniae when it was added subsequent to PEG insertion. When synbiotic feeding was commenced prior to PEG insertion, colonization by Staphylococcus aureus, Candida albicans and Candida famata was also inhibited. Lactate production increased in response the synbiotic or control (SSSM). These results indicate that the use of a synbiotic has the potential to reduce pathogen colonization on PEG tube surfaces in vivo, thereby reducing the incidence of biofilm-related infectious complications.

Vaccine development in Staphylococcus aureus: taking the biofilm phenotype into consideration.

Vaccine development against pathogenic bacteria is an imperative initiative as bacteria are gaining resistance to current antimicrobial therapies and few novel antibiotics are being developed. Candidate antigens for vaccine development can be identified by a multitude of high-throughput technologies that were accelerated by access to complete genomes. While considerable success has been achieved in vaccine development against bacterial pathogens, many species with multiple virulence factors and modes of infection have provided reasonable challenges in identifying protective antigens. In particular, vaccine candidates should be evaluated in the context of the complex disease properties, whether planktonic (e.g. sepsis and pneumonia) and/or biofilm associated (e.g. indwelling medical device infections). Because of the phenotypic differences between these modes of growth, those vaccine candidates chosen only for their efficacy in one disease state may fail against other infections. This review will summarize the history and types of bacterial vaccines and adjuvants as well as present an overview of modern antigen discovery and complications brought about by polymicrobial infections. Finally, we will also use one of the better studied microbial species that uses differential, multifactorial protein profiles to mediate an array of diseases, Staphylococcus aureus, to outline some of the more recently identified problematic issues in vaccine development in this biofilm-forming species.

Effect of pH on an in vitro model of gastric microbiota in enteral nutrition patients.

Patients with dysphagia due to oropharyngeal disease or cerebrovascular accident require long-term nutritional support via enteral feeding, which often results in microbial overgrowth in the upper gastrointestinal (GI) tract. Gastric acid is the primary innate defense mechanism in the stomach and has been assumed to provide an effective barrier to microbial colonization at pH values of <4. To evaluate the efficacy of gastric acid as a barrier to overgrowth, the microbiota of gastric and duodenal aspirates was assessed by culturing methods. Additionally, a fermentor-based model incorporating enteral nutrition tubing of the gastric microbiota of enteral nutrition (EN) patients was constructed to assess the effect of pH on the microbiota. Results showed that gastric acidity had a relatively small effect on the numbers of microorganisms recovered from intestinal aspirates but did influence microbiota composition. Similarly, at pH 3 in the fermentor, a complex microbiota developed in the planktonic phase and in biofilms. The effect of pH on microbiota composition was similar in aspirates and in the fermentors. Candidas and lactobacilli were aciduric, while recoveries of Escherichia coli and Klebsiella pneumoniae decreased as pH was reduced, although both were still present in significant numbers at pH 3. Only Staphylococcus aureus and Bifidobacterium adolescentis persisted at higher pH values both in vitro and in vivo. Lactate and acetate were the main organic acids detected in both aspirates and fermentors. These data show that the simulator used in this investigation was capable of modeling the effects of environmental influences on the upper GI microbiota of EN patients and that gastric pH of <4 is not sufficient to prevent microbial overgrowth in these individuals.

Effect of pH and antibiotics on microbial overgrowth in the stomachs and duodena of patients undergoing percutaneous endoscopic gastrostomy feeding.

Enteral nutrition via a percutaneous endoscopic gastrostomy (PEG) tube is often part of management in patients with dysphagia due to neurological or oropharyngeal disease. Gastrostomy placement can affect normal innate defense mechanisms in the upper gut, resulting in bacterial overgrowth. In this study microbiological investigations were done with gastric and duodenal aspirates from 20 patients undergoing PEG tube placement and PEG tubes from 10 patients undergoing tube replacement. Aspirate and PEG tube microbiotas were assessed by using viable counts and selective solid media followed by aerobic and anaerobic incubation to assess cell viabilities. The antibiotic susceptibility profiles of the isolates were determined by the disk diffusion method, and gas chromatography was used to study the bacterial metabolic products in the aspirates. The aspirates and PEG tubes contained mainly streptococci, staphylococci, lactobacilli, yeasts, and enterobacteria. Enterococci were detected only in PEG tube biofilms and not in aspirates. Gastric pH affected the composition of the aspirate microbiotas but not the total microbial counts. Staphylococci, Escherichia coli, and Candida spp. were isolated only from antibiotic-treated patients, despite the sensitivities of the bacteria to the agents used. Antibiotic treatment had no effect on the incidence of infection or the length of hospital stay in these patients.