Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single-and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2. In nature, most biofilms are not composed of one bacterial species but instead contain multiple species (24). These multispecies communities can be responsible for the fouling of ships (9, 44), the corrosion of liquid-carrying vessels (3, 14), and chronic infections in higher organisms (41,42,57). Recent research has demonstrated that in order for multispecies biofilm communities to develop, interbacterial communication is often essential (62) and facilitates the coordination of bacterial activities to promote the formation and to maintain the integrity of multispecies biofilm communities (28,32,60). Interspecies communication can be mediated by chemical or physical means. Mechanisms for chemical communication between different species include the secretion and uptake of metabolic by-products (11, 19), the exchange of genetic material (40), and the production and recognition of interspecies signal molecules such as short peptides (36) and autoinducer-2 (10). Mechanisms for interspecies physical communication can involve cell surface structures such as flagella or fimbriae (31, 48) and also include nonspecific adhesion between bacterial species (5) as well as highly specific coaggregations mediated by lectin-saccharide interactions (48).Coaggregation, the highly specific recognition and adhesion of different bacterial species to one another, was first discovered to occur between human oral bacteria in 1970 (23). Since then, research has shown that coaggregation occurs between specific bacterial species in environments other than the human oral cavity (48). Coaggregation interactions have been detected between bacteria isolated from canine dental plaque (21), the crop of chickens (61), the human female urogenital tract (30), the human intestine (34), and wastewater and freshwater biofilms (27,37,53). In particular, Buswell et al. (8) first demonstrated that coaggregation occurred between 19 freshwater strains that were isolated from a drinking water biofilm. Further studies by Rickard et al. demonstrated that coaggregation between these 19 strains was mediated by growth-phasedependent lectin...

Diabetic foot ulcers (DFUs) lead to nearly 100,000 lower limb amputations annually in the United States. DFUs are colonized by complex microbial communities, and infection is one of the most common reasons for diabetes-related hospitalizations and amputations. In this study, we examined how DFU microbiomes respond to initial sharp debridement and offloading and how the initial composition associates with 4 week healing outcomes. We employed 16S rRNA next generation sequencing to perform microbial profiling on 50 samples collected from 10 patients with vascularized neuropathic DFUs. Debrided wound samples were obtained at initial visit and after one week from two DFU locations, wound bed and wound edge. Samples of the foot skin outside of the wounds were also collected for comparison. We showed that DFU wound beds are colonized by a greater number of distinct bacterial phylotypes compared to the wound edge or skin outside the wound. However, no significant microbiome diversity changes occurred at the wound sites after one week of standard care. Finally, increased initial abundance of Gram-positive anaerobic cocci (GPAC), especially Peptoniphilus (p < 0.05; n = 5 subjects), was associated with impaired healing; thus, GPAC's abundance could be a predictor of the wound-healing outcome.

Despite continuous efforts to control cariogenic dental biofilms, very few effective antimicrobial treatments exist. In this study, we characterized the activity of the novel synthetic cyclic lipopeptide 4 (CLP-4), derived from fusaricidin, against the cariogenic pathogen UA159. We determined CLP-4's MIC, minimum bactericidal concentration (MBC), and spontaneous resistance frequency, and we performed time-kill assays. Additionally, we assessed CLP-4's potential to inhibit biofilm formation and eradicate preformed biofilms. Our results demonstrate that CLP-4 has strong antibacterial activity and is a potent bactericidal agent with low spontaneous resistance frequency. At a low concentration of 5 μg/ml, CLP-4 completely inhibited UA159 biofilm formation, and at 50 μg/ml, it reduced the viability of established biofilms by>99.99%. We also assessed CLP-4's cytotoxicity and stability against proteolytic digestion. CLP-4 withstood trypsin or chymotrypsin digestion even after treatment for 24 h, and our toxicity studies showed that CLP-4 effective concentrations had negligible effects on hemolysis and the viability of human oral fibroblasts. In summary, our findings showed that CLP-4 is a potent antibacterial and antibiofilm agent with remarkable stability and low nonspecific cytotoxicity. Hence, CLP-4 is a promising novel antimicrobial peptide with potential for clinical application in the prevention and treatment of dental caries.

The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3-10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.

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