The diagnosis of bacterial infections remains a major challenge in medicine. Although numerous contrast agents have been developed to image bacteria, their clinical impact has been minimal because they are unable to detect small numbers of bacteria in vivo, and cannot distinguish infections from other pathologies such as cancer and inflammation. Here, we present a family of contrast agents, termed maltodextrin-based imaging probes (MDPs), which can detect bacteria in vivo with a sensitivity two orders of magnitude higher than previously reported, and can detect bacteria using a bacteria-specific mechanism that is independent of host response and secondary pathologies. MDPs are composed of a fluorescent dye conjugated to maltohexaose, and are rapidly internalized through the bacteria-specific maltodextrin transport pathway, endowing the MDPs with a unique combination of high sensitivity and specificity for bacteria. Here, we show that MDPs selectively accumulate within bacteria at millimolar concentrations, and are a thousand-fold more specific for bacteria than mammalian cells. Furthermore, we demonstrate that MDPs can image as few as 10(5) colony-forming units in vivo and can discriminate between active bacteria and inflammation induced by either lipopolysaccharides or metabolically inactive bacteria.

Escherichia coli produces biofilms in response to the small molecule autoinducer-2 (AI-2), a product of the LuxS enzyme. LuxS is part of the activated methyl cycle and could also affect biofilm development by AI-2-independent effects on metabolism. A luxS deletion mutant of E. coli W3110 and an inducible plasmid-luxS-complemented strain were used to identify AI-2-independent phenotypes. Differential interference contrast microscopy revealed distinct surface colonization patterns. Confocal microscopy followed by quantitative image analysis determined differences in biofilm topography correlating with luxS expression; deletion mutant biofilms had a 'spreading' phenotype, whereas the complement had a 'climbing' phenotype. Addition of exogenous 4,5-dihydroxy-2,3-pentanedione (DPD), an AI-2 precursor, to the deletion mutant increased biofilm height and biomass, whereas addition of the methyl donor S-adenosyl methionine or aspartate prevented the luxS-complemented strain from producing a thick biofilm. The luxS-complemented strain autoaggregated, indicating that fimbriae production was inhibited, which was confirmed by transmission electron microscopy. DPD could not induce autoaggregation in the deletion mutant, demonstrating that fimbriation was an AI-2-independent phenotype. Carbon utilization was affected by LuxS, potentially contributing to the observed phenotypic differences. Overall, the work demonstrated that LuxS affected E. coli biofilm formation independently of AI-2 and could assist in adapting to diverse conditions.

Indirect pathogenicity (IP), the commensal protection of antibiotic-sensitive pathogens by resistant microorganisms of low intrinsic virulence, can prevent the eradication of polymicrobial infections. The contributions of antibiotic resistance mechanisms and biofilm structure to IP within polymicrobial biofilms were investigated using a model two-member consortium. Escherichia coli ATCC 33456 was transformed with vectors conferring either ampicillin or spectinomycin resistance, creating two distinct populations with different resistance mechanisms. Each strain alone or the consortium was grown as biofilms in flow cells and planktonically in chemostats. Comparisons in survival and activity were made on the basis of MICs and minimum biofilm preventative concentrations, a newly introduced descriptor. In ampicillin-containing medium, commensal interactions were evident during both modes of cultivation, but the sensitive strain experienced a greater benefit in the chemostat, indicating that the biofilm environment limited the commensal interaction between the Amp r and Spt r strains. In spectinomycin-containing medium, growth of the sensitive strain in chemostats and biofilms was not aided by the resistant strain. However, green fluorescent protein expression by the sensitive strain was greater in mixed-population biofilms (9% ؎ 1%) than when the strain was grown alone (2% ؎ 0%). No comparable benefit was evident during growth in the chemostat, indicating that the biofilm structure contributed to enhanced activity of the sensitive strain.The concept of indirect pathogenicity (IP) describes the circumstance where, in a mixed infection, an antibiotic-resistant microorganism of low intrinsic virulence protects an antibiotic-sensitive pathogen from eradication. IP was recognized during treatment failure of polymicrobial infections, and numerous examples involving beta-lactamaseproducing bacteria have been reported (3,15,16). IP is a form of commensalism, which is an interactive association where one organism benefits while the other is not affected (2). In the case of beta-lactamase-producing bacteria, susceptible antibiotics including penicillins and cephalosporins are inactivated via hydrolysis of the cyclic amide bond (25), leading to commensal protection of the sensitive strain. The occurrence of IP in microbial biofilms, or surface-attached communities of microorganisms, could have broad significance, because diverse kinds of polymicrobial infections are biofilm based, including device-related infections (8), ventilator-associated pneumonia (1), wounds (9), and abscesses (4). Presently, little information is available on the role of biofilm structure in the severity of IP or the development of IP in the presence of non-beta-lactam antibiotics.In a polymicrobial infection exhibiting IP, the efficiency of antibiotic inactivation by a resistant microorganism should determine the extent of the commensal protection provided to the sensitive microorganism. To investigate the role of antibiotic detoxification mechanisms in...

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Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.