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.

The specific biofilm formation (SBF) assay, a technique based on crystal violet staining, was developed to locate plant essential oils and their components that affect biofilm formation. SBF analysis determined that cinnamon, cassia, and citronella oils differentially affected growth-normalized biofilm formation by Escherichia coli. Examination of the corresponding essential oil principal components by the SBF assay revealed that cinnamaldehyde decreased biofilm formation compared to biofilms grown in Luria-Bertani broth, eugenol did not result in a change, and citronellol increased the SBF. To evaluate these results, two microscopy-based assays were employed. First, confocal laser scanning microscopy (CLSM) was used to examine E. coli biofilms cultivated in flow cells, which were quantitatively analyzed by COMSTAT, an image analysis program. The overall trend for five parameters that characterize biofilm development corroborated the findings of the SBF assay. Second, the results of an assay measuring growth-normalized adhesion by direct microscopy concurred with the results of the SBF assay and CLSM imaging. Viability staining indicated that there was reduced toxicity of the essential oil components to cells in biofilms compared to the toxicity to planktonic cells but revealed morphological damage to E. coli after cinnamaldehyde exposure. Cinnamaldehyde also inhibited the swimming motility of E. coli. SBF analysis of three Pseudomonas species exposed to cinnamaldehyde, eugenol, or citronellol revealed diverse responses. The SBF assay could be useful as an initial step for finding plant essential oils and their components that affect biofilm formation and structure.Selected natural products that originate in plants can influence microbial biofilm formation. For example, halogenated furanones, a class of compounds that inhibit biofilm formation by interfering with bacterial quorum sensing, were identified in a marine alga and are thought to have evolved to reduce biofouling (34). Other plant-derived compounds inhibit peptidoglycan synthesis (24), damage microbial membrane structures (10), modify bacterial membrane surface hydrophobicity (35), and modulate quorum sensing (14), all of which could influence biofilm formation. Terrestrial plants also support populations of surface-attached bacteria (3, 23) and could potentially produce phytochemicals that attenuate biofilm development through specific mechanisms. However, many plant essential oils, which are mixtures of numerous organic chemicals, contain compounds that inhibit microbial growth (2,8,32). Thus, a screening procedure to identify phytochemicals with specific antibiofilm activity must take into account the cytotoxicity of plant essential oils.Crystal violet (CV) staining, a colorimetric method, has been used widely to measure biofilm formation in part because of its amenability to large screening procedures (25, 26). For many applications, particularly screening assays for surface adhesion-deficient mutants (25,27), measuring the absolute amount of biofi...

Aims:  To investigate the effect of cinnamaldehyde (CA) on transcription from selected quorum sensing (QS) promoters. Methods and Results:  The action of CA on QS was assayed using three E. coli green fluorescent protein (GFP) based bioreporters (two inducible and the other constitutive) and two Vibrio harveyi bioluminescent reporter strains. LuxR‐mediated transcription from the PluxI promoter, which is induced by 3‐oxo‐C6‐homoserine lactone (HSL), was reduced by 70 per cent following exposure to 200 μmol l−1 CA (26 ppm). The bioluminescence of Vibrio harveyi BB886, which is mediated by 3‐hydroxy‐C4‐HSL, was reduced by 55 per cent after exposure to 60 μmol l−1 CA (8 ppm), and 100 μmol l−1 CA (13 ppm) inhibited the bioluminescence of the autoinducer‐2 (AI‐2) responsive reporter strain V. harveyi BB170 by nearly 60 per cent. CA did not inhibit the growth of the bioreporter strains at these concentrations. CA had a minimal effect on LasR promoter activity, induced by 3‐oxo‐C12‐HSL. Conclusions:  Low concentrations of CA were effective at inhibiting two types of acyl homoserine lactone mediated QS, and also autoinducer‐2 mediated QS. Significance and Impact of Study:  Because CA is widely used in the food and flavour industries, its potential to affect bacterial QS regulated processes should be recognized.

A consortium comprised of two engineered microorganisms was assembled for biodegradation of the organophosphate insecticide parathion. Escherichia coli SD2 harbored two plasmids, one encoding a gene for parathion hydrolase and a second carrying a green fluorescent protein marker. Pseudomonas putida KT2440 pSB337 contained a p-nitrophenol-inducible plasmid-borne operon encoding the genes for p-nitrophenol mineralization. The co-culture effectively hydrolyzed 500 microM parathion (146 mg l(-1)) and prevented the accumulation of p-nitrophenol in suspended culture. Kinetic analyses were conducted to characterize the growth and substrate utilization of the consortium members. Parathion hydrolysis by E. coli SD2 followed Michaelis-Menten kinetics. p-Nitrophenol mineralization by P. putida KT2440 pSB337 exhibited substrate-inhibition kinetics. The growth of both strains was inhibited by increasing concentrations of p-nitrophenol, with E. coli SD2 completely inhibited by 600 microM p-nitrophenol (83 mg l(-1)) and P. putida KT2440 pSB337 inhibited by 1,000 microM p-nitrophenol (139 mg l(-1)). Cultivation of the consortium as a biofilm indicated that the two species could cohabit as a population of attached cells. Analysis by confocal microscopy showed that the biofilm was predominantly comprised of P. putida KT2440 pSB337 and that the distribution of E. coli SD2 within the biofilm was heterogeneous. The use of biofilms for the construction of degradative consortia may prove beneficial.

Plant compounds that induced Arthrobacter sp. strain B1B to cometabolize polychlorinated biphenyls (PCBs) were identified by a screening assay based on the formation of a 4,4-dichlorobiphenyl ring fission product. A chemical component of spearmint (Mentha spicata), l-carvone, induced Arthrobacter sp. strain B1B to cometabolize Aroclor 1242, resulting in significant degradation of 26 peaks in the mixture, including selected tetraand pentachlorobiphenyls. Evidence for PCB biodegradation included peak disappearance, formation of a phenylhexadienoate ring fission product, and chlorobenzoate accumulation in the culture supernatant. Carvone was not utilized as a growth substrate and was toxic at concentrations of greater than 500 mg liter ؊1. Several compounds structurally related to l-carvone, including limonene, p-cymene, and isoprene, also induced cometabolism of PCBs by Arthrobacer sp. strain B1B. A structure-activity analysis showed that chemicals with an unsaturated p-menthane structural motif promoted the strongest cometabolism activity. These data suggest that certain plant-derived terpenoids may be useful for promoting enhanced rates of PCB biodegradation by soil bacteria.