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Karleigh Huff, Amornrat Aroonnual,
Amy E Fleishman Littlejohn,
Bartek Rajwa,
Euiwon Bae,
Padmapriya P Banada,
Valery Patsekin,
E Daniel Hirleman,
J Paul Robinson,
Gary P Richards,
Arun K Bhunia
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ABSTRACT: The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for ∼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.
Microbial Biotechnology 05/2012; 5(5):607-20. · 2.53 Impact Factor
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ABSTRACT: The translocation of proteins across the bacterial cell wall is carried out by the general secretory (Sec) system. Most bacteria have a single copy of the secA gene, with the exception of a few Gram-positive bacteria, which have an additional copy of secA, designated secA2. secA2 is present in Listeria monocytogenes and is responsible for secretion and translocation of several proteins including virulence factors; however, little is known about the secA2 gene and its genetic organization in nonpathogenic members of the genus Listeria. The goal of this study was to determine the presence of secA2 locus and analyze the genetic relatedness among pathogenic and nonpathogenic Listeria species. Cloning experiments revealed that secA2 is present in all analyzed pathogenic (L. monocytogenes and L. ivanovii) and nonpathogenic (L. welshimeri, L. innocua, L. seeligeri, L. grayi and L. marthii) Listeria species except L. rocourtiae. Likewise, SecA2 transcripts were also detected in all species. Sequence analysis further revealed that 2331 nucleotides (776 amino acids) are conserved in L. monocytogenes, L. welshimeri, L. innocua and L. marthii. Three nucleotides are deleted in L. ivanovii and L. seeligeri and six in L. grayi, resulting in amino acid counts of 775, 775 and 774, respectively. secA2 is flanked upstream by iap (encoding p60) and downstream by a putative membrane protein (lmo0583, lmo f2365_0613) in all analyzed Listeria species, demonstrating conserved genetic organization of the secA2 locus in pathogenic and nonpathogenic species. Deletion of secA2 in L. innocua impaired accumulation of SecA2 substrate, N-acetyl muramidase (NamA) in the cell wall, providing evidence for the presence of functional SecA2 in nonpathogenic Listeria.
Gene 09/2011; 489(2):76-85. · 2.34 Impact Factor
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ABSTRACT: Morphology of colonies is important for taxonomy and diagnostics in microbiology where the response to environmental factors is sensitive enough to support discrimination. In this research, we analyzed the forward scattering patterns of individual Escherichia coli K12 colonies when agar hardness and nutrition levels were varied from the control sample. As the agar concentration increased from 1.2% to 1.8%, the diameter of the forward scattering patterns also increased for the same experimental condition which reflects that the colony thickness at the apex is greater for increased agar concentrations. Regarding nutrition, increasing dextrose resulted in smaller mean colony diameters while the mean diameters of the colonies were proportional to the yeast extract concentration up to 0.5%. The result reveals that ±0.3% agar concentration from the control sample is sufficient to create variations in the scattering patterns. For nutrition -0.25% of yeast extract showed significant variations while +0.25% from control sample showed minimal variations.
Journal of Biophotonics 04/2011; 4(4):236-43. · 4.34 Impact Factor
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ABSTRACT: Label-free microcolony identification via elastic light scattering was investigated for three different genera: Salmonella enterica serovar Montevideo, Listeria monocytogenes F4244, and Escherichia coli DH5α. Microcolonies were defined as bacterial colonies in their late-lag phase to early-exponential phase with the diameter range of 100-200 µm. To link biophysical characteristics with corresponding scattering patterns, a phase contrast microscope and a confocal displacement meter were used to measure the colony diameter and its 3D height profile. The results indicated that the growth characteristics of microcolonies were encoded in their morphologies which correlated to the characteristic diffraction patterns. Proposed methodology was able to classify three genera based on comprehensive phenotypic map which incorporated growth speed, ring count, and colony diameter. While the proposed method illustrated the possibility of discriminating microcolonies in their early growth stage, more thorough biophysical understanding is needed to expand the technology to other species.
Biotechnology and Bioengineering 03/2011; 108(3):637-44. · 3.95 Impact Factor
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ABSTRACT: Listeria adhesion protein (LAP), an alcohol acetaldehyde dehydrogenase (lmo1634), interacts with host-cell receptor Hsp60 to promote bacterial adhesion during the intestinal phase of Listeria monocytogenes infection. The LAP homologue is present in pathogens (L. monocytogenes, L. ivanovii) and non-pathogens (L. innocua, L. welshimeri, L. seeligeri); however, its role in non-pathogens is unknown. Sequence analysis revealed 98 % amino acid similarity in LAP from all Listeria species. The N-terminus contains acetaldehyde dehydrogenase (ALDH) and the C-terminus an alcohol dehydrogenase (ADH). Recombinant LAP from L. monocytogenes, L. ivanovii, L. innocua and L. welshimeri exhibited ALDH and ADH activities, and displayed strong binding affinity (K(D) 2-31 nM) towards Hsp60. Flow cytometry, ELISA and immunoelectron microscopy revealed more surface-associated LAP in pathogens than non-pathogens. Pathogens exhibited significantly higher adhesion (P<0.05) to Caco-2 cells than non-pathogens; however, pretreatment of bacteria with Hsp60 caused 47-92 % reduction in adhesion only in pathogens. These data suggest that biochemical properties of LAP from pathogenic Listeria are similar to those of the protein from non-pathogens in many respects, such as substrate specificity, immunogenicity, and binding affinity to Hsp60. However, protein fractionation analysis of extracts from pathogenic and non-pathogenic Listeria species revealed that LAP was greatly reduced in intracellular and cell-surface protein fractions, and undetectable in the extracellular milieu of non-pathogens even though the lap transcript levels were similar for both. Furthermore, a LAP preparation from L. monocytogenes restored adhesion in a lap mutant (KB208) of L. monocytogenes but not in L. innocua, indicating possible lack of surface reassociation of LAP molecules in this bacterium. Taken together, these data suggest that LAP expression level, cell-surface localization, secretion and reassociation are responsible for LAP-mediated pathogenicity and possibly evolved to adapt to a parasitic life cycle in the host.
Microbiology 09/2010; 156(Pt 9):2782-95. · 3.06 Impact Factor
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ABSTRACT: Technologies for rapid detection and classification of bacterial pathogens are crucial for securing the food supply. This report describes a light-scattering sensor capable of real-time detection and identification of colonies of multiple pathogens without the need for a labeling reagent or biochemical processing. Bacterial colonies consisting of the progeny of a single parent cell scatter light at 635 nm to produce unique forward-scatter signatures. Zernike moment invariants and Haralick descriptors aid in feature extraction and construction of the scatter-signature image library. The method is able to distinguish bacterial cultures at the genus and species level for Listeria, Staphylococcus, Salmonella, Vibrio, and Escherichia with an accuracy of 90-99% for samples derived from food or experimentally infected animal. Varied amounts of exopolysaccharide produced by the bacteria causes changes in phase modulation distributions, resulting in strikingly different scatter signatures. With the aid of a robust database the method can potentially detect and identify any bacteria colony essentially instantaneously. Unlike other methods, it does not destroy the sample, but leaves it intact for other confirmatory testing, if needed, for forensic or outbreak investigations.
Biosensors & bioelectronics 10/2008; 24(6):1685-92. · 5.43 Impact Factor
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ABSTRACT: Bacterial colonies play an important role in the isolation and identification of bacterial species, and plating on a petri dish is still regarded as the gold standard for confirming the cause of an outbreak situation. A bacterial colony consists of millions of densely packed individual bacteria along with matrices such as extracellular materials. When a laser is directed through a colony, complicated structures encode their characteristic signatures, which results in unique forward scattering patterns. We investigate the connection between the morphological parameters of a bacterial colony and corresponding forward scattering patterns to understand bacterial growth morphology. A colony elevation is modeled with a Gaussian profile, which is defined with two critical parameters: center thickness and diameter. Then, applying the scalar diffraction theory, we compute an amplitude modulation via light attenuation from multiple layers of bacteria while a phase modulation is computed from the colony profile. Computational results indicate that center thickness plays a critical role in the total number of diffraction rings while the magnitude of the slope of a colony determines the maximum diffraction angle. Experimental validation is performed by capturing the scattering patterns, monitoring colony diameters via phase contrast microscope, and acquiring the colony profiles via confocal displacement meter.
Journal of Biomedical Optics 15(4):045001. · 3.16 Impact Factor
