Cocultures of Enterococcus faecium and Aeromonas veronii Induce the Secretion of Bacteriocin-like Substances against Aeromonas.

Lactic acid bacteria (LAB) were screened from Lutjanus russellii (red sea bass), and their antimicrobial activities were evaluated against two Aeromonas species isolated from the Nile tilapia, namely, Aeromonas veronii (AV) and Aeromonas jandaei (AJ). Three LAB isolates, Enterococcus faecium MU8 (EF_8), Enterococcus faecalis MU2 (EFL_2), and E. faecalis MU9 (EFL_9), were found to inhibit both AV and AJ; however, their cell-free supernatant (CFS) did not do so. Interestingly, bacteriocin-like substances (BLS) induced by cocultures of EF_8 with AV exhibited the highest antimicrobial activity against both Aeromonas sp. The size of BLS was less than 1.0 kDa; the purified BLS were susceptible to proteinase K digestion, indicating that they are peptides. BLS contained 13 identified peptides derived from E. faecium, as determined by liquid chromatography-tandem mass spectrometry. Cocultures of Gram-positive-producing and -inducing LAB strains have been used to increase bacteriocin yields. To our knowledge, this is the first report describing inducible BLS produced by cocultures of Gram-positive-producing and Gram-negative-inducing strains.

Associations between the rs5498 (A > G) and rs281432 (C > G) polymorphisms of the ICAM1 gene and atherosclerotic cardiovascular disease risk, including hypercholesterolemia.

Background: Atherosclerotic cardiovascular disease (ASCVD) originates from complex risk factors, including age, gender, dyslipidemia, obesity, race, genetic and genetic variation. ICAM1 gene polymorphisms are a significant risk factor for ASCVD. However, the impact of the rs5498 and rs281432 polymorphisms on the prevalence of hypercholesterolemia (HCL) has not been reported. Therefore, we determine the relationships between single nucleotide polymorphisms (SNPs), including rs5498 and rs281432 on Intercellular adhesion molecule 1 gene (ICAM1) and ASCVD susceptibility in patients with HCL. Methods: The clinical characteristics of 278 participants were assessed, and classified to groups having HCL and without HCL. ICAM1 SNPs genotyping was performed by DNA sequencing, and ICAM1 expression was measured using real-time PCR. Results: Positive dominant model rs5498 participants had twice the risk of HCL (95% confidence interval (CI): [1.24-3.23], P = 0.005). The frequency of the G allele in rs5498 was 1.69 times higher in participants with HCL than in controls (95% CI [1.15-2.47], P = 0.007). Participants with the rs5498 AG or GG variants and high ICAM1 mRNA expression (≥3.12) had 2.49 times the risk (95% CI [1.42-4.38], P = 0.001), and those with a high LDL-C concentration (≥3.36 mmol/L) had 2.09 times the risk (95% CI [1.19-3.66], P = 0.010) of developing ASCVD compared with those with low ICAM1 mRNA and LDL-C levels. Interestingly, participants carrying the rs5498 AG or GG variants who had tachycardia (resting heart rates (RHRs) >100 beats/min) had a 5.02-times higher risk than those with a lower RHR (95% CI [1.35-18.63], P = 0.016). Conclusions: It may consider the G allele in ICAM1 rs5498 is associated with a higher risk of ASCVD in Thai people with HCL, and is also positively associated with ICAM1 mRNA expression, LDL-C concentration, and RHR.

Effective inhibition of Clostridioides difficile by the novel peptide CM-A.

Clostridioides difficile infection is the most common cause of nosocomial and antibiotic-associated diarrhea. C. difficile treatment is increasingly likely to fail, and the recurrence rate is high. Antimicrobial peptides are considered an alternative treatment for many infectious diseases, including those caused by antibiotic resistant bacteria. In the present study, we identified a CM peptide, a hybrid of cecropin A and melittin, and its derivative which possesses potent antimicrobial activity against C. difficile strain 630. CM peptide exhibited antibacterial activity with minimum inhibitory concentration of 3.906 µg/ml (2.21 µM). A modified derivative of CM, CM-A, exhibited even greater activity with a minimum inhibitory concentration of 1.953 µg/ml (1.06 µM) and a minimum bactericidal concentration of 7.8125 µg/ml (4.24 µM), which indicates that CM-A peptide is more efficient than its parent peptide. A fluorescence-activated cell sorter analysis revealed that the membrane of C. difficile 630 could be an important target for CM-A. This peptide induced high levels of cell depolarization and cell permeability on C. difficile cell membrane. Moreover, electron microscopy imaging showed that CM-A interferes with the C. difficile cell membrane. Hence, the antimicrobial peptide CM-A may represent a promising novel approach for the treatment of C. difficile infections.

Temporal Variations in Patterns of Clostridioides difficile Strain Diversity and Antibiotic Resistance in Thailand.

Clostridioides difficile has been recognized as a life-threatening pathogen that causes enteric diseases, including antibiotic-associated diarrhea and pseudomembranous colitis. The severity of C. difficile infection (CDI) correlates with toxin production and antibiotic resistance of C. difficile. In Thailand, the data addressing ribotypes, toxigenic, and antimicrobial susceptibility profiles of this pathogen are scarce and some of these data sets are limited. In this study, two groups of C. difficile isolates in Thailand, including 50 isolates collected from 2006 to 2009 (THA group) and 26 isolates collected from 2010 to 2012 (THB group), were compared for toxin genes and ribotyping profiles. The production of toxins A and B were determined on the basis of toxin gene profiles. In addition, minimum inhibitory concentration of eight antibiotics were examined for all 76 C. difficile isolates. The isolates of the THA group were categorized into 27 A-B+CDT- (54%) and 23 A-B-CDT- (46%), while the THB isolates were classified into five toxigenic profiles, including six A+B+CDT+ (23%), two A+B+CDT- (8%), five A-B+CDT+ (19%), seven A-B+CDT- (27%), and six A-B-CDT- (23%). By visually comparing them to the references, only five ribotypes were identified among THA isolates, while 15 ribotypes were identified within THB isolates. Ribotype 017 was the most common in both groups. Interestingly, 18 unknown ribotyping patterns were identified. Among eight tcdA-positive isolates, three isolates showed significantly greater levels of toxin A than the reference strain. The levels of toxin B in 3 of 47 tcdB-positive isolates were significantly higher than that of the reference strain. Based on the antimicrobial susceptibility test, metronidazole showed potent efficiency against most isolates in both groups. However, high MIC values of cefoxitin (MICs 256 µg/mL) and chloramphenicol (MICs ≥ 64 µg/mL) were observed with most of the isolates. The other five antibiotics exhibited diverse MIC values among two groups of isolates. This work provides evidence of temporal changes in both C. difficile strains and patterns of antimicrobial resistance in Thailand.

Isolation and characterization of thermophilic cellulose and hemicellulose degrading bacterium, Thermoanaerobacterium sp. R63 from tropical dry deciduous forest soil.

Thermophilic microorganisms and their enzymes have been utilized in various industrial applications. In this work, we isolated and characterized thermophilic anaerobic bacteria with the cellulose and hemicellulose degrading activities from a tropical dry deciduous forest in northern Thailand. Out of 502 isolated thermophilic anaerobic soil bacteria, 6 isolates, identified as Thermoanaerobacterium sp., displayed an ability to utilize a wide range of oligosaccharides and lignocellulosic substrates. The isolates exhibited significant cellulase and xylanase activities at high temperature (65°C). Among all isolates, Thermoanaerobacterium sp. strain R63 exhibited remarkable hydrolytic properties with the highest cellulase and xylanase activities at 1.15 U/mg and 6.17 U/mg, respectively. Extracellular extract of Thermoanaerobacterium sp. strain R63 was thermostable with an optimal temperature at 65°C and could exhibit enzymatic activities on pH range 5.0-9.0. Our findings suggest promising applications of these thermoanaerobic bacteria and their potent enzymes for industrial purposes.

Probiotic Activity of Enterococcus faecium and Lactococcus lactis Isolated from Thai Fermented Sausages and Their Protective Effect Against Clostridium difficile.

Lactic acid bacteria, Enterococcus faecium and Lactococcus lactis, previously isolated from Thai fermented sausages were elucidated their probiotic properties especially in the control of Clostridium difficile 630. Both isolates survived in simulated gastric solution at pH 3 followed in simulated intestinal solution at pH 8. The presence of skimmed milk also helped the bacteria to survive through acidic and alkaline in gastrointestinal conditions. The adhesion properties of both isolates were tested using a human colon adenocarcinoma cell line. The result showed that both isolates exhibited desirable probiotic properties which adhered to Caco-2 cells. The neutralized cell-free supernatant of both isolates demonstrated that no cytotoxicity toward Caco-2 cells vice versa cell-free supernatant of C. difficile 630 toward Caco-2 cell demonstrated high toxicity. The immunomodulation effect in response to bacterial neutralized cell-free supernatant and cell-free supernatant was also studied. The expression level of pro-inflammatory cytokine of Caco-2 cell which are tumor necrosis factor-α and interleukin-8 was evaluated using quantitative reverse transcriptase PCR. Both isolates were able to diminish the expression level of TNF-α and IL-8 induced by the cell-free supernatant of C. difficile 630. Hence, these isolates would be able to improve the gut health through counteracting the C. difficile-associated intestinal inflammation in human cell lines. These results may contribute to the development of the isolates using as probiotics.

Associations among serum folate, waist-to-hip ratio, lipid profile, and eating habits with homocysteine in an elderly Thai population.

A lower serum folate level is common in older populations and is associated with increased serum homocysteine concentration. In turn, an elevated homocysteine level is associated with increased risk of cardiovascular disease and age-related diseases. Contemporary studies of folate and dietary risk factors for cardiovascular disease among the elderly population in Thailand are lacking. This cross-sectional study aimed to investigate the relationships among serum folate, homocysteine level, and nutritional status in the elderly Thai. Three hundred individuals, aged 60 years and over, underwent anthropometric and physiological measurements, and biochemical parameters, and eating habits were also determined. Folate insufficiency was found in approximately 35% of subjects. Folate and homocysteine showed a significant inverse correlation. Serum homocysteine levels rose with increasing age. Folate deficiency and high waist-to-hip ratio were associated with 7-fold and 2.5-fold increased risk for hyperhomocysteinemia, respectively. There were positive correlations between homocysteine and waist-to-hip ratio and systolic blood pressure, but a negative correlation between homocysteine and high-density lipoprotein (r = -0.239, p < 0.01), which are markers for cardiovascular disease risk. Folate negatively correlated with body mass index, waist-to-hip ratio, and diastolic blood pressure, but positively with high-density lipoprotein (r = 0.162, p < 0.01). Investigation of eating habits showed that low consumption of green leafy vegetables and high consumption of sugary foods were associated with high homocysteine levels. Given associations between nutritional status and cardiovascular disease confirmed in this study, nutrition education, holistic health promotion, and appropriate behavioral modification of eating habits represent important measures for preventing premature cardiovascular disease in the elderly Thai population.

Maoberry (Antidesma bunius) ameliorates oxidative stress and inflammation in cardiac tissues of rats fed a high-fat diet.

BACKGOUND: Chronic fat-rich diets consumption is increased risk associated with cardiovascular diseases (CVD). Prevention or reduction the progression of cardiac tissue deterioration could benefit in CVD. This study aimed to examine the effects of maoberry (Antidesma bunius), a antioxidant-rich tropical fruit, supplementation on oxidative stress and inflammation in cardiac tissues of rats fed a high-fat diet (HFD). METHODS: The male rats orally received HFD with maoberry extract doses of 0.38, 0.76 or 1.52 g/kg or simvastatin (10 mg/kg) for 12 weeks. At the end of the experimental period, the rats were fasted, euthanized and harvested for the hearts. RESULTS: Significantly reduced oxidative stress (malondialdehyde levels) and enhanced antioxidant capacity (ferric-reducing activities) in cardiac tissues of the rats were found. Maoberry extract remarkably ameliorated the expressions of genes involved with pro-inflammatory such as the tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) and endothelial nitric oxide synthase (eNOS). CONCLUSIONS: Our findings suggest that maoberry extract has remarkable effects on preventing progression of cardiac tissue deterioration at least through lowering oxidative stress and inflammation.

Microfluidics: innovative approaches for rapid diagnosis of antibiotic-resistant bacteria.

The emergence of antibiotic-resistant bacteria has become a major global health concern. Rapid and accurate diagnostic strategies to determine the antibiotic susceptibility profile prior to antibiotic prescription and treatment are critical to control drug resistance. The standard diagnostic procedures for the detection of antibiotic-resistant bacteria, which rely mostly on phenotypic characterization, are time consuming, insensitive and often require skilled personnel, making them unsuitable for point-of-care (POC) diagnosis. Various molecular techniques have therefore been implemented to help speed up the process and increase sensitivity. Over the past decade, microfluidic technology has gained great momentum in medical diagnosis as a series of fluid handling steps in a laboratory can be simplified and miniaturized on to a small platform, allowing marked reduction of sample amount, high portability and tremendous possibility for integration with other detection technologies. These advantages render the microfluidic system a great candidate to be developed into an easy-to-use sample-to-answer POC diagnosis suitable for application in remote clinical settings. This review provides an overview of the current development of microfluidic technologies for the nucleic acid based and phenotypic-based detections of antibiotic resistance.

Virulence Gene-Associated Mutant Bacterial Colonies Generate Differentiating Two-Dimensional Laser Scatter Fingerprints.

UNLABELLED: In this study, we investigated whether a laser scatterometer designated BARDOT (bacterial rapid detection using optical scattering technology) could be used to directly screen colonies of Listeria monocytogenes, a model pathogen, with mutations in several known virulence genes, including the genes encoding Listeria adhesion protein (LAP; lap mutant), internalin A (ΔinlA strain), and an accessory secretory protein (ΔsecA2 strain). Here we show that the scatter patterns of lap mutant, ΔinlA, and ΔsecA2 colonies were markedly different from that of the wild type (WT), with >95% positive predictive values (PPVs), whereas for the complemented mutant strains, scatter patterns were restored to that of the WT. The scatter image library successfully distinguished the lap mutant and ΔinlA mutant strains from the WT in mixed-culture experiments, including a coinfection study using the Caco-2 cell line. Among the biophysical parameters examined, the colony height and optical density did not reveal any discernible differences between the mutant and WT strains. We also found that differential LAP expression in L. monocytogenes serotype 4b strains also affected the scatter patterns of the colonies. The results from this study suggest that BARDOT can be used to screen and enumerate mutant strains separately from the WT based on differential colony scatter patterns. IMPORTANCE: In studies of microbial pathogenesis, virulence-encoding genes are routinely disrupted by deletion or insertion to create mutant strains. Screening of mutant strains is an arduous process involving plating on selective growth media, replica plating, colony hybridization, DNA isolation, and PCR or immunoassays. We applied a noninvasive laser scatterometer to differentiate mutant bacterial colonies from WT colonies based on forward optical scatter patterns. This study demonstrates that BARDOT can be used as a novel, label-free, real-time tool to aid researchers in screening virulence gene-associated mutant colonies during microbial pathogenesis, coinfection, and genetic manipulation studies.

Light scattering sensor for direct identification of colonies of Escherichia coli serogroups O26, O45, O103, O111, O121, O145 and O157.

BACKGROUND: Shiga-toxin producing Escherichia coli (STEC) have emerged as important foodborne pathogens, among which seven serogroups (O26, O45, O103, O111, O121, O145, O157) are most frequently implicated in human infection. The aim was to determine if a light scattering sensor can be used to rapidly identify the colonies of STEC serogroups on selective agar plates. METHODOLOGY/PRINCIPAL FINDINGS: Initially, a total of 37 STEC strains representing seven serovars were grown on four different selective agar media, including sorbitol MacConkey (SMAC), Rainbow Agar O157, BBL CHROMagarO157, and R&F E. coli O157:H7, as well as nonselective Brain Heart Infusion agar. The colonies were scanned by an automated light scattering sensor, known as BARDOT (BActerial Rapid Detection using Optical scattering Technology), to acquire scatter patterns of STEC serogroups, and the scatter patterns were analyzed using an image classifier. Among all of the selective media tested, both SMAC and Rainbow provided the best differentiation results allowing multi-class classification of all serovars with an average accuracy of more than 90% after 10-12 h of growth, even though the colony appearance was indistinguishable at that early stage of growth. SMAC was chosen for exhaustive scatter image library development, and 36 additional strains of O157:H7 and 11 non-O157 serovars were examined, with each serogroup producing unique differential scatter patterns. Colony scatter images were also tested with samples derived from pure and mixed cultures, as well as experimentally inoculated food samples. BARDOT accurately detected O157 and O26 serovars from a mixed culture and also from inoculated lettuce and ground beef (10-h broth enrichment +12-h on-plate incubation) in the presence of natural background microbiota in less than 24 h. CONCLUSIONS: BARDOT could potentially be used as a screening tool during isolation of the most important STEC serovars on selective agar plates from food samples in less than 24 h.

Evaluation of multiplex PCR with enhanced spore germination for detection of Clostridium difficile from stool samples of the hospitalized patients.

Clostridium difficile poses as the most common etiologic agent of nosocomial diarrhea. Although there are many diagnostic methods to detect C. difficile directly from stool samples, the nucleic acid-based approach has been largely performed in several laboratories due to its high sensitivity and specificity as well as rapid turnaround time. In this study, a multiplex PCR was newly designed with recent accumulated nucleotide sequences. The PCR testing with various C. difficile ribotypes, other Clostridium spp., and non-Clostridium strains revealed 100% specificity with the ability to detect as low as ~22 genomic copy number per PCR reaction. Different combinations of sample processing were evaluated prior to multiplex PCR for the detection of C. difficile in fecal samples from hospitalized patients. The most optimal condition was the non-selective enrichment at 37 °C for 1 h in brain heart infusion broth supplemented with taurocholate, followed by the multiplex PCR. The detection limit after sample processing was shown as being 5 spores per gram of fecal sample. Two hundred and thirty-eight fecal samples collected from the University affiliated hospital were analyzed by the enrichment multiplex PCR procedure. The results suggested that the combination of sample processing with the high-performance detection method would be applicable for routine diagnostic use in clinical setting.

Light-scattering sensor for real-time identification of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae colonies on solid agar plate.

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.

Genetic organization and molecular characterization of secA2 locus in Listeria species.

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.

Label-free identification of bacterial microcolonies via elastic scattering.

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.

On the sensitivity of forward scattering patterns from bacterial colonies to media composition.

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.

Modeling light propagation through bacterial colonies and its correlation with forward scattering patterns.

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.

LAP, an alcohol acetaldehyde dehydrogenase enzyme in Listeria, promotes bacterial adhesion to enterocyte-like Caco-2 cells only in pathogenic species.

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.

Label-free detection of multiple bacterial pathogens using light-scattering sensor.

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.