Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water

AIMS: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment. METHODS AND RESULTS: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed. This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens. An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed. This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r2 = 0.96 and 0.93, respectively). CONCLUSION: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time. This system could be easily applied to the rapid analysis of microbes in various environments.     

Development of a multiplex PCR approach for the identification of Shiga toxin-producing Escherichia coli strains and their major virulence factor genes

AIMS: To develop and evaluate a multiplex PCR (mPCR) system for rapid and specific identification of Shiga toxin-producing Escherichia coli (STEC) and their main virulence marker genes. METHODS AND RESULTS: A series of mPCR assays were developed using primer pairs that identify the sequences of Shiga toxins 1 and 2 (stx1 and stx2, including the stx2c, stx2d, stx2e and stx2f variants), intimin (eaeA), and enterohaemorrhagic E. coli enterohaemolysin (ehlyA). Moreover, two additional genes (rfb O157 and fliC H7), providing the genotypic identification of the O157:H7 E. coli serotype, were detected. As an internal positive control, primers designated to amplify the E. coli 16S rRNA were included in each mPCR. All the amplified genes in the E. coli reference strains were sucessfully identified by this procedure. The method was then used for the examination of 202 E. coli isolates recovered from cattle and children. Among them, 25 (12.4%) were stx positive including the strains of O157:H7 serotype (six isolates) and O157:NM serogroup (four strains). Moreover, 20 STEC strains possessed the eaeA (intimin) and ehlyA (enterohaemolysin) genes. CONCLUSIONS: The developed mPCR-based system enabled specific detection of STEC bacteria and identification of their main virulence marker genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to identify STEC bacteria and the majority of their virulence gene markers, including four variants of Shiga toxin, as well as the differentiation of O157:H7 from non-O157 isolates represents a considerable advancement over other PCR-based methods for rapid characterization of STEC.     

Viability of Escherichia coli O157:H7 in natural river water determined by the use of flow cytometry

The enzymatic activity and viability of Escherichia coli O157:H7 in natural river water was determined by flow cytometry. River water was collected at two sites (an agricultural area and an industrial area) on the Aigawa River (Osaka, Japan). To facilitate estimation of the physiology of E. coli O157 in natural river water, bacterial cells in the water were stained with 6-carboxyfluorescein diacetate (6CFDA) and propidium iodide (PI). The cells were sorted into two populations, using a flow cytometer, based on their esterase activity. Each population was stained with E. coli O157:H7 fluorescent antibody (FA), and E. coli O157:H7 cells were observed in the esterase-active population. River water samples collected at the same points were incubated with yeast extract containing antibiotics to prevent cell division, and bacterial cells in the incubated samples were stained with PI and FA. Escherichia coli O157:H7 existed in both the viable (elongated and/or fattened) and inactive bacterial population determined by flow cytometry. These results indicate that E. coli O157:H7 may retain metabolic activity and growth potential in the natural aquatic environment.     

Two-dimensional liquid chromatography protein expression mapping for differential proteomic analysis of normal and O157:H7 Escherichia coli

A multidimensional chromatographic method has been applied for the differential analysis of proteins from different strains of Escherichia coli bacteria. Proteins are separated in the first dimension using chromatofocusing (CF) and further separated by nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second dimension. A 2-dimensional (2-D) expression map of bacterial protein content is created for virulent O157:H7 and nonvirulent E. coli strains depicting protein isoelectric point (pI) versus protein hydrophobicity. Differentially expressed proteins are further characterized using electrospray/ionization time-of-flight mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) determination and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting for protein identification. Using this method, no significant differential protein expression is exhibited between the two O157:H7 strains examined over a pH range of 4.0-7.0, and O157:H7 strains could be distinguished from nonvirulent E. coli. Several proteins differentially expressed between O157:H7 and nonvirulent E. coli are identified as potential markers for detection and treatment of O157:H7 infection.     

Rapid monitoring of microbial contamination on herbal medicines by fluorescent staining method

AIMS: To apply fluorescent staining method for fast assessment of microbial quality of herbal medicines. METHODS AND RESULTS: The number of total bacteria and esterase-active bacteria on powdered traditional Chinese medicines were enumerated by fluorescent staining method using 6-carboxyfluorescein diacetate (6CFDA) and 4',6-diamidino-2-phenylindole (DAPI), and they were compared with colony-forming units (CFU). The CFU was approximately 10(3) per gram in ginseng radix, and no bacterial colonies were detected from others. However, the total bacterial number (TDC) was more than 10(7) per gram, and number of bacteria possessing esterase activity ranged from 1 to 3% of TDC. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Many bacteria in each Chinese medicine had enzyme activity and most of them could not be detected by conventional plate counting technique. Enumeration of bacterial cells on traditional Chinese medicines by fluorescent staining method requires less than 1 h. The double staining method with 6CFDA and DAPI could be applicable to rapid microbial monitoring of crude drugs.     

Escherichia coli with anti-O157:H7 activity isolated from bovine colon

AIMS: To isolate bacteria from bovine gastrointestinal tract and investigate their inhibitory effect on Escherichia coli O157:H7 in vitro. METHODS AND RESULTS: A total of 2400 bacterial colonies were isolated from cattle colonic mucous membrane. Thirteen strains demonstrated the ability to inhibit the growth of E. coli O157:H7. From these, seven were screened for the presence of virulence factors as: stx(1), stx(2), ehxA, eae, st1a and lt1 by polymerase chain reaction. The selected bacteriocin-producing bacteria showed susceptibility to most of the antibiotics used. CONCLUSIONS: The strains of E. coli isolated, which exhibit inhibitory activity on E. coli O157:H7 growth by the production of inhibitory substances, may be useful in the control of this pathogen in reservoirs. An important characteristic of these strains was the absence of any of the virulence factors assayed and the susceptibility to most of the antibiotics used for Gram-negative bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: These microorganisms might be used as probiotic bacteria to reduce the carriage of E. coli O157:H7 in cattle, thus limiting the contamination of carcasses at slaughter and subsequently the contamination of foods and the transfer of this pathogen to man.     

Local bacteriophage isolates showed anti- Escherichia coli O157:H7 potency in an experimental ligated rabbit ileal loop model

Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity (P?< 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven?log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E.?coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages.     

Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR

Cattle are considered the major reservoir for Escherichia coli O157:H7, one of the newly emerged foodborne human pathogens of animal origin and a leading cause of haemorrhagic colitis in humans. A sensitive test that can accurately and rapidly detect the organism in the food animal production environment is critically needed to monitor the emergence, transmission, and colonization of this pathogen in the animal reservoir. In this study, a novel multiplex polymerase chain reaction (PCR) assay was developed by using 5 sets of primers that specifically amplify segments of the eaeA, slt-I, slt-II, fliC, rfbE genes, which allowed simultaneous identification of serotype O157:H7 and its virulence factors in a single reaction. Analysis of 82 E. coli strains (49 O157:H7 and 33 non-O157:H7) demonstrated that this PCR system successfully distinguished serotype O157:H7 from other serotypes of E. coli and provided accurate profiling of the shiga-like toxins and the intimin adhesin in individual strains. This multiplex PCR assay did not cross-react with the background bacterial flora in bovine faeces and could detect a single O157:H7 organism per gram of faeces when combined with an enrichment step. Together, these results indicate that the multiplex PCR assay can be used for specific identification and profiling of E. coli O157:H7 isolates, and may be applied to rapid and sensitive detection of E. coli O157:H7 in bovine faeces when combined with an enrichment step.     

Analysis of environmental Escherichia coli isolates for virulence genes using the TaqMan PCR system

AIMS: To assess the presence of virulence genes in environmental and foodborne Escherichia coli isolates using the TaqMan PCR system. METHODS AND RESULTS: Three TaqMan pathogen detection kits called O157:H7, StxI and StxII were used to investigate the presence of virulence genes in Escherichia coli isolates. All 54 foodborne E. coli O157:H7 isolates showed expected results using these kits. Ninety (15%) of 604 environmental isolates gave positive amplification with an O157:H7-specific kit. TaqMan PCR amplification products from these 90 isolates were analysed by agarose gel electrophoresis, and 90% (81 of 90) of the environmental samples contained the expected PCR product. Sixty-six of these 90 were chosen for serotyping tests and only 35% (23 of 66) showed agglutination with both anti-O157 and anti-H7 antibodies. Further ribotyping of 16 sero-positive isolates in an automated Riboprinter did not identify these to be O157:H7. Multiplex PCR with primers for eaeA, stxI and stxII genes was used to confirm the TaqMan results in 10 selected environmental isolates. CONCLUSIONS: All three TaqMan pathogen detection kits were useful for virulence gene analysis of prescreened foodborne O157:H7 isolates, while the O157:H7-specific kit may not be suitable for virulence gene analysis of environmental E. coli isolates, because of high false positive identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to rapidly identify the presence of pathogenic E. coli in food or environmental samples is essential to avert outbreaks. These results are of importance to microbiologists seeking to use TaqMan PCR to rapidly identify pathogenic E. coli in environmental samples. Furthermore, serotyping may not be a reliable method for identification of O157:H7 strains.     

Assessment of resistance to colicinogenic Escherichia coli by E. coli O157:H7 strains

AIMS: To assess a collection of 96 Escherichia coli O157:H7 strains for their resistance potential against a set of colicinogenic E. coli developed as a probiotic for use in cattle. METHODS AND RESULTS: Escherichia coli O157:H7 strains were screened for colicin production, types of colicins produced, presence of colicin resistance and potential for resistance development. Thirteen of 14 previously characterized colicinogenic E. coli strains were able to inhibit 74 serotype O157:H7 strains. Thirteen E. coli O157:H7 strains were found to be colicinogenic and 11 had colicin D genes. PCR products for colicins B, E-type, Ia/Ib and M were also detected. During in vitro experiments, the ability to develop colicin resistance against single-colicin producing E. coli strains was observed, but rarely against multiple-colicinogenic strains. The ability of serotype O157:H7 strains to acquire colicin plasmids or resistance was not observed during a cattle experiment. CONCLUSIONS: Escherichia coli O157:H7 has the potential to develop single-colicin resistance, but simultaneous resistance against multiple colicins appears to be unlikely. Colicin D is the predominant colicin produced by colicinogenic E. coli O157:H7 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential for resistance development against colicin-based strategies for E. coli O157:H7 control may be very limited if more than one colicin type is used.     

Detection of bacteria carrying the stx2 gene by in situ loop-mediated isothermal amplification

A new in situ DNA amplification technique for microscopic detection of bacteria carrying a specific gene is described. Loop-mediated isothermal amplification (LAMP) was used to detect stxA(2) in Escherichia coli O157:H7 cells. The mild permeabilization conditions and low isothermal temperature used in the in situ LAMP method caused less cell damage than in situ PCR. It allowed use of fluorescent antibody labeling in the bacterial mixture after the DNA amplification for identification of E. coli O157:H7 cells with an stxA(2) gene. Higher-contrast images were obtained with this method than with in situ PCR.     

Detection of Escherichia coli 0157:H7 using a surface plasmon resonance biosensor

The BIAcore biosensor was used to detect binding of Escherichia coli O157:H7 with specific antibodies. Immobilized Protein A or Protein G captured antibodies which in turn bound to the bacteria. Alternatively, immobilized antibody captured the E. coli O157:H7 and the bacteria were further probed by a second antibody which enhanced the signal. The regenerated sensor surfaces were used for at least 50 separate analyses. The surface plasmon resonance biosensor has potential for use in rapid, real-time detection and identification of bacteria, and to study the interaction of organisms with different antisera or other molecular species. © Rapid Science Ltd. 1998     

Interaction of live and dead Escherichia coli O157:H7 and fluorescent microspheres with lettuce tissue suggests bacterial processes do not mediate adherence

AIMS: The goal of this study was to determine whether any specific bacterial processes (biochemical or genetic) or cell surface moieties were required for the interaction between Escherichia coli O157:H7 and lettuce plant tissue. METHODS AND RESULTS: Escherichia coli O157:H7 and Fluospheres (fluorescent polystyrene microspheres) were used in experiments to investigate interactions with lettuce. Fluospheres were used as they are a non-biological material, of similar size and shape to a bacterial cell, but lack bacterial cell surface moieties and the ability to respond genetically. Live and glutaraldehyde-killed E. coli O157:H7 attached at levels of c. 5.8 log(10) cells per cm(2) following immersion of lettuce pieces into a suspension containing c. 8 log(10) CFU ml(-1). In a separate experiment, numbers of bacteria or Fluospheres associated with lettuce decreased by c. 1.5 log cm(-2) following a 1-min wash. Exposure times of 1 min, 1 h, or 6 h had little effect on the level of attachment for Fluospheres, and live or killed cells of E. coli O157:H7 to lettuce tissue. SIGNIFICANCE: These results indicate that bacterial processes and cell surface moieties are not required for the initial interaction of E. coli O157:H7 to lettuce plant tissue.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

APPLICATIONS OF TIME-RESOLVED FLUOROIMMUNOASSAY TO DETECT MAGNETIC BEAD CAPTURED ESCHERICHIA COLI O157:H7

A time-resolved fluorescence technique was developed to detect Escherichia coli O157:H7 in ground beef burger. After a 4.5 h enrichment period, streptavidin coated magnetic beads conjugated with biotin-labeled anti E. coli O157:H7 were used to capture the bacteria. The bacteria were, at the same time, also labeled by a nonfluorescent, europium (Eu)-tagged anti-E. coli O157:H7 antibody. The sandwiched bacterial complexes were then concentrated using a magnetic particle concentrator and washed to remove other solution components. Upon addition of an enhancement buffer, the Eu-labels were then released from the antibodies and chelated to nitrilo-triacetic acid (NTA) and trioctylphosphine oxide (TOPO) to form highly fluorescent Eu-(2-NTA)₃(TOPO)_2–3 micellar complexes. Delayed fluorescence associated with these complexes was measured and its intensity was used to estimate the original bacterial concentration spiked in hamburger. This approach was applied to detect E. coli O157:H7 spiked in hamburgers. The results indicated this method is able to detect 1 CFU/g of the bacteria after a brief enrichment for four and half hours at 37C. Specificity studies indicated that the approach exhibited no or limited cross reactivity to Salmonella typhimurium, E. coli K-12 or Shigella dysenteriae spiked in hamburgers. Thus, the developed approach may be used as a rapid screening procedure for E. coli O157 bacteria in foods.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Multilocus sequence typing and virulence factors analysis of Escherichia coli O157 strains in China

Escherichia coli O157:H7, an important food-borne pathogen, has become a major public health concern worldwide. The aim of this study was to investigate the molecular epidemiologic feature of E. coli O157:H7 strains in China. 105 E. coli O157:H7 isolates were collected from various hosts and places over 9 years. A multilocus sequence typing scheme (MLST) was applied for bacteria genotyping and polymerase chain reaction (PCR) was used for virulence factor identification. Seven new MLST sequence types (STs), namely ST836, ST837, ST838, ST839, ST840, ST841, and ST842 were identified, which grouped into two lineages. Phylogenetic analysis suggested that the most two frequent STs in China, ST837 and ST836, may be the derivatives of E. coli O157:H7 Sakai or E. coli O157:H7 EDL933. Geographical diversity and host variety of E. coli O157:H7 were observed in China. In addition, the different distribution of tccp was detected. The data presented herein provide new insights into the molecular epidemiologic feature of E. coli O157:H7, and aid in the investigation of the transmission regularity and evolutionary mechanism of E. coli O157:H7.     

Method for assessment of functional affinity of antibodies for live bacteria

A rapid and convenient method for assessment of functional affinity of antibodies against live bacteria is described. When a combination of immunomagnetic separation (IMS) with bioluminescent or fluorescent genetic labelling of the cells was employed, the method showed good correlation with plate count. However, the use of reporter bacteria allowed results to be obtained within 1 h compared with days using conventional methods. Due to its lower detection limit, the bioluminescent assay performed better than the fluorescent assay. Antibody affinities for Escherichia coli O157:H7 and Salmonella enteritidis were examined at different environmental conditions such as pH 3-7, temperature 4-25 degrees C, and sodium chloride concentrations 0-5% and compared with sensitivities of ELISA.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

Validating the use of green fluorescent-marked Escherichia coli O157:H7 for assessing the organism behaviour in foods

AIMS: Monitoring bacterial kinetics in food is of great importance in food safety. The targeted micro-organism has to be identified accurately among competitive flora. Using green fluorescent protein (GFP)-transformed strains is a possible answer to such issues. However, quantitative studies require that this transformation does not alter the micro-organism behaviour: parent and transformed organisms were thus compared. METHODS AND RESULTS: Three Escherichia coli O157:H7 strains were transformed using a GFP-plasmid expressing. Parent and transformed strains were compared according to their genetic characteristics and serotypes. Growth ability was also assessed in constant and fluctuating temperature profiles. Cardinal values of pH, water activity and temperature were computed. No differences were observed between parent and transformed strains for all these experiments. The plasmid was satisfactorily maintained within transformed strains throughout the studies. Growth was eventually monitored in beef meat. CONCLUSIONS: Using the GFP marker is of great value, as it allows easier enumeration of E. coli O157:H7 in food in the presence of natural microflora. Using transformed strains is legitimate: their behaviour does not differ from that of their parent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: GFP transformation appears to be a valuable and reliable tool for challenge testing studies and predictive microbiology.