Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35 degrees C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Rapid detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water using peptide nucleic acid probes

A new chemiluminescent in situ hybridization (CISH) method that provides simultaneous detection, identification, and enumeration of Pseudomonas aeruginosa in bottled water within 1 working day has been developed. Individual micro-colonies of P. aeruginosa were detected directly on membrane filters following 5 h of growth by use of soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeted to a species-specific sequence in P. aeruginosa rRNA. Within each micro-colony, reaction of the peroxidase with a chemiluminescent substrate generated light that was subsequently captured by film or with a digital camera system. Each spot of light represented one micro-colony of P. aeruginosa. Sensitivity and specificity for the identification of P. aeruginosa were 100% as determined by testing 28 P. aeruginosa strains and 17 other bacterial species that included closely related Pseudomonas species. Furthermore, the number of micro-colonies of P. aeruginosa represented by light spots correlated with counts of visible colonies following sustained growth. We conclude that PNA CISH speeds up traditional membrane filtration techniques and adds the specificity of PNA probe technology to generate fast and definitive results.     

Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner

A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes and an array scanner for rapid detection, identification, and enumeration of Escherichia coli is described. The test utilizes Cy3-labeled peptide nucleic acid (PNA) probes complementary to a specific 16S rRNA sequence of E. coli. Samples were filtered and incubated for 5 h, the membrane filters were then analyzed by fluorescence in situ hybridization and results were visualized with an array scanner. Results were provided as fluorescent spots representing E. coli microcolonies on the membrane filter surface. The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter. Above this level, better accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as individual colonies once they were visible by eye.     

A novel approach for the identification of bacterial taxa-specific molecular markers

AIMS: To develop and establish a methodology for an oriented and fast identification of species taxa-specific molecular markers useful for the identification of micro-organisms. METHODS AND RESULTS: From the complete microbial genomes available in Pfam database, taxa-specific protein domains were identified which lead to the selection of taxa-specific loci. This strategy was used to identify six genetic markers: four specific for Pseudomonas syringae pv. tomato, one specific for P. syringae pv. syringae and one specific for P. putida. The discriminatory potential of these loci was evaluated by Southern hybridization using several pseudomonad species and pathovars, by dot-blot hybridization and by multiplex PCR optimized for the simultaneous detection of P. putida, P. syringae pv. syringae and P. syringae pv. tomato. Sensitivity assays indicated a detection limit of approximately 10 pg of chromosomal DNA template needed for each bacterium. CONCLUSIONS: The proposed methodology was efficient on the selection of six Pseudomonas-specific markers able to discriminate Pseudomonas at the species and pathovar level. SIGNIFICANCE AND IMPACT OF THE STUDY: The oriented search of taxa-specific molecular probes described in this work, which can be easily extended to other groups of bacteria, will improve the accuracy and expedite the identification of micro-organisms by DNA-based molecular methods.     

The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria, Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S. Florida

A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, Staphylococcus aureus and Pseudomonas aeruginosa, was achieved within 6-8 h of processing. Following 5 h of incubation on TSA, soybean peroxidase-labeled peptide nucleic acid probes (Boston Probes, Boston, MA) targeting species-specific 16S rRNA sequences of P. aeruginosa and S. aureus were used to hybridize microcolonies of the target species in-situ. In addition, a universal probe for 16S rRNA sequences was used to target the eubacteria. Probes were detected after a light generating reaction with a chemiluminescent substrate and their presence recorded on Polaroid film. The probes showed limited cross-reactivity with mixed indigenous bacteria extracted from seawater and sand by shaking with phosphate-buffered saline (PBS). Specificity and cross-reactivity was tested on the reference bacterial genera Pseudomonas, Staphylococcus, Vibrio, Shigella, Salmonella, Acinetobacter, Enterobacter, Escherichia and Citrobacter. These tests confirmed that the probes were specific for the microorganisms of interest and were unaffected by high salt levels. The results of the PNA chemiluminescent in situ hybridization were compared with traditional plate count methods (PCM) for total 'freshwater' eubacteria, S. aureus and P. aeruginosa. Counts of eubacteria and S. aureus were comparable with numbers obtained from traditional plate counts but levels of P. aeruginosa were higher with PNA than with PCM. It is possible that PNA is more sensitive than PCM because it can detect microcolonies on the agar surface that never fully develop with the plate count method. We conclude that the in situ hybridization technique used here represents an important potential tool for the rapid monitoring of novel indicator organisms in beaches and recreational waters.     

Identification of indicator microorganisms using a standardized PNA FISH method.

A standardized fluorescent in situ hybridization (FISH) method using Peptide Nucleic Acid (PNA) probes for analysis of gram-negative and gram-positive bacteria, as well as yeast, has been developed. Fluorescently labeled PNA probes targeting specific rRNA sequences of Escherichia coli, Pseudomonas aeruginosa, Staphyloccocus aureus, Salmonella were designed, as well as PNA probes targeting eubacteria and eucarya. These PNA probes were evaluated by PNA FISH using 27 bacterial and 1 yeast species, representing both phylogenetically closely related species, as well as species important to both clinical and industrial settings. The S. aureus and P. aeruginosa PNA probes did not cross react with any of the organisms tested, whereas the E. coli PNA probe, as expected from sequence data, also detected Shigella species. The Salmonella PNA probe reacted with all of the 13 Salmonella strains, representing the 7 subspecies of Salmonella, however, it is also complementary to a few other bacterial species. The eubacteria- and eucarya-specific PNA probes detected all bacterial species and one yeast species, respectively. The general applicability of the PNA FISH method made simultaneous identification of multiple species, both gram-negative and gram-positive, in a mixed population an attractive possibility never accomplished using DNA probes. Four color images using differently labeled PNA probes showed simultaneous identification of E. coli, P. aeruginosa, S. aureus and Salmonella, thereby demonstrating the potential of multiplex FISH for various diagnostic applications within both clinical and industrial microbiology.     

Multicolour digital image analysis system for identification of bacteria and concurrent assessment of their respiratory activity

AIMS: To develop a rapid and simple multicolour digital image analysis system for simultaneous identification of bacteria and assessment of their metabolic activity. METHODS AND RESULTS: We developed an image analyser capable of distinguishing triple-stained bacterial cells. Bacteria were stained with a nucleic acid stain, a fluorescent antibody and a fluorescent metabolic indicator for enumeration, species identification and assessment of metabolic activity. This multicolour image analyser was used to simultaneously identify Escherichia coli O157:H7 in milk samples and assess their respiratory activity. The images of the triple-stained bacteria were captured using a combination of blue light and u.v. excitation and an epifluorescence microscope and were processed by our image analyser. We found a good correlation between the counts of actively respiring (r = 0.93) and total (r = 0.94) E. coli O157:H7 measured by digital image analysis and visual observation. CONCLUSION: The multicolour digital image analysis system described here was able to quantify active pathogenic micro-organisms within 2 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This multicolour image analysis allows the rapid and simultaneous quantification of bacteria, identification of species and assessment of metabolic activity.     

Conventional culture for water quality assessment: is there a future?

Conventional culture for the detection, enumeration and identification of micro-organisms has been the traditional tool of the microbiologist. It is, however, time-consuming and labour-intensive and confirmed results often require several days of analysis. Culture may not grow the organisms being sought and for enumeration may only detect a small proportion of the total population. However, it does have the advantage of being simple to use and relatively inexpensive. It is also a direct means of assessing cell viability. Novel fluorogenic dyes and fluorgenic and chromogenic substrates have overcome some of these problems by providing a means of rapid and specific detection and enumeration whilst removing the need for subculture and confirmation tests. Immunological tests such as ELISA have significantly reduced analysis time by providing specific target organism detection. Molecular techniques have removed the need for culture. Improvements in sensitivity, and removal of the inhibitory nature of sample matrices, have allowed analysts to detect low levels of micro-organisms but the questions of viability and comparability with cultural techniques still remain. Are we about to see a change of culture in water quality assessment, or can cultural techniques be developed that reduce analysis time to a few hours and can rapid methods be used for detecting the presence and viability of organisms?     

Identification of waterborne bacteria by the analysis of 16S-23S rRNA intergenic spacer region

AIM: In this study, we evaluated, the use of universal primers, specific for the 16S-23S rRNA intergenic region, to detect and identify nine species that are of high interest for the microbiological control of water. METHODS AND RESULTS: The analysis of the fragments was carried out using a High Resolution acrylamide/bisacrylamide gels in a fluorescent automated DNA sequencer. The results showed specific profiles for each of the nine species but this technique failed to detect simultaneously micro-organisms in samples containing a mixed population. CONCLUSION: Nevertheless, the electrophoretic profiles obtained provided a very useful tool for the rapid and specific identification of water isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible new methodology for a rapid identification of pathogens in water.     

PNA for rapid microbiology.

The acceptance of rRNA sequence diversity as a criterion for phylogenetic discrimination heralds the transition from microbiological identification methods based on phenotypic markers to assays employing molecular techniques. Robust amplification assays and sensitive direct detection methods are rapidly becoming the standard protocols of microbiology laboratories. The emergence of peptide nucleic acid (PNA) from its status as an academic curiosity to that of a promising and powerful molecular tool, coincides with, and complements, the transition to rapid molecular tests. The unique properties of PNA enable the development of assay formats, which go above and beyond the possibilities of DNA probes. PNA probes targeting specific rRNA sequences of yeast and bacteria with clinical, environmental, and industrial value have recently been developed and applied to a variety of rapid assay formats. Some simply incorporate the sensitivity and specificity of PNA probes into traditional methods, such as membrane filtration and microscopic analysis; others involve recent techniques such as real-time and end-point analysis of amplification reactions.     

Development of real-time PCR methods for the rapid detection of low concentrations of Gluconobacter and Gluconacetobacter species in an electrolyte replacement drink

AIMS: To develop a rapid real-time polymerase chain reaction (PCR) method to detect Gluconobacter and Gluconacetobacter species in electrolyte replacement drinks. METHODS AND RESULTS: Samples of electrolyte replacement drinks were artificially contaminated with Gluconobacter species and then filtered to collect cells. DNA was extracted from the filters and analysed by real-time PCR on the ABI Prism 7000 system, using commercial detection kits for lactic and acetic acid bacteria. In addition, specific primers and Taqman probe were designed and used for the detection of seven Gluconobacter and Gluconacetobacter species. All the assays tested demonstrated a linear range of quantification over four orders of magnitude, suggesting detection levels down to 1 CFU ml(-1) in the original drink. CONCLUSIONS: A real-time PCR method was developed to detect low concentrations of Gluconobacter and Gluconacetobacter sp. in an electrolyte replacement drink. SIGNIFICANCE AND IMPACT OF THE STUDY: Real-time PCR methods allow a rapid, high throughput and automated procedure for the detection of food spoilage organisms. The real-time PCR assay described is as sensitive as the conventional method that involves pre-enrichment, enumeration on a selective agar (typically malt extract agar) and identification with a differential medium (typically Wallerstein nutrient agar). The real-time PCR assay also provides a more rapid rate of detection, with results in less than 24 h following enrichment for Gluconobacter and Gluconacetobacter species.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

Oligonucleotide probes for specific detection of Giardia lamblia cysts by fluorescent in situ hybridization

AIMS: Our study focused on the design of oligonucleotide probes and a suitable hybridization protocol that would allow rapid and specific identification of potentially viable cysts of the waterborne parasite Giardia lamblia. METHODS AND RESULTS: Comparative analysis of ribosomal RNA (rRNA) sequences of Giardia lamblia and a number of closely and more distantly related species identified six regions that appear to be specific for the G. lamblia 16S rRNA. Fluorescently labelled probes targeting these regions were produced and employed in fluorescent in situ hybridization (FISH) experiments. Two of the six probes tested successfully. CONCLUSION: Our study provides the first reported probes for specific FISH detection of G. lamblia. The method depends on sufficient amounts of intact rRNA in the target organism, which is unlikely to be present in nonviable cysts that have been exposed to the environment for a prolonged period. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, detection of G. lamblia cysts is largely based on immunofluorescence assays (IFA) targeting cyst wall surface antigens. These assays lack specificity and will detect species others than G. lamblia. Further, IFA will detect nonviable cysts and cyst wall fragments that do not pose a public health risk. In contrast, FISH probes allow specific detection and are likely to only detect viable, infectious cysts.     

Development and evaluation of a reverse dot blot assay for the simultaneous detection of common toxic microalgae along the Chinese coast

Toxic microalgae currently pose a great threat to human health, ecosystem, fishery, tourism, and aquaculture along the Chinese coast. The detection of toxic microalgae by routinely monitoring natural waters is necessary to provide timely mitigation. Therefore, an effective, simultaneous detection protocol should be established for the simple, rapid, and accurate identification of causative algae. This study developed and evaluated a reverse dot blot assay (RDBA) combined with a low-density membrane-based DNA array for the rapid and simultaneous detection of toxic microalgae that are commonly distributed along the Chinese coast. The large subunit rDNA D1–D2 regions of the target species were first sequenced to design taxonomic probes. Probe specificity was validated by performing a cross-reactivity test with dot blot hybridization. The tailed probes were immobilized onto a nylon membrane to prepare a low-density DNA array for RDBA. The established detection procedure involved DNA extraction, biotin (Bio)-labeling of objective sequences by multiple polymerase chain reaction (M-PCR), RDBA, coloration, and judgment of hybridization by the naked eye. Bio-labeled primer-based labeling proved to be an economical and effective method to prepare Bio-labeled PCR products for RDBA. The detection limits of RDBA using the M-PCR-labeling products from DNA templates prepared by different methods were also compared, and a kit-based DNA extraction method displayed the lowest detection limit of 0.5 cells. Simulation results showed that RDBA can recover all target species and was not affected by background DNA. RDBA was proven effective, specific, and sensitive for the simultaneous detection of toxic microalgae in the field samples. Therefore, this method may be used in the field monitoring of natural samples.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

The use of gene probes in the rapid analysis of natural microbial communities

Summary Hybridization probes produced from DNA sequences have proven to be a powerful tool in the rapid and sensitive analysis of natural microbial communities. By using function-specific probes, such as those identifying genes coding for photosynthesis, the potential a microbial community has for performing a given function may be rapidly determined. Gene probes have also been used in the identification and isolation of a specific catabolic genotype in less than one-fourth the time required for the conventional culture enrichment technique. Species-specific probes constructed from portions of genes coding for ribosomal RNA have been used for the rapid identification and enumeration of bacterial species in environmental samples. The use of reassociation kinetics as a measure of community diversity and complexity is also discussed. The successful application of this technique to community analysis may reduce the time required from 1 year, for conventional analysis, to 2 weeks.     

Development of specific fluorescent oligonucleotide probes for in situ identification of wine lactic acid bacteria

A rapid method for the identification of lactic acid bacteria (LAB) from wine has been developed. This method is based on fluorescence in situ hybridisation (FISH), using fluorescent oligonucleotide probes, homologous to 16S rDNA of those species of LAB commonly found in wines. The protocol for the specific detection of these bacteria was established through the hybridisation of 36 reference strains. The specificity of the probes was evaluated by using pure cultures. Probes were used to identify species in different wines, making it evident that direct identification and quantification from natural samples without culturing is also possible. The results show that FISH is a promising technique for the rapid identification of LAB, allowing positive identification in a few hours (4-16 h).     

Development of a real-time PCR-based method for rapid differential identification of Mycobacterium species

AIMS: To develop a real-time PCR method for rapid differential identification of many clinically important mycobacteria to the species level. METHODS AND RESULTS: Eighteen Mycobacterium species that are considered clinically important were targeted for the identification. One primer pair and 21 pairs of hybridization probes (HybProbes) specific for the genus, species or complex were designed based on the rpoB gene sequences of mycobacteria. Twenty-five different Mycobacterium reference species were tested. In a single round of real-time PCR, all the nontuberculous mycobacteria (NTM) species tested were identified at the genus level and 16 of the 18 targeted species were differentially identified to the species or complex level during the amplification cycles; subsequent melting curve analysis allowed the specific identification of all the target species at the species or complex level without cross-reactivity with the other species. CONCLUSIONS: The developed real-time PCR assay rapidly identifies the NTM at the genus level and 18 clinically important Mycobacterium species at the species or complex level. SIGNIFICANCE AND IMPACT OF THE STUDY: This real-time PCR assay provides a useful tool for the rapid differentiation of most clinically important Mycobacterium species.     

The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique.