Direct Detection Of Pathogens Research Articles

A Simple Method for the Direct Detection of Salmonella and Escherichia coli O157:H7 from Raw Alfalfa Sprouts and Spent Irrigation Water Using PCR

The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10−1 to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10−1 CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.

Infectious Diseases Testing

Molecular methods have been applied widely for the diagnosis of infectious diseases. Beginning with solution hybridization in the early 1990s, multiple methods for nucleic acid hybridization and amplification have been introduced into the laboratory for the identification and characterization of microbial pathogens. This unit contains examples of several basic approaches for microbial detection or characterization in the laboratory. Methods in this chapter include automated nucleic acid extraction, direct detection of microbial pathogens, and characterization of pathogens by DNA sequencing or typing. Any of these methods could be customized for the characterization of bacteria, fungi, parasites, or viruses.

Direct Detection of Bacterial Pathogens in Brain Abscesses by Polymerase Chain Reaction Amplification and Sequencing of Partial 16S Ribosomal Deoxyribonucleic Acid Fragments

To evaluate the feasibility of detecting bacterial pathogens directly from the clinical brain abscess specimens by polymerase chain reaction (PCR) amplification and sequencing of bacterial 16S ribosomal deoxyribonucleic acid (rDNA). A total of 14 specimens were tested by both culture and PCR amplification, targeting the full-length or a partial region of 16S rDNA. 16S rDNA is known to be conserved in bacteria. Sequencing of partial-length and full-length 16S rDNA was performed. The sequence data were compared with known sequences of 16S rDNA in the National Center for Biotechnology Information GenBank by using the Basic Local Alignment Search Tool (BLAST) algorithm. The species with the best match of similarity were regarded as the pathogenic species in the samples. We also developed a Streptococcus-specific multiplex PCR analysis for identifying members of the Streptococcus species, the most common pathogen of brain abscesses. The 10 culture-positive specimens were all PCR-positive for partial 16S rDNA, but only seven were positive for full-length 16S rDNA amplification. Bacterial DNA was not detected in the remaining four specimens with a negative culture. Species identification by phenotypes from culture was in agreement with that by sequencing results of partial-length (or full-length) 16S rDNA. The Streptococcus-specific PCR analysis could detect Streptococcus species correctly in one step. Bacterial 16S rDNA sequences provide reliable clues to the identification of unknown pathogens. PCR analysis of 16S rDNA and sequencing may identify pathogens to the species level directly from brain abscesses. This approach is rapid and is useful especially in the identification of slow-growing and fastidious organisms.

Direct detection of bacterial pathogens in representative dairy products using a combined bacterial concentration-PCR approach

To develop a simple, rapid method to concentrate and purify bacteria and their nucleic acids from complex dairy food matrices in preparation for direct pathogen detection using polymerase chain reaction (PCR). Plain non-fat yogurt and cheddar cheese were each seeded with Listeria monocytogenes or Salmonella enterica serovar. Enteritidis in the range of 10(1)-10(6) CFU per 11-g sample. Samples were then processed for bacterial concentration using high-speed centrifugation (9700 g) followed by DNA extraction, PCR amplification, and amplicon confirmation by hybridization. Bacterial recoveries after centrifugation ranged from 53 to >100% and 71 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the non-fat yogurt samples; and from 77 to >100% and 69 to >100% for serovar. Enteritidis and L. monocytogenes, respectively, in the cheddar cheese samples. There were no significant differences in recovery efficiency at different inocula levels, and losses to discarded supernatants were always <5%, regardless of dairy product or pathogen. When followed by pathogen detection using PCR and confirmation by amplicon hybridization, detection limits of 10(3) and 10(1) CFU per 11-g sample were achieved for L. monocytogenes and serovar. Enteritidis, respectively, in both product types and without prior cultural enrichment. This study represents progress toward the rapid and efficient direct detection of pathogens from complex food matrices at detection limits approaching those that might be anticipated in naturally contaminated products.

Recommendations for microbial source tracking: Lessons from a methods comparison study

The methods comparison study described in accompanying manuscripts demonstrated the potential value of microbial source tracking (MST) techniques, but also identified a need for method refinement. This paper provides three classes of recommendations to improve MST technology: optimization, development and evaluation. Optimization recommendations focus on library-dependent methods and include improved selection of restriction enzymes or antibiotics, better definition of appropriate library size, selection of target species and choice of statistical pattern-matching algorithms. Methods development recommendations focus on identifying new genomic targets and quantification procedures for library-independent methods. Longer-term methods development recommendations include integration of microarrays and other direct pathogen detection technology with MST. Studies defining host specificity and population dynamics should aid selection of target species during methods development. Evaluation recommendations include enhancements that should be incorporated into future methods comparison studies, along with studies to assess the value of MST results for risk characterization.

Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples

The application of PCR for the direct and sensitive detection of food-borne pathogens is largely affected by the quality of the template DNA prepared from food samples. In the present study, a chemical extraction method of bacterial DNA from spiked milk samples for the direct detection of Staphylococcus aureus and Yersinia enterocolitica was evaluated by PCR. Gene specific primers were designed to target the nuclease (nuc) and the attachment invasion locus (ail) genes of S. aureus and Y. enterocolitica, respectively and used in PCR. A combination of organic solvents, detergents and alkali in the DNA extraction method permitted a detection limit of 10cfuml−1 milk for both the pathogens. When equal numbers of S. aureus and Y. enterocolitica were spiked in milk samples, the individual detection limit was determined to be 103cfuml−1 milk. Simultaneous amplification of 482 and 359bp fragments of thenuc and ail genes was obtained using the primer pairs in a single reaction. Multiplex PCR enabled the detection of 104cfuml−1 milk of S. aureus and Y. enterocolitica without any pre-enrichment step. A combination of conventional isolation technique and PCR using DNA extracted by the proposed method was used to test raw milk samples for possible contamination with S. aureus and Y. enterocolitica. The presence ofS. aureus in the tested samples was indicated by both the methods while Y. enterocolitica could not be detected in any of the samples. The template DNA extraction method developed in this study is rapid, sensitive and avoids interference from potential PCR inhibitors and demonstrates the potential of detecting multiple pathogens in milk samples without any enrichment.

Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes.

We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture. The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome. Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag. The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection. Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level. Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented. The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca. 30 CFU mg(-1) of tissue was estimated. A similar detection limit was observed for several other gram-negative pathogens. This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.

Application of molecular biological methods for diagnosis and epidemiology of human fungal infections

For mycological diagnosis molecular methods can be applied to detect the pathogen directly without prior cultivation and to identify species and subspecies. For the detection of infecting agents specific DNA probes and/or the polymerase chain reaction (PCR) are widely used, whereas normally only PCR can provide sufficient sensitivity for the direct detection of pathogens in clinical material. Prospects and limitations of PCR approaches for the detection of pathogenic fungi reported in the literature will be discussed. DNA polymorphisms which are useful for species identification and epidemiological strain typing of medically relevant fungi can be detected by such methods as the analysis of restriction fragment length polymorphisms (RFLP), and Southern hybridization with appropriate DNA probes, and as karyotyping by pulsed field gel electrophoresis (PFGE). These techniques which could be applied successfully to different epidemiological studies are, however, laborious and time consuming. By using a PCR-fingerprinting method which can be performed much simpler polymorphic DNA regions are amplified with different non-specific primers. Distinctive and reproducible sets of amplification products were observed for 26 different Candida and 8 other fungal species. The number and size of the amplification products obtained were characteristic for each species. By comparing species-specific PCR-fingerprints of clinical isolates with those of reference strains, clinical isolates could be identified to the species level even if they could not be identified by conventional typing methods. With all primers, PCR-fingerprints also displayed intraspecies variability. Therefore, PCR-fingerprinting can also be applied for epidemiological strain characterization among medically relevant fungi.

Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions.

DNA from Listeria monocytogenes was used as the model system from this investigation, with PCR primers based on the listeriolysin O gene. Under standard polymerase chain reaction (PCR) conditions and with no prior treatment, amplification failed in the presence of more than 5% milk. Since inhibition of the PCR occurred at the same milk concentrations with full fat, half fat and fat-free milk, inhibition was not attributed to the fat content of the milk. Calcium ions were, however, identified as a major source of PCR inhibition. The results demonstrated that the inhibitory effects of calcium ions and milk could be partially reversed by increasing the magnesium concentration in the reaction to well above the standard levels normally required for PCR. This work has important implications for the use of the PCR in the direct detection of food pathogens.

A Microbiological Paradox: Viable but Nonculturable Bacteria with Special Reference to Vibrio cholerae

The observation that directly-detectable bacterial cells are unable to grow on bacteriological culture media under certain conditions raises questions regarding the viability of these cells. Various terminologies have been used to describe substrate-responsive and metabolically-active bacterial cells that cannot be cultured. The currently-accepted term is “viable but nonculturable.” During the past 15 years, the viable but nonculturable phenomenon has been actively investigated. Bacterial pathogens in the viable but nonculturable state can maintain virulence and produce disease. These organisms may escape detection if bacteriological culture methods are solely used. Thus, methods for direct detection of specific pathogens in food, water and environmental samples are preferable. Viable but nonculturable Vibrio cholerae have been extensively studied, and several sensitive and reliable direct-detection kits have been developed. Viable but nonculturable forms of bacteria are now recognized as a common phenomenon, observable in many bacterial species, which suggests that standard bacteriological laboratory protocols for assessing microbiological safety of food and drinking water are less reliable than direct detection methods.

Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters.

The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible. These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification. We have developed an assay for the detection of bacterial cells in large volumes of water. Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction. This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell. This technique could be used for the detection of any bacteria or virus in water or air.

Current and possible alternate indicators of fecal contamination in tropical waters: A short review

AbstractBacteria that belong to the total and fecal coliform groups have been used for several decades as indicators of fecal pollution of waters and other systems. The prevailing idea behind this is that these two groups of bacteria cannot replicate outside the gastrointestinal tract of warm‐blooded animals. Several studies conducted by this and other laboratories have indicated that these bacteria may be naturally present in certain areas of the world. Naturally occurring fecal coliforms (the genera Escherichia and Klebsiella) have been isolated from pristine sites and from sites where there was no apparent fecal contamination. All these results lead us to the conclusion that these groups of bacteria are not adequate indicators of fecal contamination in tropical areas of the world. Alternate indicators are being tested. This laboratory and others around the world are testing the adequacy of bacteriophages (more specifically, coliphages) for this purpose. In Puerto Rico coliphages have been detected only in areas known to be contaminated with domestic sewage, but not in pristine areas, although high levels of fecal coliforms can be detected at the latter sites. All these results suggest that coliphages may be used as indicators that more realistically demonstrate fecal contamination of waters in tropical areas of the world. In addition, newer techniques, although still in the trial stages, can be used to directly detect pathogens in the environment. Of all the techniques currently available, the polymerase chain reaction is the most promising for the direct detection of pathogens and therefore for the unequivocal evaluation of the biological quality of waters.

New Rapid Detection Methods for Salmonella and Listeria

We are now very much aware of the hazards associated with salmonella and listeria in foodstuffs. Newer methodologies are compared with conventional techniques for their rapid detection. Recent developments in culture media are discussed, as is a new rapid culture test kit for salmonella. Novel techniques such as ELISA, nucleic acid probes and flow cytometry are described, and a comparative assessment is made of reported findings. Nucleic acid probe and ELISA assays are considerably quicker than traditional culture techniques but they would benefit from increased sensitivity; they show inferior specificity with listeria and are still dependent on an enrichment stage. Recent DNA amplification techniques could provide a solution to the problem of poor probe sensitivity and allow the direct detection of pathogens from food samples. Flow cytometry may have the potential for high sensitivity and specificity and could be useful for on‐line monitoring in factories.

Probe directed at a segment of Rickettsia rickettsii rRNA amplified with polymerase chain reaction

In an effort to explore a sensitive taxon-specific detection system for bacteria, we sequenced the 16S rRNA from two strains of Rickettsia rickettsii, five other rickettsiae, and Coxiella burnetti to find a probe site unique to R. rickettsii. We then synthesized a 16-mer that hybridized only to the rRNA of R. rickettsii. Using a primer complementary to a sequence found only in rickettsial rRNA, we then generated a cDNA. We amplified the probe site in a 180-base segment of the cDNA by using the cDNA primer and a second primer also unique to rickettsiae in a polymerase chain reaction. The segments of rRNA from each of the rickettsiae were amplified 10(6)- to 10(7)-fold, and the R. rickettsii probe hybridized only to the amplified segment from R. rickettsii. The rRNAs from Staphylococcus aureus, C. burnetii, and Neisseria meningitidis were not amplified and did not hybridize with the probe. The approach detailed below may prove clinically useful in the direct detection of pathogens that are difficult to cultivate.

Problems with infection in pregnancy. Sensible serology and direct detection of pathogens in pregnancy

Problems with infection in pregnancy. Sensible serology and direct detection of pathogens in pregnancy

Indicator organisms for estuarine and marine waters

The use of indicator organisms for estuarine and coastal waters has been reviewed. The natural flora of the environment must be considered in selecting an indicator organism, but, more importantly, recent work which shows a viable but non‐recoverable stage of pathogens entering the marine environment demonstrates that the conventional detection of indicator microorganisms is misleading, if not inaccurate. Results suggest that the newly developed epifluorescent/immunofluorescent direct detection of pathogens in the environment may be the most reliable method for determining public health hazards in marine and estuarine waters.

Indicator organisms for estuarine and marine waters

The use of indicator organisms for estuarine and coastal waters has been reviewed. The natural flora of the environment must be considered in selecting an indicator organism, but, more importantly, recent work which shows a viable but non-recoverable stage of pathogens entering the marine environment demonstrates that the conventional detection of indicator microorganisms is misleading, if not inaccurate. Results suggest that the newly developed epifluorescent/immunofluorescent direct detection of pathogens in the environment may be the most reliable method for determining public health hazards in marine and estuarine waters.