International Journal of Food Microbiology, 12 (1991) 67-76 © 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50
Identification of foodborne pathogens by nucleic acid hybridization W a l t e r E. Hill a n d S t a c y e P. K e a s l e r Molecular Biology Branch, Division of Microbiology, Center for Food Safe~ and Applied Nutrition. Food and Drug Administration, Washington, DC. U.S.A. (Received 16 August 1990; accepted 5 October 1990)
Nucleic acid hybridization methods have been developed and used to identify microorganisms in foods. Tests performed on mixed cultures save the time required to establish pure cultures. Enterotoxigenic or invasive strains of foodborne bacterial pathogens are detected with probes that identify genes responsible for virulence. Hybridization tests signal the presence or absence of a particular strain or an entire genus and are especially well suited for screening foods for specific pathogens. With the colony hybridization assay format, foodborue bacteria harboring a specific gene can be enumerated. However. hybridization tests require the presence of 105 to 106 cells to yield a positive result, thereby limiting sensitivity and necessitating a time-consuming growth step. In vitro DNA amplification techniques increase the amount of DNA segments 105-106-fold in 2 to 3 h, thus enhancing test sensitivity. Key words: DNA hybridization; Foodborne pathogens: Polymerase chain reaction: Gene probes
The health hazards presented by foodborne bacterial and viral pathogens far outweigh the impact on morbidity and mortality caused by chemicals and parasites in foods. Contamination of foods by pathogenic bacteria and viruses accounted for about 717o of the 910 confirmed foodborne disease outbreaks and 977o of the 54 453 cases of foodborne illness in the United States between 1983 and 1987 (Bean et al.,
1990). Of the approximately 2400 outbreaks during this period, the etiological agent was confirmed in only about 4070. These figures probably underestimate the total impact, as noted by the Centers for Disease Control, Atlanta, GA, and emphasize the need for improved investigatory tools. Rapid methods, in general, focus on reducing both the time required and the amount of labor needed to obtain a result. Such techniques are critical because they facilitate analysis and, therefore, the response to foodborne disease outbreaks. Many of these rapid tests are suitable for screening large numbers of test samples but generally are hampered by requiring enrichment and selective culturing steps. Rapid methods based on DNA hybridization techniques, however, can identify microCorrespondence address: W.E. Hill, Molecular Biology Branch, Division of Microbiology, Center for Food Safety and Appfied Nutrition, Food and Drug Administration, Washington, DC 20204, U.S.A.
68 organisms in mixed cultures, obviating the need to isolate and characterize pure strains. Recent developments in DNA amplification techniques may reduce the time required for analysis to less than 8 h.
D N A hybridization tests
A number of rapid and exquisitely specific DNA hybridization tests for identifying microorganisms have been developed. These methods are based on our ability to obtain specific segments of DNA that have known functions and to label these molecules so that their presence is observed easily. These pieces of DNA are often called gene probes because they can be used to probe the genetic information of DNA isolated from bacterial cells. General procedures for using gene probes have been recently reviewed (Keller and Manak, 1989). An important step in developing gene probes is to decide what type of microorganism needs to be identified. Gene probes can be made genus-, species-, or strain-specific. Often, when gene probes for disease-causing strains of bacteria are developed, the probes are targeted to a gene that plays a significant role in pathogenesis. Species-specific and genus-specific probes are usually targeted to variable regions found in ribosomal RNA genes. The advent of DNA sequencing techniques (Hames and Higgins, 1985) and the in vitro synthesis of oligonucleotides (Gatt, 1984) make 'synthetic' DNAs popular choices for gene probes. The sensitivity of most DNA hybridization tests used with food require 105 to 10 6 copies of the target DNA to generate a reliable, positive signal for labelling and identification systems that are based on radioactivity or enzymes. Although these copies are generally obtained by allowing cells to grow under appropriate cultural conditions, recent advances in enzymatic replication allow the production of sufficient copies in vitro at rates that far exceed that of the biological duplication of cells. Two classes of formats are commonly used for the examination of foods using nucleic acid hybridization assays: DNA colony hybridization and solution hybridization. Each technique has its own constellation of benefits and drawbacks.
Colony hybridization A popular format used with DNA hybridization tests for the analysis of foods is based on colony hybridization (Griinstein and Hogness, 1975; Maas, 1983). Small amounts (about 0.1-0.2 ml) of homogenized food are spread-plated onto appropriate media and incubated until colonies appear. A filter replica is made, the colonies are lysed in situ, and the DNA is denatured by high pH, steaming, or microwaving (Datta et al., 1987). The filters are then used in standard hybridization protocols with radioactive or enzyme-labelled gene probes. Often, autoradiography is used to identify 32p-labelled probes. Enzyme-labelled probes are observed by using chromogenic substrates. Each colony identified as positive represents a colony forming unit in the food homogenate that contains the same gene as that present in
69 the gene probe. Quantitative results are obtained by counting the number of positive colonies and multiplying by the dilution used to make the original spread plate. Colony hybridization can be used to identify small numbers of cells of particular strains even when high backgrounds of indigenous microflora are present (Hill et al.. 1983a). However, when aerobic plate counts reach 10 6 tO 10 7 per gram, efficiency is reduced because the target cells cannot complete a sufficient number of cell cycles to produce colonies containing 105 t o 10 6 copies of the target gene (Hill et al., 1985: Jagow and Hill, 1988). Fortunately, selection techniques can be used in conjunction with plating to improve sensitivity (Jagow and Hill, 1988). Colony hybridization allows the indirect selection of pure cultures from colonies that react with the gene probe. If sterile filters are used and the master plate is saved, colonies on the master plate that are probe-positive can be identified and used to establish pure cultures. Colony hybridization loses some sensitivity as a result of test sample dilution. This is unfortunate because determining whether foods harbor low numbers of particular bacterial pathogens can be important. Normally, a food is diluted ten-fold when homogenized in buffer before plating. When spread plates are used, the volume plated is usually about 0.1-0.2 ml, so that the material on a colony hybridization filter represents a 50- to 100-fold dilution of the original test sample. A single probe-positive colony observed under these conditions represents a titer of 50 to 100 cells per gram. Therefore, the sensitivity of this procedure is insufficient for identifying microorganisms that have limits, for example, of 1 cell per 25 grams. Sample concentration techniques, quantitative amplification of gene copies, or the application of hybridization techniques in a most-probable-number format are required to improve this level of sensitivity while retaining the ability to enumerate particular pathogens.
Liquid hybridization In liquid hybridization systems, both target and probe molecules are free in solution. This format has a kinetic advantage over solid support systems such as colony hybridization because both reactants are mobile (Meinkoth and Wahl, 1984). Sensitivity may also be improved over colony hybridization because fewer target molecules may be required to obtain a positive signal. This aspect of sensitivity is a n advantage over colony hybridization, in which cell debris may interfere with the ability of probes to reform a double helix with immobilized target sequences, leaving only a small fraction of the target available for hybridization and resulting in reduced sensitivity. The largest technical obstacle to liquid hybridization systems is the problem of removing a labelled probe that is not bound to the target DNA. Several multiprobe systems have been developed to deal with this troublesome aspect of homogeneous hybridization formats (Ranki et al., 1983; Chan et al., 1989). Early gene probe tests relied heavily on radioactive labels (primarily 32p) because of the non-specific background problems observed when antigen-antibody or enzymatic identification systems were used (Leary et al., 1983; Leary and Ruth, 1989). Because capture probes and detector probes do not interact with each other, background levels can be significantly reduced by performing additional capture
70 and release cycles. This format has been used to produce a relatively rapid, simple. reliable, and low background non-radioactive hybridization assay system (Curiale et al., 1990).
Gene probes for foodborne bacterial pathogens The first cloned probes used to identify pathogenic bacteria in foods were those for the genus Salmonella (Fitts et al., 1983; Fitts, 1985), enterotoxigenic Escherichia coli (Hill, 1981; Hill et al., 1983a), and pathogenic Yersinia enterocolitica (Hill et al.. 1983b). These early probes were used with target DNAs immobilized on membrane filters. The Salmonella probes were targeted to random chromosomal fragments, but the probes to pathogenic E. coli and Y. enterocolitica identified specific virulence-related genes carried on plasmids. Following the sequencing of genes involved in pathogenicity, synthetic oligonucleotide probes were used for enterotoxigertic E. coli (Hill et al., 1985), the thermostable direct hemolysin of Vibrio parahaemolyticus (Nishibuchi et al., 1986), a hemolysin of Listeria monocytogenes (Datta et al., 1988), enterotoxin B of Staphylococcus aureus (Notermans et al., 1988), an invasive factor in Y. enterocolitica (Milliotis et al., 1989), and the cytolysin of V. vulnificus (Yamamoto et al.. 1990). While the first probes used to identify all salmonellae were based on random fragments, recently developed probes target particular regions of the ribosomal RNA. These sections of the genome are well suited as phylogenetic tools because their variable and conserved regions often match particular taxonomic groups (Hogan, 1989). Since many ribosomal gene sequences are known, synthetic probes can be easily selected. Also, test sensitivity can be increased because bacterial cells usually contain more than 1000 copies of ribosomal RNA. Synthetic probes will probably continue to grow in popularity. They are readily available commercially for a relatively low cost compared to that of purifying cloned probes. Oligonucleotide probes are also more specific than the longer, cloned probes. The reliability of several probes in identifying foodborne bacteria has been determined by collaborative study (Hill and Payne, 1984; Hill et al., 1986; Curiale et al., 1990; Flowers et al., 1987). Generally, results of probe methods agreed with those of conventional methods in 95-98% of cases. In no instance were they statistically significantly worse.
Gene probes for viruses Very little work has been reported on the use of gene probes to identify viruses in foods. However, a considerable amount of research on DNA hybridization techniques for identifying viruses in clinical and environmental specimens has been
71 carried out. Some of these methods will probably be applied to the analysis of foods in the same manner that clinical tests developed for the identification of pathogenic bacteria in laboratory specimens were adapted to the screening of foods. Many of the gene probes for identifying viruses consist of viral inserts and bacterial plasmid vectors. Hepatitis A virus in fecal specimens and other enteroviruses have been identified by using cloned cDNA probes (Ticehurst et al., 1987: Tassopoulos et al., 1986; Hyypia et al., 1984). Rotavirus has also been identified with cDNA probes in fecal specimens (Lin et al., 1985: Dimitrov et al., 1985). Unfortunately, clinical specimens, especially stools, often contain DNA sequences homologous to vector sequences possibly as a result of bacterial contamination (Ambinder et al., 1986). The occurrence of false positives, therefore, can be problematic in specimens that harbor even normal microflora. Using purified inserts as probes and a control probe for the vector alone is recommended. To avoid these problems, probes not associated with bacterial plasmids have been developed. The high degree of stability of R N A - R N A hybrids makes single-stranded RNA probes made by in vitro transcription of cloned cDNA ideal for identifying viral RNA. Hepatitis A virus in sewage-polluted water and stool has been successfully identified with such an RNA probe (Jiang et al., 1987). Poliovirus-derived RNA probes have been used to identify enteroviral RNA in cell culture lysates and. with less success, in clinical specimens (Petitjean et al., 1990; Cova et al., 1988). Synthetic oligonucleotide probes for enteroviruses have been derived from coxsackievirus nucleotide sequences (Bruce et al., 1989). DNA templates suitable for the polymerase chain reaction (PCR) can be generated by reverse transcription of viral RNA. A PCR-based identification and typing test for rotavirus in fecal specimens correlates well and provides information much more quickly (Gouvea et al., 1990) than standard methods. PCR assays for hepatitis B (Baginski et al, 1990) and enteroviruses (Rotbart, 1990) have been reported.
Target amplification As stated above, current gene probe techniques for foodborne pathogens usually require 105 to 10 6 copies of the target sequence to provide a reliable, positive signal. Typically, this number of copies (cells) is obtained by allowing cells to grow either on solid media for colony hybridization or in selective enrichment broths for liquid hybridization. These growth steps usually require at least overnight incubation or longer if multistage enrichments are carried out. Such incubations often add considerably to the total time needed for analysis. Recently, several techniques have been developed for the in vitro replication of specific segments of nucleic acids. These methods may substantially shorten the time needed for conducting assays to 1-2 h, or possibly a few minutes. Furthermore, because the theoretical limit of detection is one molecule of target DNA, test sensitivity could be improved greatly. Once a large amount of target is available, non-radioactive identification systems may be used.
72 The PCR, which has been automated (Saiki et al., 1988). can generate more than a one milfion-foid increase of a particular D N A region in 1 - 2 h and has been applied to m a n y aspects of genetics and molecular biology, such as sequencing, cloning, and gene probe generation (Innis et al., 1990; Erlich et al., 1989). Because most molecules generated by P C R are of uniform size, gel electrophoresis can be used as the diagnostic step. Hybridization or D N A labels are not necessary. This reaction has been used to identify invasive strains of Shigella flexneri and E. coli in artificially contaminated lettuce (Lampel et al., 1990) and V. vulnificus in ovsters (Hill et al., 1990). PCR-based identification of bacterial pathogens in environmental test samples has been reported (Atlas and Bej, 1990). Although hepatitis A virus was responsible for 29 of 41 (71%) of the confirmed foodborne outbreaks of viral etiology, the only P C R report deals with the identification of hepatitis B virus (Baginski et al., 1990). The RNA-directed R N A polymerase, Qfl replicase, can be used to produce as m a n y as a billion copies of a replicatable sequence within 30 rain (Kramer and Lizardi, 1989). This feature can be incorporated into a rapid, and sensitive diagnostic test (Lomeli et al., 1989); however, no applications to foods have been reported. The recently published amplification method of Guatelli et al. (1990) proceeds at a single temperature and does not require thermal cycling as does PCR. The self-sustained sequence replication (3SR) system can produce a 10-million-fold amplification within 1 - 2 h. The widespread use of these methods awaits the development of simple test sample preparation protocols that produce D N A templates suitable for amplification. Unless the kinetics for these reactions can be carefully controlled, these amplification techniques are best used in p r e s e n c e / a b s e n c e tests. However, if the reactions are set up in a most-probable-number format, quantitative estimates may be obtained. Interpretation of assays based on amplification techniques must be done with caution because cells may not be viable and virions may not be functional in the test samples.
Gene probes that identify a wide range of foodborne bacterial pathogens have been developed. Probes are available to identify the bacteria that caused at least 71% of the confirmed foodborne disease outbreaks (Campylobacter, E. coil, Salmonella, Shigella, V. cholerae, and V. parahaemolyticus ) and 84% of the cases (42 398 of 50 217) from 1983 to 1987 in the United States (Bean et al., 1990). If the approximately 16 000 cases of saimonellosis from the large 1985 Chicago dairy outbreak were not included, about 77 rather than 84% of the cases could have been identified by the use of gene probes. Clearly, these figures suggest that gene probes can be important tools for the analysis of suspect foods during outbreaks of foodborne disease. Wider acceptance and implementation of these methods will occur with the development of additional safe and convenient kits based on non-isotopic labels.
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