Identification of Foodborne Baterial Pathogens by Gene Probes

Probes and Their Targets

Campylobacter jejuni: Ribosomal RNA

A probe that is specific for C. jejuni ribosomal RNA genes has been developed (86,87) and is available commercially. A pool of randomly selected and tested chromosomal fragments is also specific for C. jejuni, but the target has not been reported (83).

Escherichia coli: Heat-labile enterotoxin genes

The heat-labile enterotoxins (LT) of E. coli are a closely related group of proteins; they are distinguished from heat-stable enterotoxins (ST) by being immunogenic and are inactivated by heating at 60°C for 10 Min (31). The toxins stimulate adenylate cyclase (30) and can be detected by tissue culture assays of Chinese hamster ovary cells (30) or mouse Y-l adrenal cells (13). Using these tests, So et al. (102) localized and cloned the structural gene for LT; Dallas et al. (8) recloned a smaller fragment into plasmid pEWD299. Although there are several different genes for LT, as evidenced by their nucleotide sequences (56,73,103,110,111), they all share a significant amount of genetic similarity. The region of the LT genes chosen as a gene probe target is identical in each of these genes, so that all strains with the genetic potential to produce LTs should be detected.
The LT probe, eltA11, is a 20 base synthetic oligonucleotide that encodes amino acids 45-51 of the A subunit of the E. coli LT (111).

E. coli: Heat-stable enterotoxin genes

The heat-stable enterotoxin (ST) of E. coli is distinguished from LT (above) by heat stability and lack of immunogenicity. It can be detected by the suckling mouse bioassay (12) and acts by stimulating guanylate cyclase (22). There are at least two different types: ST I (also known as STa and STP) and ST II (also known as STb and STH). The latter toxin is not active in the infant mouse assay. These genes have been cloned and the nucleotide sequences of the region encoding STa and STb have been determined (74,82,101).
The STP probe is a 22 base synthetic oligonucleotide for the toxin type strain first isolated from pigs. It targets the region of the gene that encodes amino acids 4-12 of the toxin protein.
The STH probe is targeted to the ST elaborated by a strain of E. coli isolated from a human. The probe is also 22 bases long and targets the region of the STH that encodes amino acids 19-26 of the toxin.
Both of these probes have been tested for their specificity, and data are available on their ability to detect a few ST-producing cells against a high level of ST-negative microorganisms (35). The reliability of the colony hybridization technique with oligonucleotide probes was tested by collaborative study, using pure cultures of strains harboring the STH or STP genes (36).

Enteroinvasive Escherichia coli (EIEC) and Shigella: Invasive gene

Some strains of E. coli invade colonic epithelial cells, multiply intracellularly, and spread intercellularly, causing a dysenteric enteritis similar to that caused by Shigella (15). However, an important difference is that the infectious dose for Shigella may be as low as 1-10 organisms, whereas 108 EIEC cells are necessary to cause disease. A number of genetic determinants that encode virulence factors of EIEC and Shigella spp. are located on a large [220 kilobase (kb) pair] invasion plasmid (96). Loss of this virulence plasmid renders the bacterium avirulent (97). A 17 kb EcoRI fragment was used as a hybridization probe to detect invasive Shigella species and EIEC (4).
Small and Falkow (100) demonstrated that a 2.5 kb pair HindIII fragment of the large plasmid is required for invasion of human epithelial cells. Plasmid DNA involved in the invasion of HeLa cells by S. flexneri has also been cloned (61). These regions of the plasmid have been sequenced and are genetically similar (54). A probe from this region of the plasmid is specific for tissue culture cell-invasive EIEC and Shigella. Such probes also identify strains that are invasive in the guinea pig eye assay (98). Of 41 probe-positive isolates tested by the guinea pig method, 2 were negative, indicating that a few strains may be invasive in tissue culture assays but not in tests that require a greater number of pathogenic determinants (108). A synthetic probe of 18 bases has been constructed. Its target is within a gene that encodes for a virulence factor.

Enterohemorrhagic E. coli (EHEC): Shiga-like toxin (SLT) genes

Human illnesses ranging from simple diarrhea to hemorrhagic colitis and hemorrhagic uremic syndrome have been associated with strains of E. coli that produce moderate to high levels of Shiga-like toxins (SLTs). Strains of E. coli serotype O157:H7 are the most significant pathogens associated with hemorrhagic colitis; strains of serotype O26:H11 are also classified as EHEC. More than 50 other serotypes of E. coli that produce SLTs have been identified, but the correlation of these serotypes with disease is uncertain. Two related but distinct cytotoxins, SLT I and SLT II, have been characterized. Individual strains produce one or both cytotoxins. For example, E. coli O157:H7 produces SLT I, SLT II, or both, whereas O26:H11 produces only SLT I. The DNA sequences of SLT I and SLT II structural genes have been published, and analysis shows that SLT I has 99% homology with the Shiga toxin gene of S. dysenteriae type 1, but SLT II has only 60% homology (41). Two synthetic oligodeoxyribonucleotide probes were prepared from sequence data from the A-subunit regions of the SLT I and SLT II genes (nucleotides 473-490 and 472-490, respectively). HC agar (M62) (see ref. 105) was the selective medium chosen to screen isolates and foods for E. coli strains that carry the SLT gene because the growth of E. coli O157:H7 is less inhibited on HC agar than on other selective media. HC agar contains NaCl and a lower concentration of bile salts No. 3. Its plating efficiency of a strain of E. coli O157:H7 at 37 and 43°C for 17 h was similar to that of plate count agar. Plating efficiencies of other E. coli serotypes that carry the SLT gene have not been determined. The modified enrichment procedure of Doyle and Schoeni (14) is included in the method for detection of low level contamination of foods. For additional information about enterohemorrhagic strains of E. coli, see the review by Karmali (46).

Enterohemorrhagic E. coli (EHEC): O157:H7 serotype-specific probe

The fluorogenic MUG assay for E. coli is based on the activity of the -glucuronidase (GUD) enzyme, which is encoded by the uidA gene in E. coli. Although isolates of serotype O157:H7 are negative with the MUG assay, genetic studies have shown that this EHEC serogroup also contains uidA gene sequences for the GUD enzyme (21). Sequencing analysis has determined that the uidA gene of O157:H7 serotype contains several base mutations; therefore, it is not identical to the uidA gene of MUG assay (+) E. coli. Although the base mutations in the uidA allele of O157:H7 do not appear to be responsible for the absence of the MUG phenotype, one of the base changes was found to be conserved among the O157:H7 serogroup. An oligonucleotide probe, PF-27, directed to this base alteration was developed and determined to be specific only for EHEC isolates of serotype O157:H7. Other SLT-producing EHEC and other pathogenic E coli or enteric bacteria failed to hybridize with PF-27 (20).

Listeria monocytogenes: Invasion-associated protein (iap) and hemolysin (hly) genes

Of the seven Listeria species that have been isolated from a variety of foods, including dairy, vegetable, meat, and poultry products, only L. monocytogenes has been implicated in human disease. Genetic and physiological studies have incriminated an extracellular hemolysin as one of the virulence factors in L. monocytogenes (5,26,47). This hemolysin (also called listeriolysin O or alpha-listeriolysin) has been cloned and sequenced (11,55,64). Several oligonucleotides (including AD13) were constructed by using the sequence of the listeriolysin O gene (64) and can specifically identify L. monocytogenes in foods by colony hybridization (11,70).
A 5.3 kb DNA fragment encoding a 60 kilodalton (Kdal) protein (msp) associated with hemolytic activity has been cloned (24). Kuhn and Goebel (53) reported the cloning and sequencing of a gene (iap) whose product (a 60 Kdal protein) may be involved in the uptake of L. monocytogenes by nonprofessional phagocytes. Sequence analysis revealed that the msp and iap share extensive homology, which indicates that msp and iap may be the same gene (51). An internal region of this gene was sequenced (Datta, unpublished results) and a synthetic probe, AD07, was used to identify and enumerate L. monocytogenes in a number of foods (9,10,34). Thus, either AD07 (for the iap gene) or AD13 (for the hly gene) can be used to detect and enumerate L. monocytogenes in foods. To avoid false-negative results because of "silent" mutations in the gene (nucleotide changes that affect DNA probe binding but do not change the gene function), both probes should be used in combination (designated AD713).

Salmonella species:

Originally, several restriction endonuclease fragments selected randomly from the Salmonella chromosome were used as probes to identify members of the genus (23). Although these molecules served as specific probes, the role played by the target genes was never reported. More recently, probes were developed for regions of the bacterial ribosomal gene that are unique for salmonellae. These probes were used to develop a commercial kit that also uses a nonisotopic labeling and detection system (7).

Staphylococcus aureus: entB probe

Six groups (A, B, C1, C2, D, and E) of related enterotoxins associated with pathogenicity are elaborated by some strains of S. aureus and can cause symptoms of staphylococcal food poisoning if ingested (1). The genes for enterotoxins A, B, C1, and E (entA, entB, entC1, and entE) have been cloned and sequenced (2,3,6,43,85).
Three synthetic oligonucleotide probes were synthesized according to the sequence of the entB gene and used to test 210 strains of S. aureus (78). One probe was specific for entB; the others hybridized with strains producing enterotoxin C. The former probe was used to detect EntB-producing S. aureus in artificially contaminated crabmeat (Trucksess and Williams, manuscript in preparation). Although the nucleotide sequences of enterotoxin genes for groups A, C1, and E are known, synthetic probes have not been reported.

Vibrio cholerae: Cholera toxin

The classical cholera enterotoxin (CT) is a major virulence factor in pathogenic strains of V. cholerae. The mechanism of action and immunological reactivity is quite similar to the LT of E. coli. Genes encoding this multisubunit protein were cloned and sequenced (57,58,63). Non-O1 V. cholerae enterotoxin genes are apparently similar to classical CT (33). Two sequences from the A subunit structural gene for production of the classical enterotoxin are used as probes to detect the CT gene: ctxA11 (bases 702-721) and ctxA12 (bases 718-735).

Vibrio parahaemolyticus: Thermostable direct hemolysin

An important foodborne pathogen often associated with seafood, V. parahaemolyticus can produce a thermostable direct hemolysin (TDH) (95), also referred to as the Kanagawa phenomenon-associated hemolysin (69). This phenotype is commonly associated with strains isolated from humans with gastroenteritis but rarely found in environmental isolates (44). It is not yet known if this hemolysin is a virulence factor, but epidemiological evidence suggests that it is. The gene for the hemolysin has been cloned and sequenced (45,75,106). The specificity of both the cloned probes (76) and a synthetic oligonucleotide probe, tdh3 (77) has been established. The tdh3 probe is 18 bases long and its target encodes amino acids 122-128 of the tdh gene.

Vibrio vulnificus: Cytotoxin/hemolysin

V. vulnificus has been implicated as a cause of human infections and septicemia. The primary source of infection appears to be raw or undercooked seafood, especially raw oysters (71). This lactose-positive vibrio produces a cytotoxin/hemolysin which was implicated as a virulence factor (28), and the gene has been cloned (109). A 3.2 kb DNA fragment carrying the structural gene for this protein is a specific probe for V. vulnificus (72) and has been sequenced (112). One synthetic probe (VV6) exhibited 100% specific for 166 laboratory and environmental strains of V. vulnificus (FDA Contract No. 223-84-2031, Task XIII).

Yersinia pseudotuberculosis: Invasive gene (INV-3)

A chromosomal gene of Y. pseudotuberculosis, inv, which plays an integral part in Yersinia pathogenicity, has been cloned and sequenced (38,39). Oligonucleotide probe INV-3, based on published inv sequence (39) is 21 nucleotide bases long and targeted to a region 200 base pairs away from the 5' terminus of the inv gene of Y. pseudotuberculosis (19). Tests of INV-3 using Southern and colony hybridizations were compared with HeLa cell invasion studies and shown to be specific only for invasive Y. pseudotuberculosis isolates. Although there are homologous sequences between the inv genes of Y. enterocolitica and Y. pseudotuberculosis, this homology is not detectable by INV-3.

Yersinia enterocolitica: Chromosomal invasion gene (PF-13)

The genes responsible for mammalian cell invasion are also carried on the chromosome in Y. enterocolitica (66). Unlike Y. pseudotuberculosis, however, Y. enterocolitica has two loci that encode the invasion phenotype. The inv locus, homologous to the inv gene of Y. pseudotuberculosis, allows high level invasion of several tissue culture cell lines, whereas the ail gene shows more host specificity (66). Analysis of Yersinia serotypes and species using cloned probes from inv and ail showed that all disease-causing isolates are tissue culture-invasive; all these isolates reacted with the AIL gene probe (67). The INV probe reacted with both tissue culture-invasive and noninvasive isolates; however, recent evidence suggests that the inv in these noninvasive strains may not be expressed. The oligonucleotide probe PF13 is targeted specifically to a region 60 base pairs away from the 3' terminus of the ail gene of Y. enterocolitica. The probe is 18 nucleotides in length. A comparison of colony and Southern hybridization studies of 150 yersiniae and non-yersiniae isolates and HeLa cell invasion studies showed that PF13 hybridized only with invasive Y. enterocolitica isolates (19).

Yersinia enterocolitica: Plasmid gene (SP-12)

All pathogenic Yersinia species carry a 42-48 Mdal plasmid (pYV), which encodes for many of the virulence-associated phenotypes (84). These include Ca2+-dependent growth, mouse lethality, cytotoxicity, Sereny reaction, production of V and W antigens, serum resistance, and production of outer membrane proteins (YOPs). The pYV plasmid of Y. enterocolitica was subcloned and the region encoding for HEp-2 cell cytotoxicity and Sereny reaction was identified and sequenced (91). A 24 base oligonucleotide probe, SP12, targeted to this region was shown to be specific for the virulence plasmid. The use of SP12 for detecting pathogenic Y. enterocolitica isolates in artificially inoculated foods was also evaluated (65).

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