Multiplex PCR method for detection of three Aeromonas enterotoxin genes.

A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay.

Antibiotic Resistance of Agricultural and Foodborne Salmonella Isolates in Canada: 1986-1989.

A total of 689 Salmonella cultures isolated during 1986-1989 from Canadian agricultural products and from imported fish, shellfish, and reptiles were examined for resistance to a test panel of 11 antibiotics. The incidence of antibiotic resistance in strains from all sources seemingly increased during the study period, whereas the occurrence of resistance within individual sample categories fluctuated annually. Although poultry figured as a major reservoir of resistant salmonellae (53.4%), red meats and fish/shellfish also yielded substantial numbers of resistant strains. The range of streptomycin (27.1 to 48.7%) and tetracycline (24.3 to 37.8%) resistance among poultry and red meat isolates, and identification of meat isolates carrying chloramphenicol (0.4 to 9.1%) and ampicillin (3.4 to 11.4%) resistance codons was disquieting. Most of the multiply-resistant (≥ 2 antibiotics) strains belonged to somatic serogroups B and C, with poultry occurring as the principal reservoir of multiresistant phenotypes. Of the 27 resistance patterns encountered in this study, all but two contained a resistance determinant for streptomycin and/or tetracycline. These findings underscore a disturbing level of antibiotic resistant Salmonella in the food chain, and the need to reassess the alleged benefits of subtherapeutically medicated feeds in current animal husbandry practices.

Enzyme Immunoassay - Membrane Filter Method for Detection of Salmonellae in Foods.

An enzyme-linked immunosorbent assay (ELISA) technique using a horseradish peroxidase-protein A-Spicer Edwards antiserum complex was developed for the detection of Salmonella colonies on membrane filters. In pure culture, 64 Salmonella species tested gave a positive reaction (purple stain). Of 22 naturally contaminated food samples, there was an exact correlation between the AOAC hydrophobic grid-membrane filter procedure and the ELISA technique (40.9% positives). This technique is simple, requires little equipment and can be completed in less than 2.5 h, thus allowing the detection of Salmonella spp. in foods within 48 h from initiation of sampling.

Salmonella Detection in Foods: Present Status and Research Needs for the Future.

Standard cultural procedures generally require 4 to 5 d for presumptive evidence of Salmonella in foods. Attempts at greater method brevity have resulted in the use of selective enrichment cultures as test material for short immunological tests including fluorescent antibody (FA), enrichment serology (ES), enzyme-linked immunosorbent assay (ELISA), direct immunoenzyme (DI) and membrane filter-disc-immunoimmobilization (MFDI) assays. Nonimmunological tests such as the lysine-iron-cystine-neutral red (LICNR) broth and a 14C-dulcitol radiometric technique have also been applied to enrichment broth cultures. Sensitivity of short (4 to 6 h) incubation of selective enrichment broths has yet to be established. The need for rapid, cost-efficient preenrichment-dependent analytical schemes is clear. Investigations on the modification of the Limulus amoebocyte lysate (LAL) test to detect Salmonella cell wall antigens in preenrichment cultures or application of the ELISA, the hydrophobic-grid-membrane (HGMF) techniques or other rapid diagnostic tests to preenrichment cultures are indicated.

Effective Enrichment-Plating Conditions for Detection of Salmonella in Foods.

The performance of tetrathionate brilliant green (TBG) and selenite cystine (SC) enrichments, and bismuth sulfite (BSA) and brilliant green sulfa (BGS) plating media was assessed on the basis of data on 2085 Salmonella -contaminated low and high moisture foods that were collected during a 6-year (1977 to 1983) study involving 22 laboratories. None of the eight enrichment- plating combinations considered identified all positive samples. TBG was markedly more sensitive than SC for detection of salmonellae in high moisture foods, where enrichment in TBG and plating on both BSA and BGS identified 92% of contaminated samples. Plating of SC enrichment cultures on the two agar media was substantially less productive (63%). With low moisture foods, TBG and SC rates of isolation varied by less than 10% under homologous conditions. Plating media did not affect method sensitivity. Identification of numerous contaminated samples by TBG or SC enrichment alone underlines the value of using multiple enrichment and plating media in standard methods.

Update on Preenrichment and Selective Enrichment Conditions for Detection of Salmonella in Foods.

The search for short and sensitive cultural methods for detection of Salmonella in foods has met with limited success. Short (3-8 h) incubation of non-selective enrichment media do not provide conditions for effective resuscitation of stressed or injured salmonellae and result in unacceptably high numbers of false-negative results. Isolation of Salmonella is not dependent on the nutritional value of preenrichment media; simple media such as lactose and nutrient broths are equally reliable as highly nutritive sterility testing media. The need for detergents in non-selective enrichment of fatty foods and use of preenrichment transfer volumes greater than 1 ml is not indicated. Although selective enrichment in tetrathionate brilliant green (TBG) broth at an elevated temperature (43 C) increases method sensitivity, use of Mueller-Kauffman TBG under similar analytical conditions may be inhibitory to Salmonella . Refrigeration of preenrichment and selective enrichment broth cultures has been used successfully to provide greater analytical flexibility by increasing the number of days on which analyses can be initiated without engendering weekend work.