Evaluation of PCR-based methods and ribotyping performed with a mixture of PstI and SphI to differentiate strains of Salmonella serotype Enteritidis

Abstract

The capacity to differentiate Salmonella serotype Enteritidis strains by PCR ribotyping; RAPD typing with three arbitrary primers and ribotyping with a mixture of PstI and SphI or 'PS ribotyping', was evaluated on a series of 65 strains associated with human infections and 11 reference strains. The series had been analysed previously by phage typing and ribotyping performed with PstI and Sph I, separately. All methods typed all the strains; however, only ribotyping showed good reproducibility and sensitivity. Twenty-two PS ribotypes (discrimination index = 0.74) were identified, differentiating strains ascribed to seven phage types (PTs 1, 4, 6, 6a, 7, 8 and RDNC) as well as phage untyped strains. Conversely, some strains of PTs, 1, 4, 5a, 6, 6a, 7, 34 and RDNC showed the most frequent PS ribotype. By PCR ribotyping a single profile was found; while by RAPD typing, one, two or three RAPD types were identified with the primers MK22, OPB6 and OPB17, respectively. All Spanish strains were assigned to a single combined RAPD type, except PTl1 strains which showed a different and specific RAPD type with OPB17. The banding patterns defining the PS ribotypes were interpreted more easily and the patterns could be compared more accurately than the banding patterns defining RAPD types. A similarity dendrogram generated from the 22 PS ribotypes was traced and compared with RAPD types and phage types. Data from this work indicated that 'PS ribotyping' was the most useful genetic procedure to differentiate Enteritidis strains, and, therefore, it can be used as a complementary or alternative typing method to phage typing within this serotype.

Diversity of Strains of Salmonella enterica Serotype Enteritidis from English Poultry Farms Assessed by Multiple Genetic Fingerprinting

“…At present, there is not a consensus as to which method is best suited for differentiation of serotype Enteritidis strains. Comparison of antibiotic resistance patterns (19), plasmid profiles (32,38), IS200 restriction fragment length polymorphism (31), ribotyping (16)(17)(18), and pulsed-field gel electrophoresis (PFGE) (20,27,34,35) have been used to differentiate strains for epidemi-ological purposes with various degrees of success. Also several PCR-based methods, such as random amplification of polymorphic DNA, have been used for this purpose, but these methods may lack sensitivity and reproducibility for routine use, and they do not appear to be suitably discriminatory due to the inability to separate artefactual variation and true polymorphism (18,36).…”

Subtyping of Salmonella enterica Serotype Enteritidis Strains by Manual and Automated Pst I- Sph I Ribotyping

“…While the stability of the ribotype patterns was not probed extensively here, repeat analysis of each isolate gave identical results in all but one instance. Similarly, Landeras and Mendoza (12) found that only a single band of about 7 kb, similar to the weak band that migrated at about 6.6 kb in this study, was not always reproducible and, hence, did not include that band in pattern analysis.…”

“…In the absence of phage typing data, PFGE has not been effective for distinguishing S. enterica serotype Enteritidis PT 1 isolates from serotype Enteritidis PT 4 isolates due to pattern similarities (16). Randomly amplified polymorphic DNA (RAPD) analysis, PCR ribotyping, and typing by PCR for repetitive elements (M13 and the enterobacterial repetitive intergenic consensus sequence [ERIC]) have low indices of discrimination (9,11,12,15). This can create problems when clusters of similar isolates are detected within a short period of time and can result in extensive and costly epidemiologic investigations that are ultimately unproductive.…”

“…However, some strains remain untypeable because their DNA was not restricted (7). Recently, Landeras and colleagues (10,12,13) developed a method for ribotyping of S. enterica serotype Enteritidis isolates using a mixture of restriction enzymes, PstI and SphI (PS; PS ribotyping), and this appeared to be more discriminatory than other methods (10,11,14). However, the efficacy of the PS ribotyping method for the differentiation of S. enterica serotype Enteritidis PT 8 strains has been tested with only a relatively few isolates.…”

Investigation of a food-borne Salmonella Oranienburg outbreak in a Mexican prison

“…Nath et al [40] obtained a D value of 0.8978 for the serotype Typhi. In the case of the serotype Enteritidis, Landeras and Mendoza [44] reported a D value of 0.31, Soto et al [43] of 0.52, and Fernandez et al [45] of 0.68.…”