Characterization of recurrent and sporadic Listeria monocytogenes isolates from raw milk and nondairy foods by pulsed-field gel electrophoresis, monocin typing, plasmid profiling, and cadmium and antibiotic resistance determination

Abstract

Following previous surveys to assess the incidence of Listeria monocytogenes in raw milk and nondairy foods processed in Northern Ireland, isolates were characterized as recurrent or sporadic on the basis of multilocus enzyme electrophoresis (MEE) analysis and restriction fragment length polymorphism typing. In the present study, 45 representative recurrent and sporadic electrophoretic types (ETs) previously identified by MEE were subjected to pulsed-field gel electrophoresis (PFGE) of genomic DNA macrorestriction fragments, monocin typing, plasmid profiling, and an examination of resistance to cadmium and nine different antibiotics. Although PFGE proved to be capable of subdividing a number of recurrent and sporadic ETs, the grouping of strains arrived at by PFGE and MEE were in broad agreement, and previous conclusions regarding the designation of L. monocytogenes strains as recurrent or sporadic remained unaltered. It is considered that PFGE was able to detect minor genetic changes in recurrent ETs which occurred during the time period in which food surveys were carried out. Production of type E monocin (Types A to E were found among the 45 strains), plasmid carriage, and resistance to cadmium occurred more frequently in recurrent than in sporadic strains and may be important with regard to the ability of L. monocytogenes to persist in food and food-processing environments. Only 2 of 45 strains showed resistance to any of the nine antibiotics tested: two sporadic strains were resistant to tetracycline (MIC, 64 microg x ml(-1)).

FIG. 1

PFGE resolution of ApaI (A) and AscI (B) restriction fragments of genomic DNA from 12 L. monocytogenes strains (lanes 2 to 7 and 9 to 14). Lanes 1, 8, and 15 of each gel contained XbaI restriction fragments of genomic DNA from an E. coli reference strain (G5244) as molecular size markers. PFGE was performed at 200 V with pulse times ramped from 4 to 40 s over 22 h. The dendrograms showing the relationships between ApaI (C) and AscI (D) profiles for the 12 strains were created using software supplied by Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom. The software uses the neighbor-joining method of Saitou and Nei (17) to compare profiles on the same gel. The numbers on the branches of the dendrograms denote the lengths of the branches. The shorter the distance, the more similar the lanes.

FIG. 2

Dendrogram showing the relatedness of band patterns derived from PFGE of AscI-restricted genomic DNA from 23 L. monocytogenes strains isolated sporadically and recurrently in successive raw-milk samples obtained from processors A to D. PFGE was performed at 200 V with pulse times ramped from 4 to 40 s over 22 h. When the same ET was recovered on successive visits to a processor (ET 27, ET 35, and ET 40), these were regarded as recurrent strains. The dendrograms and depiction of band patterns were created using software supplied by Nonlinear Dynamics. The software uses the UPGMA algorithm of Sneath and Sokal (20) to compare profiles between gels.

FIG. 3

Dendrogram showing the relatedness of band patterns derived from PFGE of AscI-restricted genomic DNA from 22 L. monocytogenes strains isolated sporadically and recurrently in nondairy food samples obtained from processors J, K, M, and N. PFGE was performed at 200 V with pulse times ramped from 4 to 40 s over 22 h. When the same ET was recovered on successive visits to a processor (ET 09 and ET 42), these were regarded as recurrent strains. The dendrograms and depiction of band patterns were created using software supplied by Nonlinear Dynamics. The software uses the UPGMA algorithm of Sneath and Sokal (20) to compare profiles between gels.

FIG. 4

Subtyping of recurrent L. monocytogenes ETs by PFGE of AscI restriction fragments of genomic DNA. PFGE was performed at 200 V with pulse times ramped from 4 to 40 s over 22 h. The recurrent strains ET 35 (lanes 2 to 4), ET 40 (lanes 6 to 8), and ET 42 (lanes 10 to 12) were isolated from food samples obtained on successive visits to processors B, D (milk), and M (nondairy food), respectively. XbaI restriction fragments of genomic DNA from an E. coli reference strain (G5244) were used as molecular size markers (lanes 1, 5, and 9).