Molecular analysis of Pseudomonas aeruginosa: epidemiological investigation of mastitis outbreaks in Irish dairy herds

Abstract

Pseudomonas aeruginosa is a pathogen in both humans and animals. This bacterium, most often associated with respiratory infections in cystic fibrosis patients, was found to be the causative agent in bovine mastitis outbreaks among 11 Irish dairy herds. Epidemiological findings suggested that the infection was spread to all herds by teat wipes that had been contaminated with this organism. Two molecular-typing strategies were used in an attempt to determine the genomic relationship(s), if any, of the P. aeruginosa strains isolated from the various herds and to verify whether the same strain was responsible for each outbreak. Thirty-six isolates from the mastitis outbreaks were tested and compared to fourteen clinical isolates from Cork University Hospital. With one exception, all outbreak-linked strains produced identical patterns when ribotyped with ClaI and PvuII enzymes. Eight of the clinical isolates gave the same ClaI ribotype pattern as the mastitis-causing strains. However, PvuII proved more discriminatory, with only the outbreak isolates producing identical patterns. Similar results were obtained with RW3A-primed DNA amplification fingerprinting, with all outbreak isolates except one displaying the same fingerprint array. The clinical strains produced several fingerprint patterns, all of which were different from those of the mastitis-causing isolates. Fine-resolution DNA fingerprinting with a fluorescence-labelled RW3A primer also identified a number of low-molecular-weight polymorphisms that would have remained undetected by conventional methods. These data support the view that the same P. aeruginosa strain was responsible for the mastitis outbreaks in all 11 herds.

FIG. 1

Ribotype patterns of a representative selection of Pseudomonas isolates after digestion with ClaI. Molecular weight markers ([DIG]-labelled lambda phage DNA fragments digested with HindIII [Boehringer]) are shown in lanes M. Lane 1, ATCC 27853 (group I); Lane 2, CIT-V2 (II); lane 3, CIT-V3 (II); lane 4, CIT-V4 (II); lane 5, CIT-V5 (II); lane 6, CIT-V6 (II); lane 7, CIT-V15 (III); lane 8, CIT-H1 (IV); lane 9, CIT-H2 (II); lane 10, CIT-H3 (VI); lane 11, CIT-H4 (II); lane 12, CIT-H5 (IV); lane 13, CIT-H6 (IV); lane 14, CIT-H7 (II); lane 15, CIT-H8 (II); lane 16, CIT-H9 (II); lane 17, CIT-H10 (V); lane 18, CIT-H11 (VI); lane 19, CIT-H12 (II); lane 20, CIT-H13 (VI); and lane 21, CIT-H14 (II). Numbers in parentheses denote ribotype groups.

FIG. 2

DNA fingerprint patterns of representative Pseudomonas isolates typed with the primer RW3A. PCR products (10-μl volumes) were loaded onto a 2% agarose gel in 1× Tris-acetate-EDTA buffer containing 0.1 μg of ethidium bromide per ml. Samples were electrophoresed at 100 V for 90 min. Lanes M contain molecular weight markers (grade III; Boehringer). Lane M* contains a mixture of molecular weight markers (grades III and V). Lane 1, ATCC 27853 (group I); lane 2, CIT-V2 (II); lane 3, CIT-V3 (II); lane 4, CIT-V4 (II); lane 5, CIT-V15 (III); lane 6, CIT-H1 (IV); lane 7, CIT-H2 (V); lane 8, CIT-H3 (VI); lane 9, CIT-H5 (VII); lane 10, CIT-H6 (VIII); lane 11, CIT-H7 (IX); lane 12, CIT-H8 (X); lane 13, CIT-H9 (VI); lane 14, CIT-H10 (XI); lane 15, CIT-H11 (XII); lane 16, CIT-H12 (XIII); and lane 17, CIT-H13 (XIV). Numbers in parentheses denote DAF groups.

FIG. 3

GeneScan traces of Pseudomonas isolates. Blue peaks represent 6-carboxyfluorescine-labelled RW3A-generated DNA fragments which were sized by direct comparison with the 6-carboxy-X-rhodamine-labelled internal standards (red peaks). The size range is indicated by the solid red arrowheads below the final trace. Regions of interest within the Pseudomonas genome are indicated by hatched and open bars and a solid blue bar for some isolates. Polymorphic DNA fragments are denoted by the solid green peak with corresponding molecular lengths.

FIG. 3

GeneScan traces of Pseudomonas isolates. Blue peaks represent 6-carboxyfluorescine-labelled RW3A-generated DNA fragments which were sized by direct comparison with the 6-carboxy-X-rhodamine-labelled internal standards (red peaks). The size range is indicated by the solid red arrowheads below the final trace. Regions of interest within the Pseudomonas genome are indicated by hatched and open bars and a solid blue bar for some isolates. Polymorphic DNA fragments are denoted by the solid green peak with corresponding molecular lengths.