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. 2003 Apr;41(4):1673-80.
doi: 10.1128/JCM.41.4.1673-1680.2003.

Tandem tetramer-based microsatellite fingerprinting for typing of Proteus mirabilis strains

Tomasz Cieślikowski  1 , Dobrosława Gradecka, Magdalena Mielczarek, Wiesław Kaca
Affiliations

Tandem tetramer-based microsatellite fingerprinting for typing of Proteus mirabilis strains

Tomasz Cieślikowski et al. J Clin Microbiol. 2003 Apr.

Abstract

Two microsatellite tandem repeated tetramers, (GACA)(4) and (CAAT)(4), were used for Proteus mirabilis strain differentiation. The microsatellite-based PCR tests were applied for the examination of interstrain diversity for 87 P. mirabilis strains. Forty-six of the investigated strains were clinical isolates (5 were hospital isolates and 39 were outpatient clinic isolates); 42 strains were derived from the Kauffmann-Perch collection of laboratory strains. Fingerprinting done with the tetramers had a high discrimination ability [0.992 and 0.940 for (GACA)(4) and (CAAT)(4), respectively]. The distributions of clinical isolates among well-defined laboratory strains, determined by numerical analysis (unweighted pair-group method with arithmetic averages; Dice similarity coefficient), proved their genetic similarity to reference strains in the Kauffmann-Perch collection. This analysis also indicated that it is possible to estimate some phenotypic properties of P. mirabilis clinical isolates solely on the basis of microsatellite fingerprinting.

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Figures

FIG. 1.
FIG. 1.
Electrophoretic resolution by (GACA)4 PCR of six representative P. mirabilis strains derived from the Kauffmann-Perch collection. The fingerprinting procedure accuracy was tested three times for each strain. Lanes: 1 to 3, PrK 75/57; 4 to 6, PrK 66/57; 7 to 9, PrK 62/57; 10 to 12, PrK 34/57; 13 to 15, PrK 18/57; and 16 to 18, PrK 15/57. The repeated procedures included culturing, DNA extraction, and amplification. Experiments were done with 3 ml of inoculum (lanes 3, 6, 9, 12, 15, and 18), 1 liter of secondary inoculum (lanes 2, 5, 8, 11, 14, and 17), and 8 liters of culture (lanes 1, 4, 7, 10, 13, and 16). Lanes M, molecular weight markers. The white arrows on the left indicate bands applied as internal references for normalization procedure in the UPGMA analysis.
FIG. 2.
FIG. 2.
Accuracy of (CAAT)4 PCR fingerprinting for six representative P. mirabilis strains derived from the Kauffmann-Perch collection. Lanes: 1 to 3, PrK 66/57; 4 to 6, PrK 62/57; 7 to 9, PrK 38/57; 10 to 12, PrK 34/57; 13 to 15, PrK 18/57; and 16 to 18, PrK 15/57. The testing procedures included culturing, DNA extraction, and amplification. See the legend to Fig. 1 for a further description of lanes. Lanes M, molecular weight markers. The white arrows on the left indicate bands used as internal reference standards for normalization in the UPGMA analysis.
FIG. 3.
FIG. 3.
Reproducibility of (GACA)4 PCR (left) and (CAAT)4 PCR (right) in UPGMA band pattern analysis (the Dice similarity coefficient was used). An 0.8% position tolerance value was used. The calculation program GelCompar, version 4.0, was used. Three probes of each strain (A, B, and C) resolved on the same gel and one probe of some of the same strains from another gel (not labeled) are compared. The scales represent the level of homology between the investigated probes.
FIG. 4.
FIG. 4.
Electrophoretic resolution by (CAAT)4 PCR of representative P. mirabilis strains derived from the Kauffmann-Perch collection. Lanes: 1, PrK 74/57 (O48); 2, PrK 69/57 (O43); 3, PrK 64/57 (O38); 4, PrK 61/57 (O35); 5, PrK 58/57 (O32); 6, PrK 56/57 (O31) (P. vulgaris); 7, PrK 53/57 (O30); 8, PrK 52/57 (O29); 9, PrK 51/57 (O28); 10, PrK 50/57 (O27); 11, PrK 49/57 (O26); 12, PrK 47/57 (O24); 13, PrK 46/57 (O24); 14, PrK 45/57 (O24); 15, PrK 43/57 (O23); 16, PrK 41/57 (O23); 17, PrK 38/57 (O20); and 18, PrK 34/57 (O18). Lanes M, molecular weight markers.
FIG. 5.
FIG. 5.
UPGMA (Dice) cluster analysis of 35 P. mirabilis laboratory strains by (CAAT)4 PCR. The calculated values for clustering errors are boxed. The scale at the left represents the homology level. The broken line indicates the accuracy of the method. The arrows indicate the main groups of indistinguishable strains for the cutoff value established at 90% similarity.
FIG. 6.
FIG. 6.
Representative electrophoretic patterns of (GACA)4 PCR for out-of-clinic isolates of P. mirabilis. Lanes: 1, ZOZ 63b; 2, ZOZ 42; 3, ZOZ 203; 4, ZOZ 216; 5, ZOZ 256; 6, ZOZ 58; 7, ZOZ 352; 8, ZOZ 304; 9, ZOZ 303; 10, ZOZ 302; 11, ZOZ 87; 12, ZOZ 253; 13, ZOZ 191; 14, ZOZ 72; 15, ZOZ 14; 16, ZOZ 13; 17, ZOZ 19; 18, ZOZ 630; 19, ZOZ 670; 20, ZOZ 168; 21, ZOZ 173; 22, ZOZ 367; and 23, ZOZ 105. Lanes M, molecular weight markers.
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