We used DNA restriction fragments, derived from the exotoxin A gene and surrounding sequences, as an epidemiological marker for Pseudomonas aeruginosa. Using these DNA fragments as probes in Southern blot hybridizations and/or total genomic digestions, we were able to distinguish greater than 100 different strains of P. aeruginosa. The stability of the marker in vitro was established by using well-characterized strains, which were stored under different conditions and subjected to chemical mutagenesis. The stability of the marker within a given strain in vivo was established during experimental infection in the chronic rat lung model of pseudomonas pneumonia. P. aeruginosa serially cultured from individual patients with cystic fibrosis were examined by using this marker. Isolates that varied in colonial morphology, serotype, and biotype were identical when analyzed by Southern blot hybridization using the fragment as a probe. Indistinguishable isolates (by serotyping, biotyping, and antibiograms) cultured from two unrelated patients were easily distinguished by using Southern blot analysis.

Characterization and Use of a DNA Probe as an Epidemiological Marker for Pseudomonas aeruginosa

(1972). Pseudomonas Aeruginosa Infections. CRC Critical Reviews in Clinical Laboratory Sciences: Vol. 3, No. 3, pp. 291-347.

Pseudomonas Aeruginosa Infections

In the Gillies and Govan method of pyocin typing for Pseudomonas aeruginosa a cross-streaking technique was used, and 105 main types and 25 subtypes were identified by the patterns of inhibition observed on 13 indicator strains. Disadvantages of the technique included the need to remove test strain growth before application of the indicator strains, the 48-h period needed to obtain a result, and the inability to reliably type mucoid P. aeruginosa. Recent studies have enabled us to overcome these disadvantages and significantly improve the speed and application of pyocin typing. Our revised technique utilizes the same 13 indicator strains which are already used internationally. Test strains were rapidly applied to the surface of agar plates with a multiple inoculator. After incubation for 6 h and exposure to chloroform, the indicator strains were applied in agar overlays without prior removal of the test strain growth. After 18 h of incubation, the pyocin type was recognized by inhibition of particular indicator strains. Additionally, the activity of particulate (R and F) and nonparticulate (S) pyocins could be distinguished on the basis of inhibition zone size, which thus allowed further discrimination. The revised technique allows typing within 24 h, increases the number of identifiable types, and can be used to type mucoid strains. Images

Revised pyocin typing method for Pseudomonas aeruginosa.

Similarity of bacteriocins of Streptococcus mutans from mother and infant.

Two clinical isolates of Pseudomonas aeruginosa, one a pyocin type 5 strain from Atlanta, could transfer gentamicin resistance by conjugation. Donor and recipient strains inactivated gentamicin by acetylation. The R plasmids, pMG1 and pMG2, also determined resistance to sisomicin, another substrate of gentamicin acetyltransferase I, sulfonamides, and streptomycin, but not resistance to kanamycin, neomycin, tobramycin, butirosin, or BB-K 8. They were transmissible to many strains of P. aeruginosa, including a Rec(-) strain, but not to Escherichia coli or other enterobacteriaceae. These R plasmids were compatible with R plasmids transmissible to P. aeruginosa from E. coli, including members of C, N, P, and W incompatibility groups. From a strain carrying pMG1 and a compatible plasmid, pMG1 was transferred independently but transfer of the second plasmid often resulted in cotransfer of pMG1. In contrast, pMG1 and pMG2 were incompatible with pseudomonas R plasmids R931 and R3108, and with R931 they readily formed recombinant plasmids. The four plasmids in this incompatibility group determine additional biological properties, including resistance to inorganic and organic mercury compounds, to ultraviolet light, and to certain deoxyribonucleic acid phages. pMG1 and pMG2 also phenotypically inhibited pyocin production. Consequently such R plasmids alter the phage and pyocin types of their host strains.

Properties of R plasmids determining gentamicin resistance by acetylation in Pseudomonas aeruginosa.

Summary
Further experience of the typing of strains of Pseudomonas pyocyanea (Ps. aeruginosa) by their production of pyocines has confirmed the value of pyocine typing for epidemiological studies.
The introduction of five new indicator strains has made it possible to distinguish eight subtypes of the commonly occurring pyocine type 1. The subtyping of the type-1 strains shows the same dependence on use of the correct temperature and duration of incubation of the producer strain as does the main typing procedure.
In studies of the reliability of the typing and subtyping methods for epidemiological purposes it was noted that not all strains of Ps. pyocyanea isolated from the same site in the same patient were of the same pyocine type or subtype. When several colonies of Ps. pyocyanea from individual diagnostic plates were typed it was found that more than one pyocine type or subtype was more frequently present in the same lesion in patients in hospital than in patients being treated at home. This finding suggests that the occurrence of more than one pyocine type or subtype of Ps. pyocyanea in a patient is generally due to his having been separately infected with strains of more than one type, rather than to instability of pyocine production in a single infecting strain.

R. R. Gillies

Papers

Further studies in the pyocine typing of Pseudomonas pyocyanea.