Pyocin Typing of Pseudomonas aeruginosa: a Simplified Method

Abstract

A simplified method has been devised for typing Pseudomonas aeruginosa by pyocin production. Pyocins are produced as strains grow overnight in Trypticase soy broth (without glucose) plus 1% potassium nitrate. Because P. aeruginosa can use nitrate instead of oxygen as a terminal electron acceptor, mechanical shaking is not necessary, nor is induction by mitomycin C. Pyocins can now be produced in screw-cap tubes in a water bath or incubator. A total of 250 strains were tested as possible pyocin indicators, which included 60 strains already used in pyocin-typing systems. The final set contained 18 indicators which were chosen because (i) they had clear positive or clear negative reactions, thus eliminating reactions difficult to read, (ii) they had few zones due to bacteriophage lysis, and (iii) they were most sensitive in differentiating clinical isolates of P. aeruginosa. The final typing method was tested in several studies and the results were clear; thus definitive epidemiological conclusions could be made. Because it is simple to perform and easily automated, the new method should have application in many hospitals; however, it should be used only in carefully planned epidemiological studies. The method and its application are described in detail, and some pitfalls are discussed. Nosocomial infections caused by Pseudomonas aeruginosa continue to be a problem in many hospitals (3, 5, 8, 12, 16, 23). Within any institution there can be hundreds of different strains because it is often found in the human gut, and because it can survive and multiply in moist environments of the hospital. In any epidemiological study, a sensitive technique is required to compare these numerous strains. A number of methods have been used to differentiate P. aeruginosa in epidemiological studies. These epidemiological markers include

Improved, computer-generated system for pyocin typing of Pseudomonas aeruginosa

“…The revised method allows for typing of multiple isolates on the same set of typing plates. This method also has the advantage over the cross-streaking and broth methods (16,25) of reducing the time required to obtain typing results by 24 h and of the ability to type mucoid strains of P. aeruginosa. Researchers who already perform pyocin typing by the cross-streaking technique need no additioplal equipment other than a multipoint inoculator to perform the revised method.…”

“…These cultures were lyophilized and stored at 4°C. The 18 Farmer strains, labeled ALA 1 to 18, were used because they had been chosen originally by use of a computer program (25) and because we hoped to use them in conjunction with or instead of certain Govan strains to obtain a better set of indicator strains. These cultures were frozen in defibrinated sheep blood at -70°C.…”

“…Epidemiologic typing of strains of Pseudomonas aeruginosa can be done by a variety of techniques, including biotyping (7,27), serologic typing (9,27,29), bacteriophage typing (5,6,14,16), pyocin typing (3,8,14,16,20,25,30), and antimicrobial susceptibility patterns (7,8).…”

“…The two major techniques used for typing by pyocin production are those of Gillies and Govan (18,21) and Jones et al (25). In their original form, these two systems shared a common problem in that at least 48 h was needed to produce results.…”

Preliminary Studies of Fluorescent Pseudomonads Capable of Growth at 41 C in Swimming Pool Waters

“…All isolates were examined further to determine the pyocine types, immunotypes, and antibiotic susceptibility patterns. Pyocine typing was performed employing the 18 indicator strains of Jones et al (13). The procedure is highly sensitive, and some variations in typing patterns of isolates were found to occur which disappear upon repetition of the test.…”