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Strains of Pseudomonas aeruginosa exposed to subinhibitory concentrations of either polymyxin B, gentamicin, or carbenicillin were compared with untreated organisms for changes in sensitivity to pyocin. Pretreatment with polymyxin B or gentamicin resulted in a decreased sensitivity to pyocin, while carbenicllin pretreatment did not alter pyocin sensitivity.
A new pyocin typing method for the identification of specific strains of Pseudomonas aeruginosa is described. The method involves a bilayer agar technique in which the pyocin produced by the organism diffuses from the top agar layer number 2 through a filter paper into the bottom agar layer number 1. The filter paper permits the removal of agar layer number 2 and exposes layer number 1 which is then streaked with the indicator strains. Pyocin typing of fifty clinical isolates using both the bi-layer method and the scrap-chloroform reference method of Gillies and Govan produced identical typing results. A total of 375 clinical isolates has been typed by this inexpensive and time-saving bi-layer agar method.
One hundred randomly collected Psuedomonas aeruginosa isolates were examined during a two months period of time in order to determine their origin in a hospital environment. Initially, neither the source of the isolates, nor the patients' names were known. The serogroups were determined with commercial antisera, and the pyocin patterns were established with the simplified method of mitomycin C induced pyocin production. The results were analysed in the chi2 test. A highly significant probability value (p less than 00004) was taken as evidence of the identity of the isolates, especially among strains originating from the neurosurgical, surgical and medical intensive care units investigated. The simplified method of the originally devised mitomycin C induced pyocin production with a selected number of 'indicator' strains in conjunction with the initial serogroup determination by the commercially available antisera, allowed a rapid tracing of all Pseudomonas aeruginosa isolates. This combined method is recommended as an easy and useful epidemiological tool for the study of Pseudomonas aeruginosa hospital acquired infections.
191 strains of Pseudomonas aeruginosa, isolated from clinical specimens, were tested by passive and active pyocin typing. The results of passive typing were computerized and a final dendrogram, with its peculiar similarity levels between the single strains, was obtains. This study will permit to build a system, to mark out the Pseudomonas strains, through active pyocin typing or through passive pyocin typing.
Pyocine typing was used for the characterization of 134 Pseudomonas aeruginosa strains isolated from bovine mastitis. The scheme of Gillies & Govan (1966) was adopted with some modifications, and the procedure gave 89.6% typability. Pyocine type 1 strains were most commonly encountered and were followed in frequency by types 10 and 3. The introduction of two additional indicator strains allowed for division of these types into subtypes.In spite of some limitations, discussed in the paper, the pyocine typing scheme proved to be useful in ;marking' P. aeruginosa strains and in following their association with bovine mastitis in various herds.
The bacteriocin from Pseudomonas aeruginosa, pyocin, consists of a contractile sheath and inner core reminiscent of T-even coliphage tails. Contraction of the outer sheath was found to be promoted by 0.5 m magnesium chloride, 1% Formalin, low pH, sonic treatment, and freezing or thawing or both. The contraction caused by 0.5 m magnesium chloride, however, was found to be reversible and occurred upon reduction of the salt concentration from 0.5 to 0.02 m. In addition, direct assay showed that pyocin activity was nearly proportional to the percentage of only uncontracted forms. Initial studies suggested that the adsorption of purified pyocin onto cell wall fragments from the sensitive indicator strain of P. aeruginosa occurs with the relaxed particle only and not with the contracted form. However, after adsorption, contraction occurred. Various morphological structures, such as tail fibers and base-platelike appendages, were also observed. Upon contraction, six tail fibers were observed on many particles, four of which appeared to originate from the sheath and two from the inner core. Polysheaths and polycores several hundred nanometers in length were also occasionally observed.
The growth-inhibitory effect of a small group of different pyocines has been used to differentiate Ps. aeruginosa isolates. A method is described which can be carried out with ordinary media and is easy for routine application. One hundred and one isolates of Ps. aeruginosa were differentiated by four pyocines into 10 pyocine patterns.
Pseudomonas infections continue to be an important problem in the hospital environment. Serious infections are always invariably associated with severe underlying conditions or with diminished host resistance. The increasingly resistance of strains and hospital epidemics favour the organism prevalence. During 1978, Pseudomonas aeruginosa was isolated in 154 cultures from a variety of biological samples in the hospital. Tests of biochemical identification, serological typing, and typing by pyocine production were carried out. Susceptibility to aminoglycosides and beta-lactamic antibiotics was also tested. Serological study demonstrated a higher incidence of 4 and 11 serotypes; 69.4 percent corresponded to type I when typing by pyocine production was carried out. No relationship between serotypes and pyocine-types has been found. Ticarcillin showed a greater activity than carbenicillin (minimal inhibitory concentration less than or equal to 16 micrograms/ml). Amikacin, tobramicin, sisomicin and gentamicin inhibited 83.7 percent, 73.6 percent, 70.7 percent and 69.4 percent of the isolated strains, respectively.
The periplasmic protein banding patterns (PPBPs) of thirteen strains of Pseudomonas aeruginosa pyocin type 10/b implicated in two nosocomial outbreaks in the neonatal unit of Rafeidia Hospital, Nablus, Palestine, were examined. In addition, five strains from sporadic cases from the same unit occurring in 1996 and 1997 were also studied. Despite different sources of the strains, PPBPs generated by PAGE suggested a clonal nature of the strains obtained during each of the two outbreaks. Although they had very similar PPBPs, the two outbreak clones were not identical. In contrast, sporadic strains of P. aeruginosa pyocin type 10 appeared to be much more heterogeneous than those of the two outbreaks. PPBP analysis appeared to be a useful tool that may be of value for epidemiological purposes.
Pyocin R1, a bacteriocin of Pseudomonas aeruginosa, is a protein particle shaped like a bacteriophage tail composed of a contractile sheath, core, baseplate and tail fibers. Alkaline treatment with sodium carbonate caused sheath contraction without considerable disassembly of other components. Circular dichroism (CD) spectra of pyocin R1 before and after the treatment, and of isolated sheath, were measured in wavelength regions around 220 and 290 nm at neutral pH. The alkaline treatment caused a red shift of the minimum from 208 nm to 212 nm. A marked difference in the CD spectrum was found in the near-ultraviolet region. THe difference is considered to be mainly due to a CD spectra change of tryptophan residues in the sheath subunits.
Two hundred sixty-seven strains of Pseudomonas aeruginosa isolated from clinical specimens were immunotyped using seven anti-Pseudomonas rabbit sera, and pyocin typed using eight indicator strains. Of the 230 consecutive isolates tested, 83% were immunotypable and 90% were pyocin typable. When both typing methods were used concurrently, 261 strains were typed, giving a 98% overall typability. An attempt was made to correlate sensitivity to carbenicillin and gentamicin with various immunotypes. Using growth from sensitivity plates directly for immunotyping as an added advantage for early surveillance and treatment is discussed.
The physiochemical properties of pyocin F1 were studied. Pyocin F1 consists of flexuous rod-like particles homogenous in size. Each particle was composed of rod and fiber parts. The rod part was 105.5 +/- 9.5 nm long and 10.0 +/- 1.4 nm wide, and showed regular striations amounting to 23 layers. The fiber part was composed of several filaments; the length of the longest filament was 43.0 +/- 12.0 nm. The amino acid composition, the partial specific volume (0.720 ml/g), the sedimentation coefficient (S020,W = 35.1S), and the translational diffusion constant (0.94 +/- 0.01 x 10(-7) cm2/s) were determined. The particle weight was calculated to be 3.23 x 10(6) daltons.
By a mild alkaline treatment, pyocin R1 was disassembled into its structural parts, a contracted sheath (and its fragments), a core, and fibers. An alkaline sucrose density gradient centrifugation after this treatment was effective in obtaining fiber-density fractions. The pooled fractions were treated with IgGs against isolated sheaths and isolated cores, simultaneously, and then chromatographed on DEAE-Sepharose CL-6B. The final preparation of fibers purified in this way was confirmed to be homogeneous by electron microscopic observation and an immuno-precipitation reaction. The isolated fiber was found to consist of two major subunit proteins, No. 2 and No. 9, with molecular weights of 71,000 and 31,000, respectively. The fiber exhibited the ability to be adsorbed on sensitive bacterial cells (pseudomonas aeruginosa P14), and to protect against the inactivation of pyocin R1 by a lipopolysaccharide preparation from the bacteria.
A laboratory-grown strain of Neisseria gonorrhoeae, selected in vivo, BS4 (agar), is susceptible to complement-mediated killing by fresh human serum but is relatively resistant to killing by human phagocytes. It can be induced to serum resistance by incubation with a small molecular weight fraction of guinea pig serum. The serum-susceptible and induced-resistant forms show differences in pyocin sensitivity tests. This indicates either differences in the structure of their lipopolysaccharides or masking of some determinant(s). The pyocin sensitivity pattern of BS4 (agar) is only slightly different from that of a closely related strain, BSSH, which is more susceptible to killing by human phagocytes.
The plasmid profiles of 112 clinical isolates of Pseudomonas aeruginosa were determined by two reproducible, rapid, plasmid screening methods. Plasmid DNA was present in 15% of isolates examined. Plasmids varied in size from 1.2 X 10(6) to 60.2 X 10(6) mol. wt. The dominant serotypes encountered were O:11 and O:4, which comprised 38% and 12% of strains, respectively. Four pyocin types (1, 10, 3 and 5) dominated (respective frequencies: 56, 15, 12 and 6%). Reproducibility of pyocin typing was distinctly inferior to both plasmid profiling and serotyping. Strains of identical serotypes could be further differentiated by dissimilar plasmid profiles. Serologically untypable or polyagglutinable strains were successfully characterised by plasmid profile patterns. Thus, plasmid profiling was shown to be a useful adjunct to serotyping for the epidemiological typing of P. aeruginosa.
We sought evidence to determine if particular strains of Pseudomonas aeruginosa have a predilection for pulmonary colonisation in patients with cystic fibrosis (CF). The incidence of common pyocin types in non-CF isolates (74%) was similar to that noted in previous reports but differed significantly (X2 = 16.7, p less than 0.001) from the incidence of 40% observed in CF isolates. A retrospective analysis of respiratory isolates also indicated a relatively low incidence of common pyocin types (44%) in isolates from non-CF patients with chronic airways diseases and this incidence also differed significantly from that observed (73%) in other respiratory isolates from patients in the same hospital. These observations suggest that a subpopulation of P. aeruginosa exists which has a predilection for pulmonary colonisation in CF and other chronic pulmonary diseases and may assist in identification of factors affecting bacterial colonisation.
We have used Tn5 mutagenesis to obtain a mutant resistant to pyocin Sa. When grown in iron-deficient succinate medium this mutant lacked an 85-kDa iron-regulated outer membrane protein (IROMP), and expression of a 75-kDa IROMP was increased compared with that in the parent strain. The mutant was deficient in pyoverdin biosynthesis and showed a 95% decrease in transport of ferripyoverdin purified from the parent strain, suggesting that the 85-kDa IROMP is the specific receptor for ferripyoverdin and pyocin Sa. The mutant compensated for the deficiency in pyoverdin biosynthesis and transport by exhibiting a fourfold increase in ferripyochelin transport. The low-level transport of ferripyoverdin in the Sa-resistant mutant, which extended to heterologous pyoverdins from other strains, suggests that Pseudomonas aeruginosa has a second ferripyoverdin uptake system of lower affinity and broader specificity.
Pyocin AP41 degrades the chromosomal DNA in sensitive strains of Pseudomonas aeruginosa but has little effect on RNA, protein, and lipid syntheses. In vitro experiments showed that the carboxyl-terminal part of the large subunit of pyocin AP41 carries an inherent DNase that is responsible for its killing action.