Epidemiology of Pseudomonas aeruginosa infections investigated by pyocin typing.

All strains of Pseudomonas aeruginosa isolated in a large Canadian hospital over a 3-year period were typed by their pyocin production. Smaller collections of P. aeruginosa from other hospitals were also typed. Almost 3000 strains were examined. The typing method did not require use of complex reagents and was successful in subdividing P. aeruginosa into numerous types. No single type was restricted to infections of one particular kind. Infections of all kinds were associated with a wide variety of pyocin types. Extensive crossinfection with one particular pyocin type was observed only in urinary infection of patients with urologic disorders. The four pyocin types that were most frequent in our entire series have been reported as the commonest types causing infections in many other parts of the world.     

Nursery Outbreak of Pseudomonas aeruginosa: Epidemiological Conclusions from Five Different Typing Methods

In April 1971, nine cases of Pseudomonas aeruginosa septicemia occurred in a high-risk nursery. The epidemiology of the outbreak was studied by pyocin production, pyocin sensitivity, serological typing, antibiotic susceptibility, and phenotypic properties such as colonial morphology, pigment, and hemolysis. Ten isolates of P. aeruginosa were recovered from 9 newborn infants and from 13 environmental sources. Twenty-one of the 23 isolates had identical pyocin production patterns against 60 different indicator strains and were of the same serotype. These 21 isolates were designated as the "outbreak strain"; the other 2 isolates had no epidemiological significance. The results of pyocin sensitivity, antibiotic susceptibility tests, and phenotypic properties were dissimilar. They would yield incorrect epidemiological conclusions if used alone. The outbreak strain dissociated in vitro and these phenotypic changes accounted for the variable results by the latter three typing methods. Although the precise mode of introduction of the organism into the nursery could not be determined in retrospect, the epidemiological data strongly suggested that one infant contracted a P. aeruginosa infection, and this strain spread throughout the nursery by means of contaminated resuscitation equipment.     

Experience with epidemiologic studies of pyocyaneus infections. I. Feasibility of using serotypes and antibiotic resistance as a epidemiologic label for clinical strains]

The authors carried out serological typing of 98 Pseudomonas aeruginosa strains, isolated from patients of burn department of the Sklifosovsky First Aid Institute in January-July, 1974, and of 215 strains obtained from other sources; their sensitivity to 13 antibiotics was determined. Pseudomonas aeruginosa cultures isolated from the patients were typed with O-sera of 10 serological types. The presence of several hospital strains of Pseudomonas aeruginosa was found by means of serological typing; along with these there were revealed cultures of this causative agent sporadically appearing in the department. Sensitivity to some antibiotics could serve as an additional criterion for differentiation of Pseudomonas aeruginosa strains of the same serological type.     

Cytotoxin-converting phages, φCTX and PS21, are R pyocin-related phages

Abstract φCTX is a temperate phage of Pseudomonas aeruginosa harbouring the ctx gene that encodes cytotoxin (CTX). We identified φCTX as an R pyocin-related phage, by serological and molecular analysis, based on the findings that the infectivity of the phage was inhibited with the antisera directed R pyocins and R pyocin-related phages and that the φCTX genome showed DNA homology to the genome of PS17 (a representative of the R pyocin-related phages) as well as to the pyocin R2 genes. Another new CTX-converting, R pyocin-related phage named PS21 was isolated from a CTX-producing strain of P. aeruginosa , suggesting the distribution of the ctx gene by certain members of R pyocin-related phage family.     

Epidemiology of Pseudomonas aeruginosa in a Burns Hospital: Surveillance by a Combined Typing System

For 3 months, 259 cultures of Pseudomonas aeruginosa isolated from nonpatient environmental sources and 262 cultures from 16 infected patients in the Intensive Care Unit (ICU) of Shriners Burns Hospital were typed by a combined system with a high degree of reliability. Sinks were major sources of environmental contamination. Serotypes 1 and 2 were the predominant types found in patients, and they were most prevalent among typable strains from sinks. Strain designations were made on the basis of similarities in data from serological and phage typing. All nontypable strains were typed by pyocin production. Two infected patients carried different strains of P. aeruginosa that remained the same type for 45 days, even though their beds in ICU were approximately 6 feet apart. Cross-contamination from patient to patient and spread of infection by nursing personnel were eliminated as major modes of transmission because nasopharyngeal swabs, hair samples, and hands of nursing staff were consistently negative. Splashing of water from contaminated sinks to fomites was suggested as a possible mode of transfer for this infectious agent.     

Simplified Method for Producing Pyocins from Pseudomonas aeruginosa

Good yields of pyocin are obtained when Pseudomonas aeruginosa is grown in Trypticase soy broth (without glucose) (BBL) plus 1% potassium nitrate. As a result, pyocin production for routine epidemiological typing can be done in screw-cap tubes, without mechanical agitation or mitomycin C induction.     

Role of Pseudomonas aeruginosa in the etiology of postoperative suppurative inflammatory complications in patients with chronic suppurative lung diseases

The study of P. aeruginosa strains isolated from patients and various objects of the environment in one of the clinics of Yerevan, carried out by the methods of serotyping, pyocin typing and phage typing, has allowed the authors to establish the circulation of hospital strains and their role in the appearance of post-operative suppurative inflammatory complications in patients with chronic suppurative diseases. The study has also allowed them to reveal the sources of infection which is spread by patients serving either as its reservoirs or as intermediate hosts.     

Comparison of silver sulfadiazine and gentamicin for topical prophylaxis against burn wound sepsis.

Daily prophylactic application of either 1.0% silver sulfadiazine cream or 0.1% gentamicin cream was compared for effectiveness in preventing bacterial colonization of burn wounds and sepsis. Pseudomonas aeruginosa colonized the wounds of 37% of the 38 patients treated with silver sulfadiazine and 30% of the 33 patients treated with gentamicin; gentamicin-resistant P. aeruginosa colonized the wounds of 21% of the patients treated with gentamicin. Staphylococcus aureus colonization occurred in 55% of the patients treated with silver sulfadiazine, whereas colonization with Candida species occurred in 58% of the patients treated with gentamicin. Although gentamicin-resistant organisms caused no deaths their repeated appearance resulted in discontinuation of prophylaxiz with gentamicin cream. The next year P. aeruginosa strains resistant to gentamicin were isolated from burn wounds of only two patients who had not previously received parenteral therapy with gentamicin or tobramycin. Gentamicin cream should be reserved for treating patients with wounds infected by gentamicin-sensitive P. aeruginosa and those allergic to sulfa drugs. For most patients with burn wounds silver sulfadiazine is safe and effective as an antibacterial agent for topical prophylaxis.     

Pyocin production by bacillus pyocyaneus and sensitivity of strains of different origin to them

Using the method proposed by Gillies and Govan and their indicator strains, 342 P. aeruginosa strains isolated from the patients were studied in respect to their pyocinogenicity and typed according to the production of different types of pyocins. Besides, in 206 cultures the pyocin sensitivity of 16 standard P. aeruginosa strains (5 strains obtained from Govan and 11 strains provided by the authors) was determined. All the tested cultures fell into 23 pyocin types; of these, types I and X occured most frequently, 56 strains identified by means of indicators could not be typed due to the fact that the corresponding pyocin types were absent in Govan's scheme. The cultures isolated from the patients and the environmental objects during the outbreak of P. aeruginosa in a hospital were proved to belong to the same pyocin type (III). The double typing of the cultures, according to pyocin production and pyocin sensitivity, allowed to determine individual characteristics of 75% of the tested cultures.     

Microbiological monitoring for the causative agents of nosocomial infections (exemplified by resuscitation and intensive therapy units)]

More than 900 isolates from at least 1500 patients were tested within 1996-1998. Gram-negative organisms were the main pathogens isolated from patients with different forms of nosocomial complications such as late pneumonia, associated with artificial ventilation of the lungs, and various secondary wound or urinary tract infections. The prevalence of Pseudomonas aeruginosa was stated. Antibioticograms showed that the most active drugs were imipenem (more than 90 per cent of the susceptible isolates) and ticarcillin/clavulanate (48-58 per cent of the susceptible isolates). The activity of ticarcillin/clavulanate (Timentin) was practically the same as that of imipenem against 21 strains of P.aeruginosa isolated from the blood and cerebrospinal fluid of 21 patients with sepsis and 3 patients with secondary purulent meningitis.     

The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage

Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.     

Enhancement of dendritic cell production by fms-like tyrosine kinase-3 ligand increases the resistance of mice to a burn wound infection

Fms-like tyrosine kinase-3 ligand (Flt3L) is a hemopoietic cytokine that stimulates the production of dendritic cells. This study evaluated the ability of Flt3L-enhanced dendritic cell production to increase the resistance of mice to a burn wound infection with Pseudomonas aeruginosa, a common source of infections in burn patients that have impaired immunity and are susceptible to opportunistic microorganisms. Treatment of mice with Flt3L for 5 days caused a significant increase in dendritic cell numbers in the spleen and significantly increased survival upon a subsequent burn wound infection. Improved survival in Flt3L-treated mice was associated with limited bacterial growth and spread within the burn wounds and a decrease in systemic dissemination of P. aeruginosa. Resistance to burn wound infection could also be conferred to recipient mice by the adoptive transfer of dendritic cells that had been isolated from spleens of Flt3L-treated mice. Adoptive transfer of the same number of splenic dendritic cells from nontreated mice did not confer resistance to burn wound infection. These data indicate that Flt3L can increase the resistance of mice to a P. aeruginosa burn wound infection through both stimulation of dendritic cell production and enhancement of dendritic cell function.     

Flagella, motility and invasive virulence of Pseudomonas aeruginosa

The role of motility as a virulence factor in Pseudomonas aeruginosa burn wound sepsis was examined using mutants deficient in the Fla or Mot phenotype. Physiological profiles of parental strains and Fla- and Mot- mutants were similar with respect to antibiograms, O antigen types, growth rates, and proteolytic, exotoxin A and phospholipase activities, providing evidence for isogenicity. Lethality studies using a subcutaneous mouse burn model showed that three Fla- mutants and one Mot- mutant were much less virulent (10(2) to 10(5) times) than the parent wild-type. Topical challenges in the flame burn model showed that a Fla- mutant of strain M-2 was approximately tenfold less virulent. A reduction in virulence, although somewhat less than tenfold, was also observed in the scald burn model for M-2 Fla-, and Mot- strains. Tissue colonization experiments revealed a characteristic, rapidly systemic infection in burned mice challenged with wild-type organisms. Nonmotile mutants similarly proliferated in the burn wound, but the characteristic bacteraemia and systemic invasion were markedly absent. The infection remained localized in the skin wound and the mice survived. The pattern of infection by nonmotile mutants in the colonization studies was very similar to that obtained with Fla+ cells in burned animals passively treated with antiflagellar antibody. These results add substantial support to the concept of motility as a P. aeruginosa virulence factor in invasive infections.     

Pyocin typing of Pseudomonas aeruginosa strains

The possibility of using the typing of P. aeruginosa strains by their pyocins as one of the epidemiological markers in the study of P. aeruginosa hospital infections has been established. As this method of typing is characterized by certain variability, the authors propose that the method of the "cross analysis" of pyocins produced by P. aeruginosa strains be used simultaneously. This method is based on the following phenomenon: if the cultures to be compared are different, the pyocin produced by one strain suppresses the growth of the other one, and if the cultures are identical, no suppression of their growth by pyocins is observed.     

Pyocin Typing of Pseudomonas aeruginosa: a Simplified Method

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.     

Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.     

Epidemiological Fingerprinting of Pseudomonas aeruginosa by the Production of and Sensitivity to Pyocin and Bacteriophage

A new method has been devised to trace cross-infection by Pseudomonas aeruginosa. Unknown strains growing logarithmically in liquid media were treated with mitomycin C to induce the liberation of pyocin and phage. The lysates were then tested against 27 selected indicator strains, and the zones of clearing were differentiated as to killing by pyocin or lysis by phage. Twenty-four standard pyocin-phage lysates were then applied to each of the unknowns, and the sensitivity pattern was recorded. Thus, an “epidemiological fingerprint” consisting of 51 operational characteristics was established for each isolate. Organisms from the same source had identical or similar fingerprints, but organisms from different origins could easily be distinguished. Pyocin production, pyocin sensitivity, and phage production were found to be stable genetic characters; however, spontaneous mutations in phage sensitivity were frequently encountered. The epidemiological fingerprint has proven to be a sensitive tool in establishing the identity or dissimilarity of unknown strains. This method has been of great value in tracing the epidemiology of P. aeruginosa in the hospital environment. Each of the 157 P. aeruginosa strains tested has been typable by this method.     

Retargeting R-type pyocins to generate novel bactericidal protein complexes

R-type pyocins are high-molecular-weight bacteriocins that resemble bacteriophage tail structures and are produced by some Pseudomonas aeruginosa strains. R-type pyocins kill by dissipating the bacterial membrane potential after binding. The high-potency, single-hit bactericidal kinetics of R-type pyocins suggest that they could be effective antimicrobials. However, the limited antibacterial spectra of natural R-type pyocins would ultimately compromise their clinical utility. The spectra of these protein complexes are determined in large part by their tail fibers. By replacing the pyocin tail fibers with tail fibers of Pseudomonas phage PS17, we changed the bactericidal specificity of R2 pyocin particles to a different subset of P. aeruginosa strains, including some resistant to PS17 phage. We further extended this idea by fusing parts of R2 tail fibers with parts of tail fibers from phages that infect other bacteria, including Escherichia coli and Yersinia pestis, changing the killing spectrum of pyocins from P. aeruginosa to the bacterial genus, species, or strain that serves as a host for the donor phage. The assembly of active R-type pyocins requires chaperones specific for the C-terminal portion of the tail fiber. Natural and retargeted R-type pyocins exhibit narrow bactericidal spectra and thus can be expected to cause little collateral damage to the healthy microbiotae and not to promote the horizontal spread of multidrug resistance among bacteria. Engineered R-type pyocins may offer a novel alternative to traditional antibiotics in some infections.     

Epidemiological Tracing of Pseudomonas aeruginosa: Antibiogram and Serotyping

The spread of a particular strain of Pseudomonas aeruginosa through a pediatric burn unit was monitored using serological typing and antibiotic susceptibility data.     

A major Pseudomonas aeruginosa clone common to patients and aquatic habitats.

The genomic relatedness of 573 Pseudomonas aeruginosa strains from environmental and clinical habitats was examined by digesting the genome with the rare-cutting enzyme SpeI. Thirty-nine strains were collected from environmental habitats mainly of aquatic origin, like rivers, lakes, or sanitary facilities. Four hundred fifty strains were collected from 76 patients with cystic fibrosis (CF) treated at four different centers, and 25 additional clinical isolates were collected from patients suffering from other diseases. Twenty-nine P. aeruginosa isolates were collected from the environment of one CF clinic. Thirty strains from culture collections were of environmental and clinic origin. A common macrorestriction fingerprint pattern was found in 13 of 46 CF patients, 5 of 29 environmental isolates from the same hospital, in a single ear infection isolate from another hospital, and 8 of 38 isolates from aquatic habitats about 300 km away from the CF clinic. The data indicate that closely related variants of one major clone (called clone C) persisted in various spatially and temporally separated habitats. Southern analysis of the clonal variants with six gene probes and two probes for genes coding for rRNA revealed almost the same hybridization patterns. With the exception of the phenotypically rapidly evolving CF isolates, the close relatedness of the strains of the clone was also shown by their identical responses in pyocin typing, phage typing, and serotyping. Besides clone C, three other P. aeruginosa clones were isolated from more than one clinical or environmental source.