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.     

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.     

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.     

Cytotoxic effects of pyocin S2 produced by Pseudomonas aeruginosa on the growth of three human cell lines

Pyocin typing of 82 Pseudomonas aeruginosa strains, collected from different Iranian clinical sources, revealed that one isolate, P. aeruginosa 42A, produced pyocin S2, a protease-sensitive bacteriocin. Pyocin S2 production was induced by mitomycin C (2 micro g/mL) in the pyocin S2 producer P. aeruginosa 42A. Pyocin S2 was purified using ion exchange chromatography with CM-Sepharose CL-6B and sodium phosphate buffer (pH 8) from an 80% ammonium sulfate precipitate of whole-cell lysates. Pyocin activity of the fractions was detected using the Govan spot testing method. The purity of the active fraction was confirmed by SDS-PAGE, where a single band with a molecular mass of 74 kDa was detected. Cytotoxic effects of purified pyocin S2 and partially purified pyocin from P. aeruginosa 42A on the human tumor cell lines HepG2 and Im9 and the normal human cell line HFFF (Human Foetal Foreskin Fibroblast) were studied by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The results demonstrated that partially purified pyocin and pyocin S2 exhibited substantial inhibitory effects on the growth of the tumor cell lines HepG2 and Im9, while no inhibitory effects were observed on the normal cell line HFFF. Pure lipopolysaccharide was used as a control and was found to have no inhibitory effect on any of the cell lines tested.     

Pyocin S2 (Sa) kills Pseudomonas aeruginosa strains via the FpvA type I ferripyoverdine receptor

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa strains via a specific receptor. The genes coding for pyocin Sa (consisting of a killing protein and an immunity protein) were cloned and expressed in Escherichia coli. Sequence analysis revealed that Sa is identical to pyocin S2. Seventy-nine strains of P. aeruginosa were tested for their sensitivity to pyocins S1, S2, and S3, and their ferripyoverdine receptors were typed by multiplex PCR. No strain was found to be sensitive to both S2 and S3, suggesting that the receptors for these two pyocins cannot coexist in one strain. As expected, all S3-sensitive strains had the type II ferripyoverdine receptor fpvA gene, confirming our previous reports. S1 killed strains irrespective of the type of ferripyoverdine receptor they produced. All S2-sensitive strains had the type I fpvA gene, and the inactivation of type I fpvA in an S2-sensitive strain conferred resistance to the S2 pyocin. Accordingly, complementation with type I fpvA in trans restored sensitivity to S2. Some S2-resistant type I fpvA-positive strains were detected, the majority (all but five) of which had the S1-S2 immunity gene. Comparison of type I fpvA sequences from immunity gene-negative S2-sensitive and S2-resistant strains revealed only a valine-to-isoleucine substitution at position 46 of type I FpvA. However, both type I fpvA genes conferred the capacity for type I pyoverdine utilization and sensitivity to S2. When these two type I fpvA genes were introduced into strain 7NSK2 carrying mutations in type II fpvA (encoding the type II pyoverdine receptor) and fpvB (encoding the alternative type I receptor), growth in the presence of type I pyoverdine was observed and the strain became sensitive to S2. We also found that type I pyoverdine could signal type II pyoverdine production via the type I FpvA receptor in 7NSK2.     

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.     

Epidemiology of Pseudomonas aeruginosa in a Burns Hospital: Evaluation of Serological, Bacteriophage, and Pyocin Typing Methods

In a retrospective study 36 cultures of Pseudomonas aeruginosa, isolated from patients with fatal Pseudomonas burn wound sepsis and from burned patients with nonfatal P. aeruginosa infections, were used to evaluate the consistency and reliability of serological, phage, and pyocin typing as epidemiological tools. Frequency distributions of positive reactions were analyzed by a computer in a 3-way chi-square test, and a high degree of consistency was demonstrated for each method. From these data, 75% of the cultures were differentiated by serological, 90% by phage, and 100% by pyocin typing. There was no significant difference among organisms isolated from fatal cases of burn wound sepsis and organisms from patients with nonfatal infections (chi(2) = 0.3418; P = 0.9870). The combined typing system was a sensitive and reliable epidemiological tool for intraspecific differentiation of P. aeruginosa.     

Genetic determinant of pyocin AP41 as an insert in the Pseudomonas aeruginosa chromosome.

The genetic determinant for pyocin AP41 , a bacteriocin produced by Pseudomonas aeruginosa PAF, was transferred to P. aeruginosa PAO and analyzed. By conjugation experiments, the pyocin determinant was found to be located on the chromosome, being closely linked to argG at about 45 min on the genetic map. Cloning of the pyocin AP41 gene into the plasmid R68.45 was attempted in vivo by taking advantage of its linkage at argG. R' argG+ plasmids were isolated by interspecific conjugation between P. aeruginosa and Escherichia coli recA argG strains. Some of the R' argG+ plasmids did contain the pyocin AP41 determinant. Genetic and physical analyses of these R' plasmids indicated that the pyocin AP41 determinant was located within a 2.9-kilobase extra segment found at a certain position of the chromosome of various pyocin AP41 producer strains.     

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.     

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.     

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.     

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.     

Pyocine Typing of Clinical Strains of Pseudomonas aeruginosa

A total of 954 clinical isolates of Pseudomonas aeruginosa were typed by their ability to produce pyocines. The strains of Pseudomonas were isolated from urines, bloods, sputa, stools, and miscellaneous infectious exudates or tissue of patients of the Mayo Clinic and four associated hospitals. About 80% of the typable strains could be grouped into three major pyocine types: A (30.9%), B (34.8%), and D (14.1%). These large groups could be divided into subtypes by using additional indicator strains. There was no significant difference in the distribution of types by either institutional or specimen source, except that urine specimens yielded the highest percentage of one type. By this procedure, 93% of all isolates could be typed. Repeated typing of serially transferred strains indicated that the procedure has a high degree of reliability. Several strains exhibited extreme fluctuation in inhibition pattern. The procedure is a simple and reliable method to monitor the patterns of nosocomial infections due to P. aeruginosa.     

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.     

Comparison of some adhesive properties of Pseudomonas aeruginosa strains isolated from respiratory and urinary tract infections

The aim of the paper was the comparison of adhesive properties concerning pathogenic potential of P. aeruginosa strains isolated from the patients with respiratory tract infections and from the patients with urinary tract infections. It was stated that P. aeruginosa strains had no haemagglutinating properties when cultured on a solid medium. Bacteria cultured in a liquid medium showed an increase of these properties in 48 h cultures as compared with 24 h cultures. They were not sensitive to heating. The haemagglutinating properties of the most strains were inhibited by D-mannose. These results seem to suggest that in P. aeruginosa strains fimbriae play an important role in adhesion. On the other hand, the mechanism of adhesion is not uniform as shows mannose-sensitivity of some strains and its lack in the other haemagglutinating strains. The most effective agglutination of human erythrocytes seems to be caused by the species specificity of the individual strains isolated from humans. The higher attachment of P. aeruginosa strains isolated from the urinary tract infections than those from respiratory tract infections to "Vero" cells suggests that these two strains populations may differ in their pathogenic potential to various tissues.     

Defective pyocin particles produced by some mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, defective in the production of active R-type pyocins, were isolated from pyocinogenic strains and their products were characterized. Polysheath-like structures were found in induced lysates of 29 out of 42 mutants. Two mutants (strain P15-16 and M189) were found to produce special defective particles, which were characterized in detail. The other 11 mutants did not produce significant amounts of any structure visible under an electron microscope. Serum blocking powers were found in lysates from P15-16 and M189 to significant amounts. Defective particle produced by strain P15-16 lacked the sheath component, whereas M189 had morphological defects at the junction between sheath and baseplate, and also in the architecture of baseplate. Both defective particles could adsorb to the surface of bacteria, that were sensitive to pyocin, at the tip of their fibers without killing cells. All M189 particles attached to the bacteria had the extended sheaths. Therefore, attachment to the bacteria by fibers is not sufficient to kill cells, and contraction of sheath must occur after the initial adsorption by fibers for pyocin to express its biological activity. Defective particles of strain P15-16, which was derived from strain P15 (a pyocin R1 producer), could be converted to active forms by an in vitro complementation reaction with extracts from certain mutants originated from strain PAO (a pyocin R2 producer). This result indicated the exchangeability of components between R-type pyocins belonging to the different groups.     

Hospital infection due to Pseudomonas aeruginosa in resuscitation departments

The bacteriological survey of resuscitation and intensive care units has revealed the presence of P. aeruginosa strains in the microflora in 69.4% of cases. The circulation of 1-2 P. aeruginosa strains, identical in their serovar and pyocin type, is indicative of the presence of hospital infection and, therefore, the endogenous character of the contamination of patients. P. aeruginosa hospital strains are the main causative agents of infectious complications in the patients treated in resuscitation units.     

R-type pyocin is required for competitive growth advantage between Pseudomonas aeruginosa strains

R-type pyocin is a bacteriophage tail-shaped bacteriocin produced by Pseudomonas aeruginosa, but its physiological roles are relatively unknown. Here we describe a role of R-type pyocin in the competitive growth advantages between P. aeruginosa strains. Partial purification and gene disruption revealed that the major killing activity from the culture supernatant of PA14 is attributed to R-type pyocin, neither F-type nor S-type pyocins. These findings may provide insight into the forces governing P. aeruginosa population dynamics to promote and maintain its biodiversity.     

Frequency and susceptibility patterns of non-fermenting gram-negative rods isolated from patients hospitalised in intensive care units of a tertiary care hospital

The aim of the study was to assess frequency and susceptibility to antimicrobial agents of non-fermenting gram-negative rods isolated from clinical specimens obtained from patients requiring intensive care, with emphasis on profile of the unit. Identification of cultured isolates was done using automated VITEK and API systems (bioMerieux, France). Susceptibility to antimicrobial agents was tested by a disk-diffusion method according to the NCCLS recommendations. In total the analysis comprised 425 strains of non-fermenting gram-negative rods, constituting 58.9% of all isolates of gram-negative bacteria. In blood cultures predominated strains of A. baumannii (46.8%) and P. aeruginosa (40.4%), while in cultures of other clinical specimens these bacteria comprised 42.9% and 43.9% of isolates. Major differences were observed in frequency of these species on both ICU units. Strains of non-fermenting rods isolated from blood cultures comprised a lower percentage of strains susceptible to antimicrobials (particularly cefepime and carbapenems) than isolates cultured from other specimens. Strains of A. baumannii resistant to imipenem and meropenem were detected with a frequency of 12.5% and 26.7%, respectively. Resistance of P. aeruginosa strains to carbapenems was 62.2% and 44.3%, respectively. There was a relatively high percentage of strains susceptible to cefepime (82.0%), ceftazidime (78.9%), amikacin (77.8%) and piperacillin/tazobactam (69.7%). Conclusions: 1. There was a predominance (58.9%) of strains of gram-negative non-fermenting rods. 2. Isolates from blood cultures were characterised by a much higher percentage of resistant strains in comparison to other specimens. 3. Strains of A. baumannii resistant to carbapenems were recorded. 4. There were differences in frequency and antimicrobial susceptibility among the strains of P. aeruginosa and A. baumannii depending on the type of clinical specimen and ICU profile.     

In vitro PYOCIN activity ofPseudomonas aeruginosa strains pretreated with antibiotics

Fifty-six selected strains of Pseudomonas aeruginosa belonging to 8 different pyocin types (H, I, 15, 6, PTI-1, PTI-2, PTI-3, PTI-4) were treated with subinhibitory concentrations (MIC/2) of either gentamicin or carbenicillin. Both treatments induced changes in pyocin patterns for all types but at different levels. The percentage of strains that retained their pyocin pattern were more or less equal in both treatments. In treated and untreated producers, the growth inhibition ability for 5 different strains of Enterobacteriaceae (Escherichia coli K12, E. coli EB, Proteus vulgaris, Salmonella typhi, Shigella flexneri) was also investigated. In all pyocin patterns the number of producers that inhibit the growth of these strains was lower after treatment with gentamicin or with carbenicillin, a smaller decrease was detected in the latter treatment. It appeared that the subinhibitory concentrations of these antibiotics are capable of protecting the Enterobacteriaceae strains from the action of the pyocins.