Heterogenous Susceptibility to R-Pyocins in Populations of Pseudomonas aeruginosa Sourced from Cystic Fibrosis Lungs.

Bacteriocins are proteinaceous antimicrobials produced by bacteria that are active against other strains of the same species. R-type pyocins are phage tail-like bacteriocins produced by Pseudomonas aeruginosa Due to their antipseudomonal activity, R-pyocins have potential as therapeutics in infection. P. aeruginosa is a Gram-negative opportunistic pathogen and is particularly problematic for individuals with cystic fibrosis (CF). P. aeruginosa organisms from CF lung infections develop increasing resistance to antibiotics, making new treatment approaches essential. P. aeruginosa populations become phenotypically and genotypically diverse during infection; however, little is known of the efficacy of R-pyocins against heterogeneous populations. R-pyocins vary by subtype (R1 to R5), distinguished by binding to different residues on the lipopolysaccharide (LPS). Each type varies in killing spectrum, and each strain produces only one R-type. To evaluate the prevalence of different R-types, we screened P. aeruginosa strains from the International Pseudomonas Consortium Database (IPCD) and from our biobank of CF strains. We found that (i) R1-types were the most prevalent R-type among strains from respiratory sources, (ii) a large number of strains lack R-pyocin genes, and (iii) isolates collected from the same patient have the same R-type. We then assessed the impact of intrastrain diversity on R-pyocin susceptibility and found a heterogenous response to R-pyocins within populations, likely due to differences in the LPS core. Our work reveals that heterogeneous populations of microbes exhibit variable susceptibility to R-pyocins and highlights that there is likely heterogeneity in response to other types of LPS-binding antimicrobials, including phage.IMPORTANCE R-pyocins have potential as alternative therapeutics against Pseudomonas aeruginosa in chronic infection; however, little is known about the efficacy of R-pyocins in heterogeneous bacterial populations. P. aeruginosa is known to become resistant to multiple antibiotics and to evolve phenotypic and genotypic diversity over time; thus, it is particularly difficult to eradicate in chronic cystic fibrosis (CF) lung infections. In this study, we found that P. aeruginosa populations from CF lungs maintain the same R-pyocin genotype but exhibit heterogeneity in susceptibility to R-pyocins from other strains. Our findings suggest there is heterogeneity in response to other types of LPS-binding antimicrobials, such as phage, highlighting the necessity of further studying the potential of LPS-binding antimicrobial particles as alternative therapies in chronic infections.

Epidemiologic characterization of Pseudomonas aeruginosa in patients with cystic fibrosis.

OBJECTIVE: To determine persistence and variability of colonization with Pseudomonas aeruginosa in cystic fibrosis patients over long time periods, and to look for possible cross-colonization. METHODS: In total, 469 Pseudomonas aeruginosa isolates were obtained from 30 patients during the period from April 1994 to April 1996. The sources were mainly sputum and a few deep throat swabs. All grown strains dissimilar in macromorphology were processed separately. Typing with PFGE was carried out by contour-clamped homogeneous electric field electrophoresis. Genomic DNA was subjected to the rare-cutting restriction enzyme SpeI. For pyocin typing, the procedure described by Fyfe was applied. RESULTS: After typing with PFGE, we observed 40 restriction profiles. Eighteen different pyocin types were found. The most frequent pyocin type was type 3, followed by types 1 and 5. Twenty-two patients were persistently colonized by one clone specific and different for each patient, and four were co-colonized by a second clone also different for each of these patients. Cross-colonization had apparently been rare in the cystic fibrosis center of Leipzig. CONCLUSIONS: Typing with PFGE is well suited for detailed investigations of colonization with Pseudomonas aeruginosa in cystic fibrosis patients. Pyocin typing can provide additional information for epidemiologic purposes.

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157.

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.

Comparison of three typing methods for Pseudomonas aeruginosa.

Fifty-seven independent isolates of Pseudomonas aeruginosa from blood specimens were typed with 3 different methods: ribotyping, random amplified polymorphic DNA (RAPD) typing, and pyocin typing. Ribotyping was performed by probing the rRNA genes of genomic DNA that was digested separately with 4 different restriction enzymes. Digestion of DNA from 57 P. aeruginosa isolates with BamHI, ClaI, EcoRI, and PstI produced 4, 4, 6, and 7 patterns, respectively. As a result, ribotyping classified the 57 isolates into 22 types. Six new ribotypes that had not been described previously were found. One BamHI, 1 ClaI, 2 EcoRI, and 2 PstI patterns were novel. RAPD typing was performed with two different polymerase chain reaction (PCR) primers (RAPD1 and RAPD2). Both primers classified the 57 isolates into 15 RAPD types and produced identical patterns. The pyocin typing method classified the 57 isolates into 10 types. According to the results obtained in this study, the ribotyping has a discriminatory index of 0.865, RAPD, 0.785, and pyocin typing, 0.676, respectively. The ribotyping method was the most effective among the 3 methods compared for typing P. aeruginosa isolates.

[The epidemiological markers of Pseudomonas aeruginosa strains circulating in different types of hospitals]. / Epidemiologicheskie markery shtammov Pseudomonas aeruginosa, tsirkuliruiushchikh v statsionarakh raznogo profilia.

Pyocinovar and serovar characteristics of 91 P.aeruginosa strains isolated from patients and the environment in a hospital for premature children, a child survey hospital and a neonatological hospital. The leading epidemiological markers of P.aeruginosa strains, among them pyocinovar 883722, serovar 6 (a hospital for premature children), pyocinovar 888888, serovar 4 (a child surgery hospital), pyocinovars 888888 and 861322 (a neonatological hospital).

Epidemiologic typing of multiply drug-resistant Pseudomonas aeruginosa isolated from an outbreak in an intensive care unit.

From June to August 1991, there was an outbreak of Pseudomonas aeruginosa infections in an intensive care unit in a general hospital in Sao Paulo, Brazil. We obtained 14 isolates from 14 patients, 11 from tracheal aspirate, and 3 from surgical wound exudates. These strains were typed by serotyping, pyocin typing, and pulsed-field electrophoresis (CHEF) of chromosomal DNA (chrDNA), and the different typing methods were analyzed. These three methods demonstrated seven identical strains. We also performed an extensive antibiogram (33 drugs) in all 14 isolates. The incidence of resistance to aminoglycosides, extended-spectrum beta-lactams, and quinolones was very high among the seven identical isolates; however, the antibiogram profile differed significantly among the isolates. Our results suggest that a unique strain caused several cross-transmitted infections during this period of time, and the emergence of antimicrobial resistance has been occurring before and after the establishment of the epidemic strain by selective drug use. The chrDNA fingerprinting proved to be versatile and precise for epidemiologic investigations of P. aeruginosa infections.

Epidemiological typing of uropathogenic Pseudomonas aeruginosa strains from hospitalized patients.

One hundred and twenty-one clinical isolates of Pseudomonas aeruginosa from patients with hospital-acquired urinary tract infections (UTIs) were studied to determine their major epidemiological markers, including API 20NE profile, O-serotype, pyocin type, phage type, lysogenic state and antibiotic susceptibility. Serotypes O4, O12, O11, O6 and O5 were found with a high frequency, accounting respectively for 23.9%, 23.1%, 12.3%, 8.2% and 5.7% of isolates. Pyocin type 10 was most common (32.2%) followed by types 1 (10.7%), 33 (7.5%) and 105 (4.1%); subtype h was predominant being characteristic of 34.7% of isolates. Most of the strains (69.4%) were either not phage typable or sensitive to phages 68 and 119x. Resistance to gentamicin, tobramycin, amikacin, imipenem and ciprofloxacin was more frequent among strains belonging to serotype O12. The O-serotypes were combined with API 20NE profiles, pyocin and phage types, lysogenic states and antibiotic resistance in order to identify epidemiologically related clones. Within predominant serotypes--O4 and O12--most strains displayed similar but not identical type characteristics, whereas other serotypes were less homogeneous. Our results support the concept that a combination of current typing techniques allows the identification of epidemiologically related P. aeruginosa isolates.

Pyocin typing of Pseudomonas aeruginosa isolates from children with cystic fibrosis.

Pyocin typing and serotyping of 433 strains of Pseudomonas aeruginosa from children with cystic fibrosis (CF) showed that pyocin type 9 was predominant, particularly in association with polyagglutinating serotype. The common pyocin groups, 1, 5 and 10, made up only 20% of these isolates in contrast to reported rates of up to 89% in other studies using non-CF strains. No strains of pyocin type 3 were found. Polyagglutinating strains made up 72% of strains from patients colonized with P. aeruginosa for more than 12 mths. Pyocin type 9 was associated with 93% of polyagglutinating strains. The parallel between pyocin type 9 and polyagglutinating serotype suggests that these may both be characteristics acquired by P. aeruginosa colonizing patients with CF. Because of confounding between duration of colonization and exposure to cross-infection, this study does not allow definition of the role of cross-infection in determining the characteristics of these strains in most patients. In siblings, however, evidence supports a role for cross-infection either between siblings or from a common source. In 6 pairs of siblings studied, each pair had at least 1 pyocin group in common concurrently, either at entry to the study or after an interval of several months. Identical and unusual pyocin groups were recognized in samples obtained on the same day from pairs of siblings. More studies are needed to compare results of pyocin typing with methods such as genome fingerprinting to characterize these strains and determine whether the observed distribution of pyocin groups in CF isolates is related to cross-infection or whether the combination of pyocin type 9 with polyagglutinating serotype is a characteristic of CF strains.

Incidence of common pyocin types of Pseudomonas aeruginosa from patients with cystic fibrosis and chronic airways diseases.

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.

Ecology of Pseudomonas aeruginosa in patients with cystic fibrosis.

The occurrence of various Pseudomonas aeruginosa strains in the sputum of 15 patients with cystic fibrosis (CF) was monitored over periods ranging from 2 to 60 months. Isolates of P. aeruginosa were typed by four different techniques, namely serotyping, active and passive pyocin typing, and phage typing. The maximum number of different serotypes found in the patients was three (one serotype in nine patients; two serotypes in five patients; three serotypes in one patient). Pyocin and phage typing showed no marked differences between strains of the same serotype in individual patients. Exacerbations of chronic respiratory infection were not associated with changes in the sputum flora, the composition of P. aeruginosa strains in which remains constant over long periods in patients with CF.

[Routes of the transmission of Pseudomonas aeruginosa infection in resuscitation and intensive therapy departments]. / Puti peredachi sinegnoinoi infektsii v otdeleniiakh reanimatsii i intensivbnoi terapii.

To find out if the transfer of P. aeruginosa infection by droplet route is possible in resuscitation and intensive care units, the bacteriological study of air samples taken in different rooms of resuscitation units (altogether 234 air samples) was carried out with the subsequent identification and typing of isolated P. aeruginosa strains. In most cases (70.5%) the microbial contamination of the air in the main rooms of resuscitation units was found not to exceed 500 microbial cells per cu. m, and no P. aeruginosa strains were isolated. The identification and typing of six P. aeruginosa strains isolated from the air of an isolation ward for patients with infectious complications made it possible to find out intraspecific differences of these microorganisms, as all of them belonged to strains of different sero- and pyocinotypes. Thus, the results of these investigations indicate that the droplet route of the transfer of P. aeruginosa hospital infection is not characteristic of resuscitation and intensive care units, as no P. aeruginosa strains are isolated from the main rooms of such units; likewise, no circulation of this microorganism was observed in the air of an isolation ward for patients with infectious complications.

Purification and characterization of pyocins R from Pseudomonas aeruginosa.

Five types of pyocins were found in Pseudomonas aeruginosa strain 112. Production of these types was induced by UV irradiation. The pyocin activity was found to be resistant to trypsin treatment. Their molar mass was found to be 282, 251, 112, 89.1 and 54.9 kg/mol, respectively. The pyocins obtained were different from any known type (such as R, S, F) in their chemical and physical properties.

[Pseudomonas septicemia after endoscopic interventions on the bile duct system]. / Pseudomonas-Septikämie nach endoskopischen Eingriffen am Gallengangsystem.

In the first half of 1982 there was an increase in septicaemia cases, especially among patients with biliary-tract drainage. The septicaemia incidence rose from 1.25% to 4.4%. The proportion of Pseudomonas septicaemias was especially high: of 20 patients (21 episodes of septicaemia) nine had infections with Pseudomonas aeruginosa alone, three a mixed septicaemia. In ten of these twelve patients there was impaired drainage by a malignant tumour. Three patients with a tumour stenosis died, mainly of the septicaemia. The cause of this increased incidence of Pseudomonas septicaemias lay in contamination of the instruments; it was in part sustained by the Endo-Washer. The connection between the septicaemia after endoscopic-retrograde cholangiography and recontamination of the endoscope was discovered after sero- and pyocin-typing of the Pseudomonas strains. By changing the methods of disinfection the Pseudomonas incidence among endoscopies fell. However, in individual samplings Pseudomonas can still be demonstrated on instruments as well as on the Endo-Washer.

[Pyocin typing of Pseudomonas aeruginosa strains]. / Piotsinotipirovanie shtammov Pseudomonas aeruginosa.

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.

A Pseudomonas pyocin typing method applicable to the clinical laboratory.

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.