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.     

自西安地区医院分离的118株绿脓杆菌的“O”血清分型（群）

血清分型是检测绿脓杆菌交叉感染和追踪其感染源的重要工具。本文用国内生产的绿脓杆菌诊断用12群“O”血清,对自西安市11所医院临床病人创面、呼吸道和尿路感染分离的118株绿脓杆菌进行了血清学分型鉴定。结果发现,118株绿脓杆菌中有115株能被“O”血清凝集,鉴定率为97.46%;有109株能被分型,分型率为92.37%。其中以血清Ⅵ.Ⅰ.Ⅲ三群菌多见,表明西安地区医院绿脓杆菌感染以这三个血清群为主。     

绿脓杆菌分子分型方法研究进展

绿脓杆菌是一种常见的人畜共患机会致病菌,广泛存在于自然界,是造成实验动物污染和医院内感染的重要病原菌之一。分子分型方法是病原菌流行病学分析的重要手段,对于确定感染来源和途径,检测交叉污染和流行菌株方面非常有效。本文主要对绿脓杆菌分子分型方法的研究进展进行综述。     

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.     

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.     

Typing of Pseudomonas aeruginosa strains in Turkish cystic fibrosis patients

The majority of cystic fibrosis (CF) patients suffer from chronic respiratory infection with the opportunistic bacterial pathogen Pseudomonas aeruginosa. The virulence of P. aeruginosa is associated with the presence of various extracellular factors, like alginate, elastase, alkaline protease which contribute tissue destruction and assist bacterial invasion. Virulence factor production of P. aeruginosa strains isolated from 46 CF patients followed in two cities in Turkey was detected. Strains were compared genotypically by arbitrarily primed PCR. Antimicrobial susceptibilities to 12 antibiotics were determined by broth microdilution method. Evaluation of virulence factor results revealed that 95.8% of the strains were alginate, 71.7% elastase and 52.1% alkaline protease producers. AP-PCR analysis revealed 35 genotypes indicated almost a complete discrepancy among the strains. The most effective drugs were penems and quinolones. Among aminoglycosides amikacin was the most effective one and a high level resistance to beta lactams was observed. Alginate is the most important virulence factor in the chronic colonisation of CF patients with P. aeruginosa. No evidence for cross infection between patients and for relationship between phenotypes and genotypes of the strains was found.     

Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.The metabolically versatile Pseudomonas aeruginosa is an opportunistic pathogen of plants, animals, and humans and is ubiquitously distributed in soil and aquatic habitats. The common reference strain is P. aeruginosa PAO1, a spontaneous chloramphenicol-resistant mutant of the original PAO strain (earlier called “P. aeruginosa strain 1”) that had been isolated in 1954 from a wound in Melbourne, Australia (9, 10). This PAO1 strain from Bruce Holloway''s laboratory has become the reference strain for Pseudomonas genetics and functional analyses of the physiology and metabolism of this gammaproteobacterium. A genetic map of its chromosome was generated by exploiting the mechanisms of gene exchange in bacteria, i.e., transduction and conjugation (11). With the advent of pulsed-field gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). By 2000 the PAO1 strain had been completely sequenced (36). Thereafter, the genome annotation has been continually updated and the database content and functionality have been expanded to facilitate accelerated discovery of P. aeruginosa drug targets and vaccine candidates (38). Two near-saturation libraries of transposon insertion mutants have been constructed in P. aeruginosa PAO1 as a global resource for the scientific community (14, 22).Comparison of the genome sequence with the physical map revealed a large, 2.2-Mb inversion between the sequenced PAO1-UW strain (36) and the original PAO1 strain (9, 10), indicating that PAO1 sublines maintained worldwide in numerous laboratories and strain collections had diversified their genomic sequence. Mutational events were already reported in the 1970s (10), and more recently sequence variations of MexT, which regulates the MexEF-OprN multidrug efflux system, were described (18, 24). Furthermore, a PAO1 subline from a German strain collection (PAO1-D) and another, independent PAO1 subline from a Japanese strain collection (PAO1-J) that had been stored by research groups in Germany and Japan, respectively, were found to be quorum-sensing-negative mutants that carried point mutations in the regulatory gene lasR (6). In addition, spontaneous secretion-defective vfr mutants from a PAO1 population were observed after several cycles of static growth (2). Similarly, we noted a difference in virulence in a mouse infection model (see below) between the MPAO1 and PAO1-DSM sublines that had been utilized for the construction of the transposon library (14) and the physical map (32), respectively. PAO1-DSM was indistinguishable in its SpeI-DpnI-SwaI-PacI physical map from the PAO1 subline that had been stored in the Holloway laboratory (12). Hence, we decided to compare the genomic sequence of the initially sequenced PAO1 subline PAO1-UW (36) with that of MPAO1 and PAO1-DSM. Combined physical mapping and DNA sequencing-by-synthesis revealed numerous single-nucleotide polymorphisms (SNPs) and insertions-deletions (indels) in the chromosomes that were associated with differences in fitness, antimicrobial susceptibility, and virulence of the sublines.     

Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.     

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.     

Pyruvate Carboxylase Deficiency in Pleiotropic Carbohydrate-Negative Mutant Strains of Pseudomonas aeruginosa

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5'-triphosphate, and Mg(2+), but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix (14)CO(2) in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C(6) and C(3) compounds. A second linkage group of similar mutant strains that grew on the C(3) compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.     

The Serological Heterogeneity of Pseudomonas syringae pv. atrofaciens Strains and Their Ecological Niches

The paper deals with a comparative analysis of the serological and ecological properties of Pseudomonas syringae pv. atrofaciens strains from the collections of microbial cultures at the Malkov Institute for Plant Genetic Resources and Zabolotny Institute of Microbiology and Virology. All of the strains from the Bulgarian collection, except for one, fall into five serogroups (II through VI) of the classification system of Pastushenko and Simonovich. The P. syringae pv. atrofaciens strains isolated from Bulgarian and Ukrainian wheats belong mainly to serogroups II and IV, respectively. The strains that were isolated from rye plants belong to serogroup I. The strains isolated from sorghum and Sudan grass belong to serogroups II, IV, and VI. Serogroup III includes the P. syringae pv. atrofaciens strains that were isolated from cereals in the United Kingdom but not in Ukraine.