Virulence and toxigenicity of Pseudomonas aeruginosa cultures of various origins

The virulence and toxigenicity of newly isolated P. aeruginosa strains have been studied in experiments on white mice. These biological properties have been shown to be most pronounced in P. aeruginosa strains isolated from proteins, sometimes greatly exceeding those in strains isolated from healthy persons and the environment. Virulence and the factors which determine it are definitely interrelated in microorganisms and can vary, depending on the conditions of their habitat.     

Pseudomonas aeruginosa bacteriophages isolated in a surgical hospital

New data on P. aeruginosa bacteriophages isolated from patients, as well as from washings obtained from various objects, in a surgical hospital are presented. 14 pure strains of P. aeruginosa bacteriophages have been isolated from 90 specimens of the material under study. The morphology of the colonies, the titer and the spectrum of action of the phages are characterized. The spectrum of action of polyvalent combination obtained by the mechanical mixture of different phages has been studied. The most active phages have been found to lyse 71.1, 63.1, 59.2 and 41.8 per cent of P. aeruginosa museum strains (225 strains).     

The hemagglutinating properties of Pseudomonas aeruginosa strains isolated from patients with nonspecific lung diseases]

To detect d-mannose-sensitive (MS) pili in 31 P. aeruginosa strains isolated from the respiratory tract of patients with inflammatory and purulent destructive pulmonary diseases, the hemagglutination (HA) test was used. The isolated Pseudomonas under study differed in the degree of manifestation of their MS adhesins. Among them microorganisms with pronounced HA activity (high HA titer) occurred, as well as those whose HA activity was less pronounced (low HA titer). P. aeruginosa strains with pronounced HA activity were more frequently isolated from patients with purulent destructive processes in the lungs. A correlation between the state of the patient at the moment of bacteriological examination and the degree of manifestation of MS pili in the P. aeruginosa strain isolated from this patients was established. The value of HA titer in the presence of d-mannose is indicative of the presence of MS adhesins in a P. aeruginosa strain.     

Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection

The present study using three isogenic mutants (F+P-, F-P+, F-P-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non-piliated (P-) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non-flagellated (F-) mutant was at least 10(5) times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P- bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P- bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.     

圈养林麝铜绿假单胞菌的分离鉴定及耐药性和病原特性分析

【背景】铜绿假单胞菌感染所致的化脓性疾病是困扰林麝驯养的重要因素,是一类较难防治的细菌性疾病,目前尚无疫苗可用来预防该病。【目的】研究林麝源铜绿假单胞菌的感染现状和分子流行病学规律。【方法】对2014年10月至2015年10月四川宝兴和陕西镇坪两个林麝养殖中心发病林麝中铜绿假单胞菌进行分离鉴定,并对其耐药情况进行分析,利用脉冲场凝胶电泳(PFGE)分型技术研究分离菌的PFGE指纹图谱,探究其流行病学趋势,并对部分菌株的致病性进行分析。【结果】分离得到60株铜绿假单胞菌,其中34株来自镇坪,26株来自宝兴。耐药结果表明:60株林麝源铜绿假单胞菌对17种抗菌药物呈现不同程度耐药性,不同地区和不同样本源间分离的铜绿假单胞菌对17种抗菌药物的耐药性总体趋于一致,多重耐药情况严重,以5耐、6耐为主。分型结果显示:60株铜绿假单胞菌PFGE图谱的相似性为49.1%-100%。经聚类分析得到A-O共15种基因簇,其中优势基因簇为C、E、G、J。致病性结果表明,流行菌株的致病力强弱依次为:动物源菌株环境源菌株,且主要流行菌株(基因簇E、F、J)的致病力大于其余菌群。【结论】铜绿假单胞菌在两地区具有水平传播的途径,本研究可为跨地区林麝化脓性炎症的防控提供理论依据。     

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.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.     

Pilus antigen 987P produced by strains of Escherichia coli serotypes O141:K--, H- and O8:K85:H--

Escherichia coli strains isolated from the alimentary tract of 68 weaned and 44 unweaned pigs with diarrhoea in various parts of Hungary, were tested for the presence of pilus antigens K88, K99 and 987P. K88 was detected in 30% of the strains from newborn pigs and in 12% of the strains isolated from weaned pigs. One strain carried K99. Based on agglutination test and immunoelectron microscopic studies with specific absorbed antisera, five non-haemolytic E. coli strains isolated from newborn pigs were found to produce so-called 987P pili. Three of these strains were designated serologically as O8:K85:H--,987P+ and two as O141: K--:H--,987P+. The Y1 cell assay, the infant mouse assay, and the ligated intestinal loop assay in less than 3-week-old pigs indicated that none of the strains produced heat-labile enterotoxin but all produced a heat-stable enterotoxin detectable in infant mice and in pig loops (STa). All the strains induced diarrhoea in newborn, colostrum deprived pigs and colonized the lower small intestine by adhesion to the villous epithelium. The results have confirmed earlier findings about adhesive virulence attributes caused by 987P pili.     

Protective properties of anatoxin obtained from a homogeneous preparation of Pseudomonas aeruginosa exotoxin A

The protective properties of formulated toxoid obtained from the highly purified preparation of P. aeruginosa exotoxin A have been studied in the test of the active immunization of mice. The study has revealed that the preparation when introduced in 1 or 2 injections in a dose of 15 micrograms, shows faint protective potency with respect to P. aeruginosa strains differing in virulence. Immunization with this toxoid in 3 and 4 injections has been found to ensure 60-100% and 50-60% protection of mice infected with P. aeruginosa toxigenic and proteolytic strains respectively. Immunization with toxoid has been found to induce the appearance of short-term antibacterial immunity which loses its capacity to protect the immunized animals, challenged with both toxigenic and proteolytic P. aeruginosa strains, as early as on day 28. The immunization of mice with toxoid in 4 injections has been shown to induce the development of antitoxic immunity capable of neutralizing up to 150 LD50 of purified exotoxin A.     

Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.     

Survey of Pseudomonas aeruginosa and its phages: de novo peptide sequencing as a novel tool to assess the diversity of worldwide collected viruses

A collection of 15 newly isolated (bacterio)phages infecting the opportunistic pathogen Pseudomonas aeruginosa was established to investigate their global diversity and potential in phage therapy. These phages were sampled in 14 different countries traversing four continents, from both natural environments and hospital sewage. They all display unique DNA and protein profiles and cluster morphologically into six groups within the three major families of the Caudovirales . Extensive host range studies on a library of 122 AFLP-genotyped clinical P. aeruginosa strains (of which 49 were newly isolated at the University Hospital of Leuven, Belgium) showed that the phages lysed 87% of the strains. Infection analysis of outer membrane mutants identified 10 phages as type IV pili-dependent. More detailed information about the evolutionary relatedness of the phages was gathered by de novo peptide sequencing of major virion proteins using tandem Matrix-Assisted Laser Desorption/Ionization Time of Flight technology. Applying this technique for the first time to viruses, seven groups of closely related phages were identified without the need of prior knowledge of genome content and/or electron microscopic imaging. This study demonstrates both the epidemic population structure of P. aeruginosa and the global spread of P. aeruginosa phage species, and points at the resistance of two clinically predominant, widespread P. aeruginosa strains against phage attack.     

Synthetic peptide vaccine and antibody therapeutic development: prevention and treatment of Pseudomonas aeruginosa

Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections.     

Comparison of virulence between clinical and environmental Pseudomonas aeruginosa isolates.

New strains of Pseudomonas aeruginosa were isolated from clinical and environmental settings in order to characterize the virulence properties of this opportunistic pathogen. P. aeruginosa was frequently recovered from oil-contaminated samples but not from non-oil-contaminated soils. The virulence of five environmental and five clinical strains of P. aeruginosa was tested using two different models, Drosophila melanogaster and Lactuca sativa var. capitata L. There was no difference in the virulence between the two groups of isolates in either of the models. Since environmental P. aeruginosa strains are used for bioaugmentation in bioremediation programs, the results presented here should be taken into account in the future design of degradative consortia and/or in establishing containment measures.     

The description of an esculin-positive biovar of Pseudomonas aeruginosa

In the study of 280 P. aeruginosa strains isolated in different hospitals of St. Petersburg for the first time 48 strains capable of hydrolyzing esculin have been detected. The hydrolysis of esculin is determined in plates with the use of the microvolume techniques the results were evaluated after 3-hour incubation at 37 degrees C. The data confirming the existence of the exculin-positive biovar of P. aeruginosa have been obtained; these data show the wide spread of esculin-positive strains in hospitals of different specialization (17.1 +/- 5.1% of P. aeruginosa strains), the characteristic combination of the sign of esculin hydrolysis with such signs as the absence of the smell of trimethylamine and the phenomenon of "iridescent lysis" of the colonies, the stability of the sign of esculin hydrolysis in strains, repeatedly isolated from patients, after the storage of the cultures and their treatment with plasmid-eliminating preparation. The name "esculinolytica" has been proposed for this biovar. The typing strain of biovar esculinolytica has been deposited in the culture collection of the Russian Research Institute of Agricultural Microbiology as P. aeruginosa ARRIAM 64-A. This biovar been found to be most widely spread in urological hospitals, where esculin-positive strains are isolated 3 times more frequently (32.2 +/- 5.1% of P. aeruginosa strains) than in surgical hospitals (10.7 +/- 2.2%).     

Expression of multiple types of N-methyl Phe pili in Pseudomonas aeruginosa

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid-borne genes of homologous pilins from Bacteroides nodosus are introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two serologically distinct populations of pili on each cell. Simultaneous production of both indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.     

Isolation and primary characteristics of elastase from a clinical strain of Pseudomonas aeruginosa

The homogeneous preparation of elastase has been obtained from P. aeruginosa clinical strain. The molecular weight of the isolated enzyme is 33,000 daltons, and its isoelectric point is 6.8. Two media manufactured in this country (dialyzed bovine heart hydrolysate and a dried semisynthetic medium) ensuring good production of the enzyme have been proposed. The optimum time for the cultivation of the producer strain (30-40 hours) has been established. Elastase has been shown to be widely spread among P. aeruginosa clinical strains.     

Effect of plasmids on the virulence of Pseudomonas aeruginosa strains in experiments on mice

A comparative study of virulence of P. aeruginosa strains PAO containing and not containing plasmids has been made. A number of plasmids which are present in strains PAO decrease their virulence for mice 3-7 times. The virulence-affecting plasmids considerably differ in their biological properties. Bacterial mutations rpm, selected as mutations stabilizing RP4 plasmid in PAO cells, have also been found to affect virulence of bacteria, decreasing its level several times. The introduction of plasmids into PAO cells carrying mutations rpm is not accompanied by decrease of virulence.     

Mutations in the Gene Encoding the Ancillary Pilin Subunit of the Streptococcus suis srtF Cluster Result in Pili Formed by the Major Subunit Only

Pili have been shown to contribute to the virulence of different Gram-positive pathogenic species. Among other critical steps of bacterial pathogenesis, these structures participate in adherence to host cells, colonization and systemic virulence. Recently, the presence of at least four discrete gene clusters encoding putative pili has been revealed in the major swine pathogen and emerging zoonotic agent Streptococcus suis. However, pili production by this species has not yet been demonstrated. In this study, we investigated the functionality of one of these pili clusters, known as the srtF pilus cluster, by the construction of mutant strains for each of the four genes of the cluster as well as by the generation of antibodies against the putative pilin subunits. Results revealed that the S. suis serotype 2 strain P1/7, as well as several other highly virulent invasive S. suis serotype 2 isolates express pili from this cluster. However, in most cases tested, and as a result of nonsense mutations at the 5′ end of the gene encoding the minor pilin subunit (a putative adhesin), pili were formed by the major pilin subunit only. We then evaluated the role these pili play in S. suis virulence. Abolishment of the expression of srtF cluster-encoded pili did not result in impaired interactions of S. suis with porcine brain microvascular endothelial cells. Furthermore, non-piliated mutants were as virulent as the wild type strain when evaluated in a murine model of S. suis sepsis. Our results show that srtF cluster-encoded, S. suis pili are atypical compared to other Gram-positive pili. In addition, since the highly virulent strains under investigation are unlikely to produce other pili, our results suggest that pili might be dispensable for critical steps of the S. suis pathogenesis of infection.     

Possible connection between the virulence of Pseudomonas aeruginosa bacteria and their ultrastructural characteristics

The results of the electron-microscopic study of P. aeruginosa isolated from patients with surgical inflammatory purulent processes and from the environment (water, soil) are presented. The morphological features of the subcellular structure of virulent and non-virulent P. aeruginosa strains, as well as their adaptation properties, appearing under unfavorable conditions have been established.