Heterogeneous virulence potential and high antibiotic resistance of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strains isolated from Korean pneumonia patients

Pseudomonas aeruginosa is an opportunistic human pathogen of clinical importance that causes airway infections in immunocompromised patients. Here, we report the virulence-associated characteristics of strains of P. aeruginosa, isolated from the sputa of 25 Korean pneumonia patients. A high degree of genomic plasticity was observed by random amplified polymorphic DNA genotype analysis, suggesting that the infections were caused by strains with diverse genomic backgrounds. Biofilm formation of each isolate was heterogeneous in terms of their relative motilities. In addition, 48% of isolates were defective in the production of 3-oxo-C₁₂-HSL (PAI-1), a quorum sensing signal molecule. In these strains, PAI-1-dependent elastase production was correspondingly decreased, suggesting that a large number of strains were presumed to be quorum sensing deficient. Multidrug resistance (MDR) was seen in 56% of the isolates tested, and 44% of the MDR strains were resistant to five or more antibiotics. Taken together, our results provide additional insights into the virulence traits of P. aeruginosa clinical isolates, which will aid in treating P. aeruginosa infections in pneumonia patients.     

Proteolytic Activity of Pseudomonas aeruginosa Isolates with TTSS-Mediated Cytotoxicity and Invasiveness to Host Cells

Over 100 of Pseudomonas aeruginosa isolates representing the two TTSS genotypes (exoU ⁻/exoS ⁺ or exoU ⁺/exoS ⁻) were cultured in different media in order to evaluate their proteolytic activities and find a relationship between proteolytic activity and the cytotoxic and/or invasive phenotypes displayed by the strains upon infection of RAW 264.7 murine macrophage-like cells and pulmonary microvascular endothelial cells (PME). The elastolytic activity, protein concentration, and total proteolytic activity (TPA) were measured in culture supernatants. No significant differences were observed in the median elastolytic activities among cytotoxic/noninvasive, noncytotoxic/invasive, and cytotoxic/invasive phenotypes displayed by P. aeruginosa strains. The only significant difference was noted when isolates of the two different TTSS genotypes were grown in a calcium-depleted minimal medium for induction of TTSS (MI). The exoU ⁻/exoS ⁺ isolates showed significant higher levels of the median elastolytic activity when compared to the exoU ⁺/exoS ⁻ isolates. These two groups of isolates secreted the elastase B (LasB) with distinct molecular masses 158 or 116 kD, respectively. The strains of the two TTSS genotypes secreted similar amount of total proteins; however, the higher values of TPA were observed for the isolates of the exoU ⁺ /exoS ⁻ genotype when grown in MI medium. We concluded that there is no direct relationship between secretion of proteases with elastolytic activity and the cytotoxic and/or invasive phenotypes of the isolates observed upon infection of both RAW 264.7 and PME monolayers. Further studies are needed to find out whether others factors beside proteases could influence the mechanism of host cells intoxication mediated by the P. aeruginosa TTSS-delivered toxins.     

Studies of Pseudomonas aeruginosa Infections Among Hospitalized Patients

A rapid identification method of glucose nonfermentative gram-negative rods was established and 320 strains isolated were divided into five groups according to their characteristics in pigmentation, acid from glucose, cytochrome oxidase activity and motility. Further characterization of the strains in each group resulted in the identification that the strains in group I were Pseudomonas aeruginosa, strains in group II, P. aeruginosa and Pseudomonas putida. Achromogenic strains of P. aeruginosa were classified into group III, Pseudomonas maltophilia, Pseudomonas alcaligenes and Alcaligenes faecalis into group IV and Acinetobacter calcoaceticus (Acinetobacter anitratus and Achromobacter lwoffii) in group V. When fluorescent pigment production was taken as a standard, 259 out of 263 chromogenic strains were identified as P. aeruginosa and the remaining four were P. putida. Whereas forty-five achromogenic strains included twenty-four A. calcoaceticus, eight P. aeruginosa, six A. faecalis, five P. maltophilia and two P. alcaligenes. From May 1970 to June 1971, 368 strains of glucose nonfermentative rods were isolated from clinical specimens sent to the Central Laboratories of Tohoku University Hospital and three fourth (286/368) of the isolates were P. aeruginosa     

Overexpressed recombinant quorum quenching lactonase reduces the virulence,motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min⁻¹ for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

     

Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition

Abstract

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.     

Molecular investigation of carbapenem resistance among multidrug‐resistant Pseudomonas aeruginosa isolated clinically in Thailand

Carbapenem resistant Pseudomonas aeruginosa were isolated among multidrug‐resistant (CR‐MDR) organisms from tertiary hospitals in Thailand. Decreased expression of oprD mRNA (93.65%) was predominant followed by increased expression of mexAB‐oprM mRNA (92.06%) and mexXY mRNA (63.49%). Interestingly, 23 of 126 (18.25%) isolates were susceptible to imipenem with down‐regulated oprD expression and non‐up‐regulated mexCD‐oprJ mRNA expression. Metallo‐β‐lactamases production was clearly positive in 24 isolates (18.46%) and weakly positive in 12 isolates (9.23%). Among both of these sets of isolates, imp‐1, imp‐14 and vim‐2 were identified. Hyperproduction of AmpC β‐lactamase had the lowest prevalence rate (3.97%). It was concluded that CR‐MDR P. aeruginosa clinical isolates in Thailand possess multifactorial resistance mechanisms.     

Pseudomonas aeruginosa-derived rhamnolipids subvert the host innate immune response through manipulation of the human beta-defensin-2 expression

Pseudomonas aeruginosa is a well‐known cause of infections especially in compromised patients. To neutralize this pathogen, the expression of antimicrobial factors in epithelial cells is crucial. In particular the human beta‐defensin hBD‐2 is especially active against P. aeruginosa. In this study, we identified rhamnolipids in P. aeruginosa culture supernatants that are able to prevent the pathogen‐induced hBD‐2 response in keratinocytes. The presence of rhamnolipids within the host cells and inhibition assays suggest that calcium‐regulated pathways and protein kinase C activation are impaired by rhamnolipids. In consequence, the induction of hBD‐2 in keratinocytes by P. aeruginosa‐derived flagellin as well as the host's own hBD‐2 mediator interleukin IL‐1β is inhibited. Strikingly, rhamnolipids did not affect the release of the proinflammatory mediator interleukin IL‐8 by flagellin. Thus, in addition to their function in establishment and persistence of P. aeruginosa infections, rhamnolipids can be engaged by P. aeruginosa for a targeted attenuation of the innate immunity to manage its survival and colonization on compromised epithelia.     

Slime Production by Pseudomonas aeruginosa

Chemical properties and compositions of slimes produced by two Pseudomonas aeruginosa strains of different colonial types were investigated. The main component of the slime from strain IFO 3445 was found to be DNA, contaminated with small amounts of protein. On the other hand, the slime from a mucoid-type strain No. 24 was an alginate-like substance consisting of mannuronic and glucuronic acids, and contained traces of protein and nucleic acid. Slimes from twenty clinical isolates of P. aeruginosa were investigated for their chemical compositions. Slimes from eighteen strains consisted of DNA, while, two strains of a mucoid-type produced slimes composed of polyuronic acid.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> from Open Ocean and Comparison with Freshwater,Clinical, and Animal Isolates

Pseudomonas aeruginosa is an opportunistic pathogen responsible for morbidity and mortality in humans, animals, and plants. This bacterium has been regarded to be widely present in terrestrial and freshwater environments, but not in open ocean environments. Our purpose was to clarify its presence in open ocean, and their genotypic and physiological characteristics were compared with those of isolates from clinical, animal, and freshwater sources. Water samples were collected from freshwater, bays, and offshore environments in Japan. Sixty-two isolates, including 26 from the open ocean, were identified as P. aeruginosa by phenotypic characteristics and the BD Phoenix System. Pulsed-field gel electrophoresis (PFGE) was performed on all strains, together with 21 clinical and 8 animal strains. The results showed that open ocean strains are composed of a few genotypes, which are separated from other strains. Although some clinical isolates made a cluster, other strains tended to mix together. Different antibiotypes were observed among marine isolates that had similar PFGE and serotyping patterns. Some were multidrug-resistant. Laboratory-based microcosm study were carried out to see the responses of P. aeruginosa toward increased NaCl concentrations in deionized water (DW). Marine strains showed better survival with the increase, whereas river and clinical strains were suppressed by the increase. These findings illustrate the potential significance of open ocean as a possible reservoir of P. aeruginosa, and there may be clones unique to this environment. To our knowledge, this is the first report on the presence and characterization of P. aeruginosa in the open ocean.     

Effect of phosphate deficiency on growth and protein profile in three strains ofPholiota nameko

Changes in mycelial dry weight and soluble protein amounts and acid phosphatase activities on a mycelial dry weight basis in the mycelia and culture supernatants during the Pi-supplied (P⁺) and Pi-depleted (P⁻) cultures of three strains ofPholiota nameko were examined. Mycelial dry weights of the three strains were lower in the P⁻ culture than in the P⁺ culture. However, soluble protein amounts in the culture supernatants and acid phosphatase activities in the mycelia and culture supernatants of the three strains were higher on a mycelial dry weight basis in the P⁻ culture than in the P⁺ culture. Total proteins of strains N2 and N4 were analyzed by two-dimensional-PAGE. Comparison of electrophoretograms of the P⁺ and P⁻ cultures showed that many polypeptides in the two strains were induced and secreted by Pi deficiency, but more than half of them were specific to each strain. Activity staining of acid phosphatase also revealed that two isozymes with the same molecular weights in the three strains were induced and secreted by Pi deficiency. Adaptive mechanisms for Pi deficiency in the three strains were discussed.     

N-butanoyl-l-homoserine lactone (BHL) deficient Pseudomonas aeruginosa isolates from an intensive care unit

Acylated homoserine lactones (AHLs) are self-generated diffusible signal molecules that mediate population density dependent gene expression (quorum sensing) in a variety of Gram-negative bacteria, and several virulence genes of human pathogens are known to be controlled by AHLs. In this study, strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae, isolated from intensive care patients, were screened for AHL production by using AHL responsive indicator strains of Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1. Positive reactions were recorded for all 50 isolates of P. aeruginosa and 10 isolates of Acinetobacter baumannii with Agrobacterium tumefaciens NT1. Surprisingly, most P. aeruginosa isolates gave negative results with C. violaceum CV026 in contrast to previous reports. This suggests that the new isolates of P. aeruginosa either failed to make short chain AHLs or the level of the signal molecule is very low.     

Chromosome-encoded inducible copper resistance inPseudomonas strains

NinePseudomonas strains were selected by their high copper tolerance from a population of bacteria isolated from heavy-metal polluted zones. Copper resistance (Cu ^r ) was inducible by previous exposure of cultures to subinhibitory amounts of copper sulfate. All nine strains possessed large plasmids, but transformation and curing results suggest that Cu ^r is conferred by chromosomal genes. Plasmid-lessPseudomonas aeruginosa PAO-derived strains showed the same level of Cu ^r as environmental isolates and their resistance to copper was also inducible. Total DNA from the environmentalPseudomonas, as well as fromP. aeruginosa PAO strains, showed homology to a Cu ^r P. syringae cop probe at low-stringency conditions but failed to hybridize at high-stringency conditions.     

Interspecific Small Molecule Interactions between Clinical Isolates of Pseudomonas aeruginosa and Staphylococcus aureus from Adult Cystic Fibrosis Patients

Pseudomonas aeruginosa and Staphylococcus aureus are the most prevalent pathogens in airway infections of cystic fibrosis (CF) patients. We studied how these pathogens coexist and interact with each other. Clinical isolates of both species were retrieved from adult CF patients. Culture supernatants from 63 P. aeruginosa isolates triggered a wide range of biofilm-stimulatory activities when added to the culture of a control S. aureus strain. The extent of biofilm formation by S. aureus was positively correlated to the levels of the 2-alkyl-4-(1H)-quinolones (AQs) Pseudomonas Quinolone Signal (PQS) and 2-heptyl-4-hydroxy quinoline N-oxide (HQNO) produced by the P. aeruginosa isolates. Supernatants from P. aeruginosa isogenic mutants deficient in PQS and HQNO production stimulated significantly less biofilm formation by S. aureus than that seen with the parental strain PA14. When studying co-isolated pairs of P. aeruginosa and S. aureus retrieved from patients showing both pathogens, P. aeruginosa supernatants stimulated less biofilm production by the S. aureus counterparts compared to that observed using the control S. aureus strain. Accordingly, some P. aeruginosa isolates produced low levels of exoproducts and also some of the clinical S. aureus isolates were not stimulated by their co-isolates or by PA14 despite adequate production of HQNO. This suggests that colonization of the CF lungs promotes some type of strain selection, or that co-existence requires specific adaptations by either or both pathogens. Results provide insights on bacterial interactions in CF.     

Isolation of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Specific Phages with Broad Activity Spectra

The aim of the study was to screen various kinds of samples for Pseudomonas aeruginosa specific phages and to isolate and partially characterize those with broad activity spectra. The Pseudomonas specific phages were isolated using an enrichment procedure with single strains or the cocktail of P. aeruginosa strains as hosts. Using the described procedure, phages were successfully isolated only from water samples, while in soil and feces no Pseudomonas specific phages were detected. The lytic spectra of isolated phages were determined by spot method on lawns of 33 P. aeruginosa strains and five species belonging to family Enterobacteriaceae. The results showed that among isolated phages, 001A, δ, and I possessed the broad activity spectra, as were able to plaque on more than 50% of tested P. aeruginosa strains, while none of the phages were able to lyse the other tested species. Significant differences in phage activity spectra were not observed when P. aeruginosa cocktail was applied for sample enrichment. The most of the phages examined by electron microscopy belonged to family Siphoviridae, while the broad activity spectra isolates, except for 001A, possessed morphological characteristics of family Podoviridae. Digested DNA of the phages δ and I showed similar patterns, indicating the prevalence and success of this phage type in the environment.     

Investigation of the biodegradation of pentachlorophenol by the predominant bacterial strains in a mixed culture

A pentachlorophenol (PCP) degrading mixed culture contained three predominant strains identified as Flavobacterium gleum, Agrobacterium radiobacter and Pseudomonas sp. The relative abilities of the three strains to degrade PCP were tested individually and in combination. Rates of PCP degradation by individual isolates were lower than that observed for the three isolates combined. Of the individual strains, Flavobacterium gleum manifested highest PCP degradation ability. A biodegradation medium inoculated with a combination of the three isolates exhibited PCP degradation patterns similar to the original mixed culture. Varying low amounts of tetrachlorophenol were found in degradation medium inoculated with individual isolates, but this intermediate was absent from media inoculated with the mixed culture.     

Susceptibility of Pseudomonas aeruginosa Urinary Tract Isolates and Influence of Urinary Tract Conditions on Antibiotic Tolerance

Pseudomonas aeruginosa is an opportunistic human pathogen, which can cause severe urinary tract infections (UTIs). Because of the high intrinsic antibiotic resistance of P. aeruginosa and its ability to develop new resistances during antibiotic treatment, these infections are difficult to eradicate. The antibiotic susceptibility of 32 P. aeruginosa isolates from acute and chronic UTIs were analysed under standardized conditions showing 19% multi-drug resistant strains. Furthermore, the antibiotic tolerance of two P. aeruginosa strains to ciprofloxacin and tobramycin was analysed under urinary tract-relevant conditions which considered nutrient composition, biofilm growth, growth phase, and oxygen concentration. These conditions significantly enhance the antibiotic tolerance of P. aeruginosa up to 6000-fold indicating an adaptation of the bacterium to the specific conditions present in the urinary tract. This reversible phenomenon is possibly due to the increased formation of persister cells and is based on iron limitation in artificial urine. The results suggest that the general high antibiotic resistance of P. aeruginosa urinary tract isolates together with the increasing tolerance of P. aeruginosa grown under urinary tract conditions decrease the efficiency of antibiotic treatment of UTIs.     

Isolation and Partial Characterization of Bacteriophages of the Phytopathogen Pseudomonas syringae

Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage were identified. The DNA from each phage was isolated and digested with the restriction endonuclease EcoRI. Eight isolates were determined to be different, with two phage isolates from sewage having restriction patterns identical to two phages from culture supernatants. The sizes of the phage DNA ranged from 24 to49 kilobases for isolates from sewage and from 39 to 52.5 kilobases for the isolates from culture supernatants. Buoyant densities of phage particles in CsCl varied from 1.498 to 1.507 g/cm³ for isolates from sewage and from 1.506 to 1.516 g/cm³ for isolates from culture supernatants. Electron microscopy revealed four morphological types. Based on plaque-forming ability of culture supernatants, 31 out of 47 strains of P. syringae are probably lysogenic.     

Molecular characterization of clinical and environmental Pseudomonas aeruginosa isolated in a burn center

In burn centers, Pseudomonas aeruginosa acts as a major cause of nosocomial infections. Therefore, this study aimed to characterize molecularly P. aeruginosa isolates collected from environmental samples and burn patients. A total of 78 strains (including 58 clinical and 20 environmental isolates) of the P. aeruginosa were collected from Beasat hospital of Hamadan, west of Iran, and was identified using API 20NE. The disk diffusion method according to the CLSI was applied for determination of the antimicrobial resistance. Moreover, the microtiter plate test was used for the quantification of Biofilm formation. The genomic features of the isolated strains was evaluated using Pulsed Field Gel Electrophoresis (PFGE). We found that 94.8% of clinical and 80% environmental isolates were capable of forming biofilm. The rate of MDR in clinical and environmental isolates was 51.7% and 40%, respectively. A significant relationship was observed between biofilm formation capability and multiple drug resistance (p < 0.05). PFGE typing showed 11 different clusters with two major clusters A with 30 (38.5%) and B with 14 (17.9%) members, containing up to 56.4% of all isolates. There was no relationship between biofilm formation ability and antibiotic resistance patterns with PFGE patterns. According to the results, the clonal spread of environmental P. aeruginosa isolates is associated with clinical isolates, and both environmental and clinical isolates are attributed to a high prevalence of the antibiotic resistance and biofilm formation ability. This study highlighted that the prevention programs should be implemented in the hospital environment to control the spread of P. aeruginosa in burn units.     

Experimental Infection with Pseudomonas aeruginosa in Mice

Comparative studies on the virulence of 22 clinical isolates of Pseudomonas aeruginosa were made by means of intraperitoneal inoculation in mice. The LD₅₀ values of these strains for mice ranged from 10^{5 0} to 10^7.5 viable cells per mouse and the average was 10^6.6. The virulence of certain strains was significantly enhanced when these strains were inoculated mixed with mucin. The highly virulent strains were often found among the strains which were serologically untypable though no relationship could be found between the virulence of test strains and their other biological characteristics such as pigments, hemolysins and extracellular enzymes. The facts suggest that pigments and extracellular enzymes play no important role in the pathogenicity of P. aeruginosa for mice. Moreover, no difference was seen on virulence among the strains isolated from the patients and healthy carriers. The susceptibility of ICR, ddN and CF#1 mice for P. aeruginosa was investigated. There was no clear difference in susceptibilities to P. aeruginosa infection.     

Targeting Acyl Homoserine Lactones (AHLs) by the quorum quenching bacterial strains to control biofilm formation in Pseudomonas aeruginosa

Navigating novel biological strategies to mitigate bacterial biofilms have great worth to combat bacterial infections. Bacterial infections caused by the biofilm forming bacteria are 1000 times more resistant to antibiotics than the planktonic bacteria. Among the known bacterial infections, more than 70% involve biofilms which severely complicates treatment options. Biofilm formation is mainly regulated by the Quorum sensing (QS) mechanism. Interference with the QS system by the quorum quenching (QQ) enzyme is a potent strategy to mitigate biofilm. In this study, bacterial strains with QQ activity were identified and their anti-biofilm potential was investigated against the Multidrug Resistant (MDR) Pseudomonas aeruginosa. A Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136-based bioassays were used to confirm the degradation of different Acyl Homoserine Lactones (AHLs) by QQ isolates. The 16S rRNA gene sequencing of the isolated strains identified them as Bacillus cereus strain QSP03, B. subtilis strain QSP10, Pseudomonas putida strain QQ3 and P. aeruginosa strain QSP01. Biofilm mitigation potential of QQ isolates was tested against MDR P. aeruginosa and the results suggested that 50% biofilm reduction was observed by QQ3 and QSP01 strains, and around 60% reduction by QSP10 and QSP03 bacterial isolates. The presence of AHL degrading enzymes, lactonases and acylases, was confirmed by PCR based screening and sequencing of the already annotated genes aiiA, pvdQ and quiP. Altogether, these results exhibit that QQ bacterial strains or their products could be useful to control biofilm formation in P.aeruginosa.