The Occurrence of 4-Amino-4-Deoxy-Arabinose in LPS of Supersusceptible Strain Pseudomonas aeruginosa Z61: Noncorrelation with Polymyxin Resistance

Lipopolysaccharides (LPS) extracted from the supersusceptible strain Pseudomonas aeruginosa Z61 were compared with LPS from other strains with varying antimicrobial susceptibilities. The presence of 4-amino-4-deoxy-arabinose (4-AraN) in P. aeruginosa Z61 LPS was confirmed by gas-liquid chromatography/mass spectrometry (GLC-MS) and quantitated by high-performance liquid chromatography (HPLC). Z61 LPS (compared with wild-type strain PAO1) has reduced amounts of rhamnose and higher concentrations of hydroxy fatty acids, 4-AraN, and phosphates. ³¹P Nuclear magnetic resonance revealed that Z61 LPS phosphates are configured in monophosphates, phosphodiesters, pyrophosphomonoesters, and glycosidic pyrophosphodiester groups. The presence of 4-AraN in P. aeruginosa LPS did not correlate with antimicrobial resistance. Received: 31 August 1998 / Accepted: 5 November 1998     

Pyocyanin biosynthesis protects Pseudomonas aeruginosa from nonthermal plasma inactivation

Pseudomonas aeruginosa is an important opportunistic human pathogen, which raises a worldwide concern for its increasing resistance. Nonthermal plasma, which is also called cold atmospheric plasma (CAP), is an alternative therapeutic approach for clinical infectious diseases. However, the bacterial factors that affect CAP treatment remain unclear. The sterilization effect of a portable CAP device on different P. aeruginosa strains was investigated in this study. Results revealed that CAP can directly or indirectly kill P. aeruginosa in a time-dependent manner. Scanning electron microscopy and transmission electron microscope showed negligible surface changes between CAP-treated and untreated P. aeruginosa cells. However, cell leakage occurred during the CAP process with increased bacterial lactate dehydrogenase release. More importantly, pigmentation of the P. aeruginosa culture was remarkably reduced after CAP treatment. Further mechanical exploration was performed by utilizing mutants with loss of functional genes involved in pyocyanin biosynthesis, including P. aeruginosa PAO1 strain-derived phzA1::Tn, phzA2::Tn, ΔphzA1/ΔphzA2, phzM::Tn and phzS::Tn, as well as corresponding gene deletion mutants based on clinical PA1 isolate. The results indicated that pyocyanin and its intermediate 5-methyl phenazine-1-carboxylic acid (5-Me-PCA) play important roles in P. aeruginosa resistance to CAP treatment. The unique enzymes, such as PhzM in the pyocyanin biosynthetic pathway, could be novel targets for the therapeutic strategy design to control the growing P. aeruginosa infections.     

Proteomics of Pseudomonas aeruginosa: the increasing role of post-translational modifications

Introduction: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen widely involved in human infections. The high occurrence of this bacterial species in the clinical field is due to its high ability to adapt to detrimental environments, in particular its strong inherent antibiotic resistance, its ability to form biofilms and to produce virulence factors. The application of proteomics to clinical microbiology is probably one of the most innovative strategies of the last decades to understand complex microbial systems, by providing individual proteome charts of pathogens.

Areas covered: In the last decade, proteomic advances have allowed in high-throughput the screening of proteins modified by diverse co- and post-translational modifications in P. aeruginosa. This review will present the current state of the art for the characterization of PTMs in P. aeruginosa by proteomics approaches. We will then discuss on the involvement of PTMs in P. aeruginosa physiology.

Expert commentary: Modified proteins and enzymes involved in the addition/removal of modifications will surely constitute targets of interest to develop new therapeutic drugs to fight against P. aeruginosa.     

A novel bacteriophage cocktail reduces and disperses Pseudomonas aeruginosa biofilms under static and flow conditions

Pseudomonas aeruginosa is an opportunistic human pathogen that forms highly stable communities – biofilms, which contribute to the establishment and maintenance of infections. The biofilm state and intrinsic/acquired bacterial resistance mechanisms contribute to resistance/tolerance to antibiotics that is frequently observed in P. aeruginosa isolates. Here we describe the isolation and characterization of six novel lytic bacteriophages: viruses that infect bacteria, which together efficiently infect and kill a wide range of P. aeruginosa clinical isolates. The phages were used to formulate a cocktail with the potential to eliminate P. aeruginosa PAO1 planktonic cultures. Two biofilm models were studied, one static and one dynamic, and the phage cocktail was assessed for its ability to reduce and disperse the biofilm biomass. For the static model, after 4 h of contact with the phage suspension (MOI 10) more than 95% of biofilm biomass was eliminated. In the flow biofilm model, a slower rate of activity by the phage was observed, but 48 h after addition of the phage cocktail the biofilm was dispersed, with most cells eliminated (> 4 logs) comparing with the control. This cocktail has the potential for development as a therapeutic to control P. aeruginosa infections, which are predominantly biofilm centred.     

Analysis of a novel class 1 integron containing metallo-β-lactamase gene VIM-2 in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Carbapenems such as imipenem are stable to most β-lactamases. Recently, increased numbers of carbapenemase producing Gram-negative bacterial strains have been isolated because of the increased use of cabapenems. In this respect, control of these infectious carbapenemase producing Gram-negative bacteria and understanding their resistance mechanism are becoming more important. These carbapenem-hydrolyzing β-lactamase genes have been reported to exist mostly as gene cassettes in an integron. This implies that antibiotic resistance genes may be transferred to other bacteria via the integron. In the present study, we identified and analyzed an integron containing VIM-2 type metallo-β-lactamase gene in a carbapenemase producing Pseudomonas aeruginosa. In addition, the possibility of resistance spread by integron located in a plasmid was tested. Among glucose non-fermenting Gram-negative bacilli with reduced imipenem susceptibility (MIC≥8 μg/ml) isolated from Korean patients, P. aeruginosa 1082 showed resistance to most β-lactams, cephalosporin, and aminoglycoside. We found that P. aeruginosa 1082 was inhibited by EDTA in EDTA double disk synergy test which means that this strain produces metallo-β-lactamase. Class 1 integron containing bla _VIM-2 (carbapenem resistance gene), qacF (quaternary ammonium compound resistance gene), aacA4 (aminoglycoside resistance gene), catB3 (chloramphenicol resistance gene), bla _oxa-30 (extended-spectrum β-lactam resistance gene), and aadAl (aminoglycoside resistance gene) gene cassettes was detected in P. aeruginosa 1082. The size of the integron was 5,246 bp and the structure and arrangement of the integron was a novel one in comparison with other integrons found in other P. aeruginosa. The integron could be transferred to Escherichia coli JM109 from P. aeruginosa 1082 possibly via self-transferable plasmid DNA. The integron and a bla _VIM-2 gene were detected in the plasmid DNA of the transconjugants whose imipenem resistance was slightly increased as a result of accepting the integron from the donor strain.     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections

Aims: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, bla_OXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla_OXA‐31 in a canine Ps. aeruginosa isolate.     

Widespread Emergence of Multidrug Resistant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from CSF Samples

Bacterial infections of the central nervous system, especially acute infections such as bacterial meningitis require immediate, invariably empiric antibiotic therapy due to the widespread emergence of resistance among bacterial species. Nosocomial infections by Pseudomonas aeruginosa have been described with an increasing trend towards multidrug resistance. P. aeruginosa isolates n = 53 (66%) isolated from the cerebrospinal fluid (CSF) were used for this study. Antibiotic resistance in 53 P. aeruginosa clinical isolates from 80 CSF samples were evaluated. Of these, n = 42 (80%) of the isolates showed multidrug resistance to more than eight antibiotics and n = 17 (32%) isolates were found to be imipenem resistant P. aeruginosa (IMPR-Pa). Genotypical examination by ERIC based PCR revealed minor genetic variations. Polymicrobial infections are common in the CSF samples. However, high prevalence of P. aeruginosa as an opportunistic pathogen has been developing with increased resistance to antimicrobial agents and thus becoming a significant threat.     

Evaluation of the ability of C. albicans to form biofilm in the presence of phage-resistant phenotypes of P. aeruginosa

Pseudomonas aeruginosa and Candida albicans are disparate microbial species, but both are known to be opportunistic pathogens frequently associated with nosocomial infections. The aim of this study was to provide a better understanding of the interactions between these microorganisms in dual-species biofilms. Several bacteriophage-resistant P. aeruginosa phenotypes have been isolated and were used in dual-species mixed-biofilm studies. Twenty-four and 48 h mixed-biofilms were formed using the isolated phenotypes of phage-resistant P. aeruginosa and these were compared with similar experiments using other P. aeruginosa strains with a defined lipopolysaccharide (LPS) deficiency based on chromosomal knockout of specific LPS biosynthetic genes. Overall, the results showed that the variants of phage-resistant P. aeruginosa and LPS mutants were both less effective in inhibiting the growth of C. albicans in mixed-biofilms compared to the wild-type strains of P. aeruginosa. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. In conclusion, the ability of strains of P. aeruginosa to inhibit the formation of a biofilm of C. albicans appears to be correlated with the LPS chain lengths of phenotypes of P. aeruginosa, suggesting that LPS has a suppressive effect on the growth of C. albicans.     

Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.     

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.     

Role of Pseudomonas aeruginosa lipopolysaccharides in modulation of biofilm and virulence factors of Enterobacteriaceae

Enterobacteriaceae members are largely distributed in the environment and responsible for a wide range of bacterial infections in hospitalized patients. Pseudomonas aeruginosa (P. aeruginosa) causes severe nosocomial infections associated with severe inflammation due to its potent virulent factors including lipopolysaccharide (LPS). The aim of this study is to assess the bacterial LPS effect on Enterobacteriaceae biofilm and other virulence factors in vitro. The effect of P. aeruginosa LPS on biofilm formation of two other species of Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae) was assessed using a standard biofilm assay. PCR was performed on genes of biofilm and virulence factors. Expression of biofilm, type-1-fimbriae and serum resistance genes in treated and untreated cells was measured with RT-PCR. P. aeruginosa LPS has the ability to stimulate biofilm formation and stabilize the already formed biofilm significantly in all tested strains. In addition, LPS significantly increased the level of expression of Bss, FimH, and Iss genes when measured by RT-PCR. P. aeruginosa LPS has a direct stimulatory effect on the biofilm formation, type-1-fimbriae, and serum resistance in both E. coli and K. pneumoniae. So, the presence of P. aeruginosa in mixed infection with Enterobactereacea leads to increase their virulence.

     

Metformin prevents the effects of Pseudomonas aeruginosa on airway epithelial tight junctions and restricts hyperglycaemia‐induced bacterial growth

Lung disease and elevation of blood glucose are associated with increased glucose concentration in the airway surface liquid (ASL). Raised ASL glucose is associated with increased susceptibility to infection by respiratory pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. We have previously shown that the anti‐diabetes drug, metformin, reduces glucose‐induced S. aureus growth across in vitro airway epithelial cultures. The aim of this study was to investigate whether metformin has the potential to reduce glucose‐induced P. aeruginosa infections across airway epithelial (Calu‐3) cultures by limiting glucose permeability. We also explored the effect of P. aeruginosa and metformin on airway epithelial barrier function by investigating changes in tight junction protein abundance. Apical P. aeruginosa growth increased with basolateral glucose concentration, reduced transepithelial electrical resistance (TEER) and increased paracellular glucose flux. Metformin pre‐treatment of the epithelium inhibited the glucose‐induced growth of P. aeruginosa, increased TEER and decreased glucose flux. Similar effects on bacterial growth and TEER were observed with the AMP activated protein kinase agonist, 5‐aminoimidazole‐4‐carboxamide ribonucleotide. Interestingly, metformin was able to prevent the P. aeruginosa‐induced reduction in the abundance of tight junction proteins, claudin‐1 and occludin. Our study highlights the potential of metformin to reduce hyperglycaemia‐induced P. aeruginosa growth through airway epithelial tight junction modulation, and that claudin‐1 and occludin could be important targets to regulate glucose permeability across airway epithelia and supress bacterial growth. Further investigation into the mechanisms regulating metformin and P. aeruginosa action on airway epithelial tight junctions could yield new therapeutic targets to prevent/suppress hyperglycaemia‐induced respiratory infections, avoiding the use of antibiotics.     

Heterologous expression of human paraoxonases in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits biofilm formation and decreases antibiotic resistance

Quorum sensing (QS) regulates virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Human paraoxonases (hPONs) are a family of closely related enzymes with multiple functions, including inactivation of the QS signal molecule in P. aeruginosa. However, there is no direct evidence to show the functions of hPONs on biofilm formation and antibiotic resistance in P. aeruginosa. In the present study, hPONs (hPON1, hPON2, and hPON3) genes were respectively cloned into the pMEKm12 shuttle vector and transformed into P. aeruginosa strain PAO1. Expression of the three recombinant proteins was confirmed by Western blotting, and growth of the recombinant strains was not affected by the hPONs gene expression. Biofilm formation and antibiotics resistance of the hPONs recombinant strains were analyzed. Our results showed that biofilm formation was significantly inhibited in all of the three hPONs recombinant strains. Interestingly, this inhibition can be reverted by addition of the corresponding hPONs polyclonal antibodies in the culture media, further indicating that the inhibition of biofilm formation was due to hPONs protein expression. In addition, we also demonstrated that hPONs expression decreased resistance of P. aeruginosa to gentamicin and ceftazidima, two antibiotics clinically used for the treatment of P. aeruginosa infection.     

Pseudomonas aeruginosa, Candida albicans, and device-related nosocomial infections: implications, trends, and potential approaches for control

For many years, device-associated infections and particularly device-associated nosocomial infections have been of considerable concern. Recently, this concern was heightened as a result of increased antibiotic resistance among the common causal agents of nosocomial infections, the appearance of new strains which are intrinsically resistant to the antibiotics of choice, and the emerging understanding of the role biofilms may play in device-associated infections and the development of increased antibiotic resistance. Pseudomonas aeruginosa and Candida albicans are consistently identified as some of the more important agents of nosocomial infections. In light of the recent information regarding device-associated nosocomial infections, understanding the nature of P. aeruginosa and C. albicans infections is increasingly important. These two microorganisms demonstrate: (1) an ability to form biofilms on the majority of devices employed currently, (2) increased resistance/tolerance to antibiotics when associated with biofilms, (3) documented infections noted for virtually all indwelling devices, (4) opportunistic pathogenicity, and (5) persistence in the hospital environment. To these five demonstrated characteristics, two additional areas of interest are emerging: (a) the as yet unclear relationship of these two microorganisms to those species of highly resistant Pseudomonas spp and Candida spp that are of increasing concern with device-related infections, and (b) the recent research showing the dynamic interaction of P. aeruginosa and C. albicans in patients with cystic fibrosis. An understanding of these two opportunistic pathogens in the context of their ecosystems/biofilms also has significant potential for the development of novel and effective approaches for the control and treatment of device-associated infections.     

Evidence for stable transformation of Pseudomonas aeruginosa with a pUC-based plasmid

Pseudomonas aeruginosa was transformed with pUC8:16, a pUC-based plasmid bearing the gene (vgb) encoding Vitreoscilla (bacterial) hemoglobin (VHb). Transformation was initially indicated by an increase in ampicillin resistance from 1500 to 2500 mg l^–1. Presence of the plasmid in P. aeruginosa was confirmed by amplification of a portion of vgb from and detection of VHb in the transformant but not the untransformed host. Southern blot analysis further indicated that pUC8:16 existed as an autonomous plasmid rather than integrated into the chromosome of the P. aeruginosa transformant.     

Influence of <Emphasis Type="Italic">Pseudomonas aeruginosa pvdQ</Emphasis> Gene on Altering Antibiotic Susceptibility Under Swarming Conditions

In Pseudomonas aeruginosa PAO1, the pvdQ gene has been shown to have at least two functions. It encodes the acylase enzyme and hydrolyzes 3-oxo-C12-HSL, the key signaling molecule of quorum sensing system. In addition, pvdQ is involved in swarming motility. It is required for up-regulated during swarming motility, which is triggered by high cell densities. As high-density bacterial populations also display elevated antibiotic resistance, studies have demonstrated that swarm-cell differentiation in P. aeruginosa promotes increased resistance to various antibiotics. PvdQ acts as a signal during swarm-cell differentiation, and thus may play a role in P. aeruginosa antibiotic resistance. The aim of this study is to examine whether pvdQ was involved in modifying antibiotic susceptibility during swarming conditions, and to investigate the mechanism by which this occurred. We constructed the PAO1pMEpvdQ strain, which overproduced PvdQ. PAO1pMEpvdQ promotes swarming motility, while PAO1ΔpvdQ abolishes swarming motility. In addition, both PAO1 and PAO1pMEpvdQ acquired resistance to ceftazidime, ciprofloxacin, meropenem, polymyxin B, and gentamicin, though PAO1pMEpvdQ exhibited a two to eightfold increase in antibiotic resistance compared to PAO1. These results indicate that pvdQ plays an important role in elevating antibiotic resistance via swarm-cell differentiation and possibly other mechanisms as well. We analyzed outer membrane permeability. Our data also suggest that pvdQ decreases P. aeruginosa outer membrane permeability, thereby elevating antibiotic resistance under swarming conditions. Our results suggest new approaches for reducing P. aeruginosa resistance.     

Dynamics of cyanophage-like particles and algicidal bacteria causing Microcystis aeruginosa mortality

The dynamics of cyanophage-like particles and algicidal bacteria that infect the bloom-forming cyanobacterium Microcystis aeruginosa was followed in a hyper-eutrophic pond from September 1998 to August 1999. The densities of M. aeruginosa ranged between 4.0 × 10⁵ and 1.9 × 10⁷ cells ml⁻¹, whereas those of algicidal bacteria were between 4.0 and 5.1 × 10² plaque-forming units (PFU) ml⁻¹ and those of cyanophage-like particles were between <5.0 × 10² and 7.1 × 10³ PFU ml⁻¹. A significant relationship was found between the densities of algicidal bacteria and M. aeruginosa (r = 0.81, n = 69, P < 0.001), suggesting that the dynamics of the algicidal bacteria may regulate the abundance of M. aeruginosa. Occasional peaks of density of cyanophage-like particles were detected in October, June, and August, when sharp declines in M. aeruginosa cell densities were also observed. The densities of cyanophage-like particles became undetectable when the abundance of M. aeruginosa was low, suggesting the density-dependent infection of M. aeruginosa by cyanophage-like particles. Thus, we suggest that infections of both algicidal bacteria and cyanophage-like particles are important biological agents that decompose blooms of M. aeruginosa in freshwater environments. Received: August 31, 2000 / Accepted: December 6, 2000     

Rhizobacterially induced protection of watermelon against Didymella bryoniae

Aims: To identify rhizobacteria from the Mekong Delta of Vietnam, which can systemically protect watermelon against Didymella bryoniae and elucidate the mechanisms involved in the protection conferred by isolate Pseudomonas aeruginosa 23_1‐1. Methods and Results: Bacteria were isolated from watermelon roots and their antagonistic ability tested in vitro. Of 190 strains, 68 were able to inhibit D. bryoniae by production of antibiotics. Four strains were able to reduce foliar infection by D. bryoniae when applied to watermelon seeds before sowing. Strain Ps. aeruginosa 23_1‐1 was chosen for investigations of the mechanisms involved in protection and ability to control disease under field conditions. In the field, the bacterium was able to significantly reduce disease in two consecutive seasons and increase yield. Furthermore, it colonized watermelon plants endophytically, with higher numbers in plants infected by D. bryoniae than in noninoculated plants. To elucidate the mechanisms involved in protection, the infection biology of the pathogen was studied in bacterially treated and control plants. Pseudomonas aeruginosa 23_1‐1 treatment inhibited pathogen penetration and this was associated with hydrogen peroxide accumulation, increased peroxidase activity and occurrence of new peroxidase isoforms, thus indicating that resistance was induced. Conclusions: The endophytic bacterium Ps. aeruginosa 23_1‐1 can control D. bryoniae in watermelon by antibiosis and induced resistance under greenhouse and field conditions. Significance and Impact of the Study: These findings suggest that rhizobacteria from native soils in Vietnam can be used to control gummy stem blight of watermelon through various mechanisms including induction of resistance.     

Monitoring microaerobic denitrification of Pseudomonas aeruginosa by online NAD(P)H fluorescence

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.