Reverting Antibiotic Tolerance of Pseudomonas aeruginosa PAO1 Persister Cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one

Background

Bacteria are well known to form dormant persister cells that are tolerant to most antibiotics. Such intrinsic tolerance also facilitates the development of multidrug resistance through acquired mechanisms. Thus persister cells are a promising target for developing more effective methods to control chronic infections and help prevent the development of multidrug-resistant bacteria. However, control of persister cells is still an unmet challenge.

Methodology/Principal Findings

We show in this report that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can restore the antibiotic susceptibility of Pseudomonas aeruginosa PAO1 persister cells at growth non-inhibitory concentrations. Persister control by BF8 was found to be effective against both planktonic and biofilm cells of P. aeruginosa PAO1. Interestingly, although BF8 is an inhibitor of quorum sensing (QS) in Gram-negative bacteria, the data in this study suggest that the activities of BF8 to revert antibiotic tolerance of P. aeruginosa PAO1 persister cells is not through QS inhibition and may involve other targets.

Conclusion

BF8 can sensitize P. aeruginosa persister cells to antibiotics.     

Duckweed (Lemna minor) as a model plant system for the study of human microbial pathogenesis

Background

Plant infection models provide certain advantages over animal models in the study of pathogenesis. However, current plant models face some limitations, e.g., plant and pathogen cannot co-culture in a contained environment. Development of such a plant model is needed to better illustrate host-pathogen interactions.

Methodology/Principal Findings

We describe a novel model plant system for the study of human pathogenic bacterial infection on a large scale. This system was initiated by co-cultivation of axenic duckweed (Lemna minor) plants with pathogenic bacteria in 24-well polystyrene cell culture plate. Pathogenesis of bacteria to duckweed was demonstrated with Pseudomonas aeruginosa and Staphylococcus aureus as two model pathogens. P. aeruginosa PAO1 caused severe detriment to duckweed as judged from inhibition to frond multiplication and chlorophyll formation. Using a GFP-marked PAO1 strain, we demonstrated that bacteria colonized on both fronds and roots and formed biofilms. Virulence of PAO1 to duckweed was attenuated in its quorum sensing (QS) mutants and in recombinant strains overexpressing the QS quenching enzymes. RN4220, a virulent strain of S. aureus, caused severe toxicity to duckweed while an avirulent strain showed little effect. Using this system for antimicrobial chemical selection, green tea polyphenols exhibited inhibitory activity against S. aureus virulence. This system was further confirmed to be effective as a pathogenesis model using a number of pathogenic bacterial species.

Conclusions/Significance

Our results demonstrate that duckweed can be used as a fast, inexpensive and reproducible model plant system for the study of host-pathogen interactions, could serve as an alternative choice for the study of some virulence factors, and could also potentially be used in large-scale screening for the discovery of antimicrobial chemicals.     

Ellagic Acid Derivatives from Terminalia chebula Retz. Downregulate the Expression of Quorum Sensing Genes to Attenuate Pseudomonas aeruginosa PAO1 Virulence

Background

Burgeoning antibiotic resistance in Pseudomonas aeruginosa has necessitated the development of anti pathogenic agents that can quench acylhomoserine lactone (AHL) mediated QS with least risk of resistance. This study explores the anti quorum sensing potential of T. chebula Retz. and identification of probable compounds(s) showing anti QS activity and the mechanism of attenuation of P. aeruginosa PAO1 virulence factors.

Methods and Results

Methanol extract of T. chebula Retz. fruit showed anti QS activity using Agrobacterium tumefaciens A136. Bioactive fraction (F7), obtained by fractionation of methanol extract using Sephadex LH20, showed significant reduction (p<0.001) in QS regulated production of extracellular virulence factors in P. aeruginosa PAO1. Biofilm formation and alginate were significantly (p<0.05) reduced with enhanced (20%) susceptibility to tobramycin. Real Time PCR of F7 treated P. aeruginosa showed down regulation of autoinducer synthase (lasI and rhlI) and their cognate receptor (lasR and rhlR) genes by 89, 90, 90 and 93%, respectively. Electrospray Ionization Mass Spectrometry also showed 90 and 64% reduction in the production of 3-oxo-C₁₂HSL and C₄HSL after treatment. Decrease in AHLs as one of the mechanisms of quorum quenching by F7 was supported by the reversal of inhibited swarming motility in F7-treated P. aeruginosa PAO1 on addition of C₄HSL. F7 also showed antagonistic activity against 3-oxo-C₁₂HSL-dependent QS in E. coli bioreporter. C. elegans fed on F7-treated P. aeruginosa showed enhanced survival with LT50 increasing from 24 to 72 h. LC-ESI-MS of F7 revealed the presence of ellagic acid derivatives responsible for anti QS activity in T. chebula extract.

Conclusions

This is the first report on anti QS activity of T. chebula fruit linked to EADs which down regulate the expression of lasIR and rhlIR genes with concomitant decrease in AHLs in P. aeruginosa PAO1 causing attenuation of its virulence factors and enhanced sensitivity of its biofilm towards tobramycin.     

Assessing the Emergence of Resistance: The Absence of Biological Cost In Vivo May Compromise Fosfomycin Treatments for P. aeruginosa Infections

Background

Fosfomycin is a cell wall inhibitor used efficiently to treat uncomplicated urinary tract and gastrointestinal infections. A very convenient feature of fosfomycin, among others, is that although the expected frequency of resistant mutants is high, the biological cost associated with mutation impedes an effective growth rate, and bacteria cannot offset the obstacles posed by host defenses or compete with sensitive bacteria. Due to the current scarcity of new antibiotics, fosfomycin has been proposed as an alternative treatment for other infections caused by a wide variety of bacteria, particularly Pseudomonas aeruginosa. However, whether fosfomycin resistance in P. aeruginosa provides a fitness cost still remains unknown.

Principal Findings

We herein present experimental evidence to show that fosfomycin resistance cannot only emerge easily during treatment, but that it is also cost-free for P. aeruginosa. We also tested if, as has been reported for other species such as Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis, fosfomycin resistant strains are somewhat compromised in their virulence. As concerns colonization, persistence, lung damage, and lethality, we found no differences between the fosfomycin resistant mutant and its sensitive parental strain. The probability of acquisition in vitro of resistance to the combination of fosfomycin with other antibiotics (tobramycin and imipenem) has also been studied. While the combination of fosfomycin with tobramycin makes improbable the emergence of resistance to both antibiotics when administered together, the combination of fosfomycin plus imipenem does not avoid the appearance of mutants resistant to both antibiotics.

Conclusions

We have reached the conclusion that the use of fosfomycin for P. aeruginosa infections, even in combined therapy, might not be as promising as expected. This study should encourage the scientific community to assess the in vivo cost of resistance for specific antibiotic-bacterial species combinations, and therefore avoid reaching universal conclusions from single model organisms.     

Evidence of MexT-independent overexpression of MexEF-OprN multidrug efflux pump of Pseudomonas aeruginosa in presence of metabolic stress

Background

The Pseudomonas aeruginosa MexEF-OprN efflux pump confers resistance to clinically significant antibiotics. Regulation of mexEF-oprN operon expression is multifaceted with the MexT activator being one of the most prominent regulatory proteins.

Methodology

We have exploited the impaired metabolic fitness of a P. aeruginosa mutant strain lacking several efflux pump of the resistance nodulation cell division superfamily and the TolC homolog OpmH, and isolated derivatives (large colony variants) that regained fitness by incubation on nutrient-rich medium in the absence of antibiotics. Although the mexEF-oprN operon is uninducible in this mutant due to a 8-bp mexT insertion present in some P. aeruginosa PAO1 strains, the large colony variants expressed high levels of MexEF-OprN. Unlike large colony variants obtained after plating on antibiotic containing medium which expressed mexEF-oprN in a MexT-dependent fashion as evidenced by clean excision of the 8-bp insertion from mexT, mexEF-oprN expression was MexT-independent in the large colony variants obtained by plating on LB alone since the mexT gene remained inactivated. A search for possible regulators of mexEF-oprN expression using transposon mutagenesis and genomic library expression approaches yielded several candidates but proved inconclusive.

Significance

Our results show that antibiotic and metabolic stress lead to up-regulation of MexEF-OprN expression via different mechanisms and that MexEF-OprN does not only extrude antimicrobials but rather serves other important metabolic functions.     

Polyphosphate Kinase 2: A Novel Determinant of Stress Responses and Pathogenesis in Campylobacter jejuni

Background

Inorganic polyphosphate (poly P) plays an important role in stress tolerance and virulence in many bacteria. PPK1 is the principal enzyme involved in poly P synthesis, while PPK2 uses poly P to generate GTP, a signaling molecule that serves as an alternative energy source and a precursor for various physiological processes. Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans, possesses homologs of both ppk1 and ppk2. ppk1 has been previously shown to impact the pathobiology of C. jejuni.

Methodology/Principal Findings

Here, we demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio. The Δppk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. However, the Δppk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Δppk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization.

Conclusions/Significance

Taken together, we have highlighted the role of PPK2 as a novel pathogenicity determinant that is critical for C. jejuni survival, adaptation, and persistence in the host environments. PPK2 is absent in humans and animals; therefore, can serve as a novel target for therapeutic intervention of C. jejuni infections.     

The Ability of Virulence Factor Expression by Pseudomonas aeruginosa to Predict Clinical Disease in Hospitalized Patients

Background

Pseudomonas aeruginosa is an opportunistic pathogen that frequently causes hospital acquired colonization and infection. Accurate identification of host and bacterial factors associated with infection could aid treatment decisions for patients with P. aeruginosa cultured from clinical sites.

Methods

We identified a prospective cohort of 248 hospitalized patients with positive P. aeruginosa cultures. Clinical data were analyzed to determine whether an individual met predefined criteria for infection versus colonization. P. aeruginosa isolates were tested for the expression of multiple phenotypes previously associated with virulence in animal models and humans. Logistic regression models were constructed to determine the degree of association between host and bacterial factors with P. aeruginosa infection of the bloodstream, lung, soft tissue and urinary tract.

Results

One host factor (i.e. diabetes mellitus), and one bacterial factor, a Type 3 secretion system positive phenotype, were significantly associated with P. aeruginosa infection in our cohort. Subgroup analysis of patients with P. aeruginosa isolated from the urinary tract revealed that the presence of a urinary tract catheter or stent was an additional factor for P. aeruginosa infection.

Conclusions

Among hospitalized patients with culture-documented P. aeruginosa, infection is more likely to be present in those with diabetes mellitus and those harboring a Type 3 secretion positive bacterial strain.     

Inhaled Lactonase Reduces Pseudomonas aeruginosa Quorum Sensing and Mortality in Rat Pneumonia

Rationale

The effectiveness of antibiotic molecules in treating Pseudomonas aeruginosa pneumonia is reduced as a result of the dissemination of bacterial resistance. The existence of bacterial communication systems, such as quorum sensing, has provided new opportunities of treatment. Lactonases efficiently quench acyl-homoserine lactone-based bacterial quorum sensing, implicating these enzymes as potential new anti-Pseudomonas drugs that might be evaluated in pneumonia.

Objectives

The aim of the present study was to evaluate the ability of a lactonase called SsoPox-I to reduce the mortality of a rat P. aeruginosa pneumonia.

Methods

To assess SsoPox-I-mediated quorum quenching, we first measured the activity of the virulence gene lasB, the synthesis of pyocianin, the proteolytic activity of a bacterial suspension and the formation of biofilm of a PAO1 strain grown in the presence of lactonase. In an acute lethal model of P. aeruginosa pneumonia in rats, we evaluated the effects of an early or deferred intra-tracheal treatment with SsoPox-I on the mortality, lung bacterial count and lung damage.

Measurements and Primary Results

SsoPox-I decreased PAO1 lasB virulence gene activity, pyocianin synthesis, proteolytic activity and biofilm formation. The early use of SsoPox-I reduced the mortality of rats with acute pneumonia from 75% to 20%. Histological lung damage was significantly reduced but the lung bacterial count was not modified by the treatment. A delayed treatment was associated with a non-significant reduction of mortality.

Conclusion

These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.     

Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

Background

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.

Results

We developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.

Conclusions

The P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-737) contains supplementary material, which is available to authorized users.     

Lipid metabolism and Type VII secretion systems dominate the genome scale virulence profile of Mycobacterium tuberculosis in human dendritic cells

Background

Mycobacterium tuberculosis continues to kill more people than any other bacterium. Although its archetypal host cell is the macrophage, it also enters, and survives within, dendritic cells (DCs). By modulating the behaviour of the DC, M. tuberculosis is able to manipulate the host’s immune response and establish an infection. To identify the M. tuberculosis genes required for survival within DCs we infected primary human DCs with an M. tuberculosis transposon library and identified mutations with a reduced ability to survive.

Results

Parallel sequencing of the transposon inserts of the surviving mutants identified a large number of genes as being required for optimal intracellular fitness in DCs. Loci whose mutation attenuated intracellular survival included those involved in synthesising cell wall lipids, not only the well-established virulence factors, pDIM and cord factor, but also sulfolipids and PGL, which have not previously been identified as having a direct virulence role in cells. Other attenuated loci included the secretion systems ESX-1, ESX-2 and ESX-4, alongside many PPE genes, implicating a role for ESX-5. In contrast the canonical ESAT-6 family of ESX substrates did not have intra-DC fitness costs suggesting an alternative ESX-1 associated virulence mechanism. With the aid of a gene-nutrient interaction model, metabolic processes such as cholesterol side chain catabolism, nitrate reductase and cysteine-methionine metabolism were also identified as important for survival in DCs.

Conclusion

We conclude that many of the virulence factors required for survival in DC are shared with macrophages, but that survival in DCs also requires several additional functions, such as cysteine-methionine metabolism, PGLs, sulfolipids, ESX systems and PPE genes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1569-2) contains supplementary material, which is available to authorized users.     

Whole-genome analysis of Fusarium graminearum insertional mutants identifies virulence associated genes and unmasks untagged chromosomal deletions

Background

Identifying pathogen virulence genes required to cause disease is crucial to understand the mechanisms underlying the pathogenic process. Plasmid insertion mutagenesis of fungal protoplasts is frequently used for this purpose in filamentous ascomycetes. Post transformation, the mutant population is screened for loss of virulence to a specific plant or animal host. Identifying the insertion event has previously met with varying degrees of success, from a cleanly disrupted gene with minimal deletion of nucleotides at the insertion point to multiple-copy insertion events and large deletions of chromosomal regions. Currently, extensive mutant collections exist in laboratories globally where it was hitherto impossible to identify all the affected genes.

Results

We used a whole-genome sequencing (WGS) approach using Illumina HiSeq 2000 technology to investigate DNA tag insertion points and chromosomal deletion events in mutagenised, reduced virulence F. graminearum isolates identified in disease tests on wheat (Triticum aestivum). We developed the FindInsertSeq workflow to localise the DNA tag insertions to the nucleotide level. The workflow was tested using four mutants showing evidence of single and multi-copy insertions in DNA blot analysis. FindInsertSeq was able to identify both single and multi-copy concatenation insertion sites. By comparing sequencing coverage, unexpected molecular recombination events such as large tagged and untagged chromosomal deletions, and DNA amplification were observed in three of the analysed mutants. A random data sampling approach revealed the minimum genome coverage required to survey the F. graminearum genome for alterations.

Conclusions

This study demonstrates that whole-genome re-sequencing to 22x fold genome coverage is an efficient tool to characterise single and multi-copy insertion mutants in the filamentous ascomycete Fusarium graminearum. In some cases insertion events are accompanied with large untagged chromosomal deletions while in other cases a straight-forward insertion event could be confirmed. The FindInsertSeq analysis workflow presented in this study enables researchers to efficiently characterise insertion and deletion mutants.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1412-9) contains supplementary material, which is available to authorized users.     

Bacteriophage-resistant mutants in Yersinia pestis: identification of phage receptors and attenuation for mice

Background

Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phage-resistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy.

Methodology/Principal Findings

The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD₅₀ and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence.

Conclusions/Significance

We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophage therapy of plague. LPS is a critical virulence factor of Y. pestis.     

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.