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.
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.
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.
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.
These results demonstrate the protective effects of lactonase SsoPox-I in P. aeruginosa pneumonia and open the way for a future therapeutic use.
The ability of soil bacteria to successfully compete with a range of other microbial species is crucial for their growth and survival in the nutrient-limited soil environment. In the present work, we ...studied the behavior and transcriptional responses of soil-inhabiting Pseudomonas fluorescens strain Pf0-1 on nutrient-poor agar to confrontation with strains of three phylogenetically different bacterial genera, that is, Bacillus, Brevundimonas and Pedobacter. Competition for nutrients was apparent as all three bacterial genera had a negative effect on the density of P. fluorescens Pf0-1; this effect was most strong during the interaction with Bacillus. Microarray-based analyses indicated strong differences in the transcriptional responses of Pf0-1 to the different competitors. There was higher similarity in the gene expression response of P. fluorescens Pf0-1 to the Gram-negative bacteria as compared with the Gram-positive strain. The Gram-negative strains did also trigger the production of an unknown broad-spectrum antibiotic in Pf0-1. More detailed analysis indicated that expression of specific Pf0-1 genes involved in signal transduction and secondary metabolite production was strongly affected by the competitors' identity, suggesting that Pf0-1 can distinguish among different competitors and fine-tune its competitive strategies. The results presented here demonstrate that P. fluorescens Pf0-1 shows a species-specific transcriptional and metabolic response to bacterial competitors and provide new leads in the identification of specific cues in bacteria-bacteria interactions and of novel competitive strategies, antimicrobial traits and genes.
Pseudomonas genomes: diverse and adaptable Silby, Mark W; Winstanley, Craig; Godfrey, Scott A.C ...
FEMS microbiology reviews,
07/2011, Volume:
35, Issue:
4
Journal Article
Peer reviewed
Open access
Abstract
Members of the genus Pseudomonas inhabit a wide variety of environments, which is reflected in their versatile metabolic capacity and broad potential for adaptation to fluctuating ...environmental conditions. Here, we examine and compare the genomes of a range of Pseudomonas spp. encompassing plant, insect and human pathogens, and environmental saprophytes. In addition to a large number of allelic differences of common genes that confer regulatory and metabolic flexibility, genome analysis suggests that many other factors contribute to the diversity and adaptability of Pseudomonas spp. Horizontal gene transfer has impacted the capability of pathogenic Pseudomonas spp. in terms of disease severity (Pseudomonas aeruginosa) and specificity (Pseudomonas syringae). Genome rearrangements likely contribute to adaptation, and a considerable complement of unique genes undoubtedly contributes to strain- and species-specific activities by as yet unknown mechanisms. Because of the lack of conserved phenotypic differences, the classification of the genus has long been contentious. DNA hybridization and genome-based analyses show close relationships among members of P. aeruginosa, but that isolates within the Pseudomonas fluorescens and P. syringae species are less closely related and may constitute different species. Collectively, genome sequences of Pseudomonas spp. have provided insights into pathogenesis and the genetic basis for diversity and adaptation.
•Promiscuous regulatory activity (crosstalk) reveals novel regulatory interactions to selection.•Crosstalk creates opportunities for genetic innovation that can facilitate adaptation.•Selection can ...reinforce binding affinity between transcription factor and binding sequences.•Gene regulatory network restructuring is frequently observed during adaptation to new niches.•Network structure is a likely key barrier to promiscuous binding potential of transcription factors.
At the level of the gene, mutation is the raw material for natural selection. However, at the level of the gene regulatory network (GRN), variation is revealed to selection via promiscuous regulator activity (‘crosstalk’), which creates opportunities for genetic innovation that can facilitate adaptation. Many genetic and environmental features can contribute to increasing potential for crosstalk by facilitating non-cognate interactions between regulatory elements. If a novel interaction provides a fitness benefit, rewired GRNs with strengthened affinity for newly forged connections can be selected. Here, we identify factors that facilitate opportunities for crosstalk and rewiring between GRNs, consider whether features of some GRNs make them more ‘rewireable’ than others and if these features might constrain evolution towards convergent outcomes. We explore patterns from laboratory and natural microbial populations that show changes within GRNs during adaptation. Finally, we discuss the prospects and open questions in the field.
The gastrointestinal (GI) tract is the reservoir for multidrug resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead ...to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals. Here, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including multidrug-resistant clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota. This study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step toward the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract.
A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the ...regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homolog of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and toward its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps.
Within soil, bacteria are found in multispecies communities, where interactions can lead to emergent community properties. Studying bacteria in a social context is critical for investigating ...community-level functions. We previously showed that cocultured Pseudomonas fluorescens Pf0-1 and
sp. V48 engage in interspecies social spreading (ISS) on a hard agar surface, a behavior which required close contact and depended on the nutritional environment. Here, we investigate whether social spreading is widespread among P. fluorescens and
isolates and whether the requirements for interaction vary. We find that this phenotype is not restricted to the interaction between P. fluorescens Pf0-1 and
sp. V48 but is a prevalent behavior found in one clade in the P. fluorescens group and two clades in the
genus. We show that the interaction with certain
isolates occurred without close contact, indicating induction of spreading by a putative diffusible signal. As with ISS by Pf0-1+V48, the motility of interacting pairs is influenced by the environment, with no spreading behaviors (or induction of motility) observed under high nutrient conditions. While Pf0-1+V48 require low nutrient but high NaCl conditions, in the broader range of interacting pairs, the high salt influence was variable. The prevalence of motility phenotypes observed here and found within the literature indicates that community-induced locomotion in general, and social spreading in particular, is likely important within the environment. It is crucial that we continue to study microbial interactions and their emergent properties to gain a fuller understanding of the functions of microbial communities.
Interspecies social spreading (ISS) is an emergent behavior observed when Pseudomonas fluorescens Pf0-1 and
sp. V48 interact, during which both species move together across a surface. Importantly, this environment does not permit the movement of either individual species. This group behavior suggests that communities of microbes can function in ways not predictable by knowledge of the individual members. Here, we have asked whether ISS is widespread and thus potentially of importance in soil microbial communities. The significance of this research is the demonstration that surface spreading behaviors are not unique to the Pf0-1-V48 interaction but rather is a more widespread phenomenon observed among members of distinct clades of both P. fluorescens and
isolates. Furthermore, we identify differences in mechanisms of signaling and nutritional requirements for ISS. Emergent traits resulting from bacterial interactions are widespread, and their characterization is necessary for a complete understanding of microbial community function.
Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and ...Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species.
Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome.
P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.
Complications arising from antibiotic-resistant bacteria are becoming one of the key issues in modern medicine. Members of drug-resistant Enterobacteriaceae spp. include opportunistic pathogens ...(e.g., Salmonella spp.) that are among the leading causes of morbidity and mortality worldwide. Overgrowth of these bacteria is considered a hallmark of intestinal dysbiosis. Microcins (small antimicrobial peptides) produced by some gut commensals can potentially cure these conditions by inhibiting these pathogens and have been proposed as a viable alternative to antibiotic treatment. In this proof-of-concept work, we leverage this idea to develop a genetically engineered prototype probiotic to inhibit Salmonella spp. upon exposure to tetrathionate, a molecule produced in the inflamed gut during the course of Salmonella infection. We developed a plasmid-based system capable of conferring the ability to detect and utilize tetrathionate, while at the same time producing microcin H47. We transferred this plasmid-based system to Escherichia coli and demonstrated the ability of the engineered strain to inhibit growth of Salmonella in anaerobic conditions while in the presence of tetrathionate, with no detectable inhibition in the absence of tetrathionate. In direct competition assays between the engineered E. coli and Salmonella, the engineered E. coli had a considerable increase in fitness advantage in the presence of 1 mM tetrathionate as compared to the absence of tetrathionate. In this work, we have demonstrated the ability to engineer a strain of E. coli capable of using an environmental signal indicative of intestinal inflammation as an inducing molecule, resulting in production of a microcin capable of inhibiting the organism responsible for the inflammation.
Herbicides are frequently released into both rural and urban environments. Commercial herbicide formulations induce adaptive changes in the way bacteria respond to antibiotics. Salmonella enterica ...sv. Typhimurium and Escherichia coli were exposed to common co-formulants of formulations, and S. enterica sv. Typhimurium was exposed to active ingredients dicamba, 2,4-D and glyphosate to determine what ingredients of the commercial formulations caused this effect. Co-formulants Tween80 and carboxymethyl cellulose induced changes in response, but the pattern of the responses differed from the active ingredients, and effect sizes were smaller. A commercial wetting agent did not affect antibiotic responses. Active ingredients induced changes in antibiotic responses similar to those caused by complete formulations. This occurred at or below recommended application concentrations. Targeted deletion of efflux pump genes largely neutralized the adaptive response in the cases of increased survival in antibiotics, indicating that the biochemistry of induced resistance was the same for formulations and specific ingredients. We found that glyphosate, dicamba, and 2,4-D, as well as co-formulants in commercial herbicides, induced a change in susceptibility of the potentially pathogenic bacteria E. coli and S. enterica to multiple antibiotics. This was measured using the efficiency of plating (EOP), the relative survival of the bacteria when exposed to herbicide and antibiotic, or just antibiotic, compared to survival on permissive media. This work will help to inform the use of non-medicinal chemical agents that induce changes in antibiotic responses.
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