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Genomic and phenotypic diversification of Pseudomonas aeruginosa during sustained exposure to a ciliate predator.

UNLABELLED: Predator-mediated selection is an important ecological force shaping bacterial evolution, but its effects on genomic adaptation and virulence in opportunistic pathogens are not fully understood. Here, we used experimental evolution to study how exposure to the ciliate predator Tetrahymena thermophila affects Pseudomonas aeruginosa. Replicate populations were evolved for 60 days with or without the predator, followed by whole-genome shotgun metagenomic sequencing and phenotypic analyses. Both treatments showed strong selection and evidence of parallel evolution at gene and nucleotide levels, indicating constrained adaptation. However, predator exposure altered evolutionary dynamics. Predator-evolved populations showed a wider distribution of mutation frequencies, with many mutations persisting at intermediate frequencies, consistent with increased clonal interference and ongoing competition among lineages. In contrast, populations evolved without predators showed more high-frequency mutations, consistent with selective sweeps, although some low-frequency variants remained. Despite substantial genomic change, phenotypic outcomes were variable. Virulence in an invertebrate host model did not consistently increase. Instead, evolved isolates showed context-dependent changes, including modest decreases or occasional increases. Competition assays also showed no consistent fitness advantage for predator-evolved isolates, suggesting trade-offs between predator resistance and growth in other environments. Overall, predator-mediated selection reshaped evolutionary dynamics by maintaining diversity and altering the balance of lineages rather than producing uniform increases in virulence. These results highlight how ecological complexity influences adaptive evolution and the context-dependent nature of pathogen traits. IMPORTANCE: Opportunistic pathogens such as Pseudomonas aeruginosa often evolve in environmental settings before infecting hosts, raising questions about how ecological interactions influence virulence. Predator-mediated selection has been suggested to increase virulence via coincidental evolution, but evidence is inconsistent. Here, we show that exposure to a eukaryotic predator does not consistently elevate virulence but does reshape evolutionary dynamics by altering how mutations spread in populations. Predator-exposed populations retained more intermediate-frequency mutations, consistent with increased clonal interference and ongoing competition among lineages, whereas non-predator populations were dominated by selective sweeps. These differences were also reflected in functional targets of adaptation, with predator exposure favoring mutations in genes involved in environmental sensing and interaction. Together, these findings suggest that ecological complexity shapes the dynamics of adaptation rather than driving a single evolutionary outcome, highlighting that virulence is an emergent property influenced by underlying evolutionary processes.

Pseudomonas aeruginosa

Outer membrane changes enable evolutionary escape from bacterial predation.

Antimicrobial resistance (AMR) is a threat to modern medicine. To combat AMR pathogens, natural predators like bacteriophages and predatory bacteria have gained interest recently. Predatory bacterium Bdellovibrio bacteriovorus is ubiquitous and has a broad prey range. It is particularly potent at killing many AMR Gram-negative bacterial pathogens featured on the WHO priority list. However, it is currently unclear whether prey bacteria can evolve genetically-determined resistance against predation by B. bacteriovorus. Here, we show that the model bacterium Escherichia coli K-12 consistently evolves resistance against B. bacteriovorus during experimental evolution. Selection for resistance scaled positively with predation pressure and was widespread after two cycles of predator exposure. Similar to antibiotics, predation resistance was costly, manifesting in a trade-off between predation resistance and fitness in the absence of predators. Genetic analysis combined with proteomics identified mutations that lead to the down-regulation of the outer membrane porin OmpF as a common resistance mechanism. In addition, a rarer mutation in cell envelope lipopolysaccharide-modifying enzyme WaaF also conferred predation resistance, likely by a pleiotropic effect, which included OmpF down regulation. While our study uncovers evolutionary and mechanistic aspects of prey escape from predation, it also highlights that the high cost of resistance reflects a handicap for the pathogen and can thus be exploited to increase treatment sustainability. Altogether, our work generates essential knowledge in ecologically important predator-prey interactions and can advance predatory bacteria as "living antibiotics" to combat AMR.

Bdellovibrio bacteriovorus

Regulation of predation by prey density: the protozoan-Rhizobium relationship.

Tetramitus rostratus and strains of Hartmanella, Naegleria, and Vahlkampfia consumed large numbers of Rhizobium meliloti cells in a salt solution, but protozoan multiplication and the bacterial decline stopped when the prey density fell to about 10-6 to 10-7 cells/ml. At higher prey densities, the maximum numbers of Hartmanella sp. and Naegleria sp. were proportional to the quantity of R. meliloti initially provided to the amoebas. When supplemental rhizobia were supplied to Hartmanella sp. or Naegleria sp. after their active feeding had terminated, presumably because the remaining 10-6 or 10-7 bacteria/ml could not be captured, replication of the protozoa was initiated. The rate of elimination of rhizobia present in large populations was proportional to the initial abundance of Naegleria sp., but the final numbers of amoebas and surviving R. meliloti cells were independent of initial numbers of predators. The surviving bacteria were not intrinsically resistant to attack because 98% of the survivors, when concentrated, were consumed. It is suggested that large populations of bacteria in nature may be reduced in size by predatory protozoa, but many of the prey cells will not be eliminated.

Animals

Chloramphenicol and tetracycline synergize with bacteriophage SeKF_13 to inactivate antimicrobial-resistant Salmonella Typhimurium.

UNLABELLED: Non-typhoidal Salmonella is estimated to cause up to 1 billion cases of global foodborne illness per year. Salmonella Typhimurium is a serovar of gravest worldwide concern as it is capable of infecting animal and human hosts and can also acquire antimicrobial resistance (AMR) determinants at a rapid rate. Recent advances in phage research have positioned them as especially useful for inactivation of Salmonella where antibiotics have proven no longer effective. Even more recently, phage-antibiotic synergy (PAS) has been proposed as a solution for AMR Salmonella, where synergistic combinations of phages and antibiotics are more effective than application of phage or antibiotic alone. Utilizing an in-house phage isolate, SeKF_13, we sought to determine the existence of PAS against a strain of Salmonella enterica serovar Typhimurium 14028 2a that is clinically resistant to bacteriostatic antibiotics chloramphenicol and tetracycline. Checkerboard assays revealed the presence of synergy when sub-lethal (sub-MIC) levels of either tetracycline or chloramphenicol were combined with phage SeKF_13 (P < 0.05; two-way ANOVA). Compared to tetracycline or chloramphenicol alone, the addition of phage also decreased the MICs of both antibiotics twofold. We also monitored the development of resistance and found that PAS significantly suppressed emergence of resistance compared to the antibacterial agents alone (P < 0.05; Tukey's HSD). Whole-genome sequencing revealed that SeKF_13 is devoid of genes encoding integrase, antimicrobial resistance, and virulence, ensuring safety in future applications. Together, our results suggest that combined treatment of phage and antibiotic can improve antimicrobial efficacy against antibiotic-resistant Salmonella enterica. IMPORTANCE: Salmonella enterica is a foodborne pathogen that causes one of the highest rates of foodborne illness worldwide. They are also capable of becoming resistant to antimicrobials very rapidly (i.e., antimicrobial resistance; AMR) due to their ability to acquire AMR determinants, undermining the effectiveness of current treatments. Bacteriophages (phages), viral predators of bacteria, have been proven to be effective in some cases, but recently, phage-antibiotic synergy has been proposed as a more effective solution than phages or antibiotics alone. We found this was, indeed, the case; using phage SeKF_13 and tetracycline or chloramphenicol (to which the Salmonella strain was resistant), we found that combination treatment was significantly more effective than either treatment alone. These results demonstrate that combined treatment of phage and antibiotic can bolster treatment efficacy against AMR Salmonella.

Salmonella typhimurium

Removal of Escherichia coli in wastewater by activated sludge.

Removal of bacteria from wastewater treated with activated sludge was studied by the use of a streptomycin-resistant Escherichia coli strain. The removal appeared to be a biphasic process. A rapid sorption of bacteria to the sludge flocs took place in the first hour after seeding mixed liquor with E. coli. Thereafter, slower elimination of E. coli was observed. The latter process was due to predation on E. coli by ciliated protozoa. This was shown by: (i) appearance of fluorescent food vacuoles of ciliates when fluorescent E. coli cells were added to mixed liquor; (ii) inhibition of predation either in the presence of cycloheximide or under anaerobic conditions; and (iii) absence of predation in bulking and washed sludge.

Anaerobiosis

Dehiscence and active spore release in pathogenic strains of the yeast Metschnikowia bicuspidata var. australis: possible predatory implication.

Strains of Metschnikowia bicuspidata var. australis, pathogenic to brine shrimp (Artemia salina), were observed to form asci which, upon reaching maturity, forcibly expelled their needle-shaped spores. The mechanical force responsible apparently originates from the formation of an ectoplasmic mucilage capable of exerting pressure over all of the ascus contents; when the apex of the peduncle ruptures, the ascospores are violently released. Cytochemical analyses indicated that the gel is a substance highly resistant to chemical and enzymatic hydrolysis. Its chemical nature is not known as yet. The morphogenetic events of this process are described, and its ecological implication, the possibility of active mechanical predation in yeast, is discussed.

Aldehyde-Lyases

Protozoa as agents responsible for the decline of Xanthomonas campestris in soil.

A streptomycin-resistant mutant of Xanthomonas campestris was used to assess the persistence of the plant pathogen in soil and the changes in populations that might be important for its survival. In soil into which large numbers of the organism were introduced, a marked decline in its abundance occurred, but after about 1 week its population density reached a level of about 105 and did not continue to fall during the test period. No such marked decline was evident in sterile soil inoculated with X. campestris. The bacterium did not lose viability if starved for carbon or inorganic nitrogen. Although abundant in soil, the numbers of propagules capable of producing antibiotics or lytic enzymes active against X. campestris did not increase coincident with the pathogen's decline, and no increase in tartrate-extractable toxins was observed. Neither bdellovibrios nor bacteriophages active against the xanthomonad were found in the soil, but a marked increase in the frequency of protozoa paralleled the phase of rapid diminution in the X. campestris population. In actidione-treated soil, in which protozoan activity was severly limited, the high cell density of the pathogen was maintained. On the basis of these data, it is concluded that predation by protozoa is responsible for the abrupt fall in frequency of the bacterium in natural soil.

Animals

Staphylococcus aureus COL: An Atypical Model Strain of MRSA That Exhibits Slow Growth and Antibiotic Tolerance due to a Mutation in PRPP Synthetase.

Methicillin-resistant Staphylococcus aureus (MRSA) has been a pathogen of global concern since its emergence in the 1960s. As one of the first MRSA strains isolated, COL has become a common model strain of S. aureus. Here we report that COL is, in fact, an atypical strain of MRSA that exhibits slow growth and multidrug tolerance. Genomic analysis identified three mutated genes in COL (rpoB, gltX and prs) with links to tolerance. Allele swapping experiments between COL and the closely-related, nontolerant Newman strain uncovered a complex interplay between these genes. However, Prs (phosphoribosyl pyrophosphate [PRPP] synthetase) accounted for most of the growth and tolerance phenotype of COL. Biochemical and transcriptomic analysis revealed that COL does not exhibit slow growth as a result of partial stringent response activation, as previously proposed. Instead, the COL Prs mutation greatly reduces the PRPP synthetase activity of the enzyme and leads to downregulation of pyrimidine, histidine, and tryptophan synthesis, three pathways that rely on PRPP. Overall, our findings indicate that COL is an atypical, antibiotic-tolerant strain of MRSA whose isolation predates the previous first report of tolerance among clinical isolates. Characterization of clinical Prs mutations and their relationship with tolerance requires further investigation.

Methicillin-Resistant Staphylococcus aureus

Myxomatosis: population dynamics of rabbits (Oryctolagus cuniculus Linnaeus, 1758) and ecological effects in the United Kingdom.

In 1953-1955, myxomatosis spread among rabbits (Oryctolagus cuniculus) in the United Kingdom, causing 99% mortality. Subsequently, there was a gradual increase in rabbit numbers. By 1955, the Ministry of Agriculture, Fisheries and Food (MAFF) had already found attenuated strains of myxoma virus. By 1970, genetic resistance had appeared. In the 1970s, mortality declined to 47-69% with only approximately 25% of rabbits infected, giving a field mortality of 12-19%. However, myxomatosis is persistent, generally showing a major prevalence peak in autumn and often a minor peak in spring. An eight-year MAFF experiment in which prevalence of the disease was artificially reduced indicates that myxomatosis remains a significant factor in population regulation. After rabbit numbers fell in the 1950s, important ecological changes took place: vegetation altered due to reduced grazing pressure, predators were affected by the reduction of a major prey species and these changes also affected many other animals. Currently, rabbit numbers have returned to approximately one-third of pre-myxomatosis levels and this is causing damage to farm and conservation habitats.

Animals

[Biological control of vectors of human and tropical diseases. Present means and prospects (author's transl)].

Biological control is "direct or indirect use of natural enemies of the injurious species to increase its mortality" (W.H.O., 1963). The more and more frequent apparition of resistant insects populations, the fears as regards the environment, the increase cost of hydrocarbur products and also some technic and operational difficulties to stop transmission by the use of only insecticide pulvérisation, impose this process. Nevertheless, practic use of natural enemies of vectors is yet unusual in spite of important research. (Identification problems, dynamic of species, insufficiency of ethology knowledge particulary of the host specificity, difficulties of application on the vectors which are the most usually widely scattered). For control of insects of medical importance (mosquitoes, black flies, tse-tse flies) it has been used either pathogen agents such as virus bacteria, microsporida or parasit agents such as fungi, mermithid nematods or at last, predators, essentially larvivorus fish. Actually, no biological agent is able to take the place of chemical and physical "traditional" means. In case of mosquito control which is more advanced, the only biological mean which is operational is the use of larvivorus fish and specially Gambusia.

Africa

The susceptibility of Marisa cornuarietis, a predator of schistosome bearing snails, to N-tritylmorpholine.

Introduction of the ampullariid snail, Marisa cornuarietis (L.), into water treated with molluscicides, in order to secure the success of chemical control of schistosome host snails, is promising. Adult Marisa can be introduced only two days after treating water of pH less than or equal to 7-9 with N-tritylmorpholine (= FresconR Shell) at a concentration of 0-03 ppm. There is considerable variation in the susceptibility of different strains: the LT50 in a concentration of 0-03 ppm Frescon at 25 degrees C was about 27-3 hours for a Puerto Rican and 44-6 hours for a Floridan strain, both 52 weeks old. At sexual maturity, i.e. approximately 18 weeks at 25 degrees C, the LT50 for the Floridan strain was approximately 31-8 hours; experiments with a hybrid stock of the two strains had an LT50 of 30-0 hours. Younger snails were significantly more susceptible to the molluscicide, and eggs were approximately four times more resistant than adults; this agrees with findings by previous authors for other snail species. In the case of the accidental uncontrolled spread of Marisa to cultivated areas it is suggested that a concentration of 0-03 Frescon is applied for at least four days.

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