Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium.

Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water.

Rapid identification of international multidrug-resistant Pseudomonas aeruginosa clones by multiple-locus variable number of tandem repeats analysis and investigation of their susceptibility to lytic bacteriophages.

The objective of this study was to determine the genetic diversity of multidrug-resistant (MDR) Pseudomonas aeruginosa strains isolated over a period of 12 months in two French hospitals and to test their susceptibility to bacteriophages. A total of 47 MDR isolates recovered from hospitalized patients were genotyped using multiple-locus variable number of tandem repeats analysis. The genotypes were distributed into five clones (including 19, 5, 5, 3, and 3 isolates, respectively) and 12 singletons. Comparison to 77 MDR strains from three other countries, and MLST analysis of selected isolates showed the predominance of international MDR clones. The larger clone, CC235, contained 59 isolates displaying different antibiotic resistance mechanisms, including the presence of the GES1, VIM-2, VIM-4, and IMP-1 ß-lactamases. Three newly isolated P. aeruginosa bacteriophages were found to lyse 42 of the 44 analyzed strains, distributed into the different clonal complexes. This pilot study suggests that systematic genotyping of P. aeruginosa MDR strains could improve our epidemiological understanding of transmission at both the local (hospital) and the national level and that phage therapy could be an alternative or a complementary treatment to antibiotics for treating MDR-infected patients.

Efficacy of bacteriophage therapy in experimental sepsis and meningitis caused by a clone O25b:H4-ST131 Escherichia coli strain producing CTX-M-15.

We evaluated phage therapy in experimental infections due to S242, a fatal neonatal meningitis Escherichia coli strain belonging to the worldwide-distributed O25b:H4-ST131 clone that produces extended-spectrum beta-lactamase CTX-M-15. A lytic phage, EC200(PP), active against S242, was isolated from environmental water. After determining in vitro and ex vivo stabilities and pharmacokinetic properties of EC200(PP) in rat pups, we assessed the therapeutic efficacy of a single dose of 10(8) PFU using models of sepsis and meningitis in which fatality was 100%. EC200(PP) was partially neutralized by human serum. In contrast to the high concentration of phage in the spleen and the kidney, low titers in urine and the central nervous system were observed. Nevertheless, in the sepsis model, EC200(PP) administered 7 h or 24 h postinfection resulted in 100% and 50% pup survival, respectively. In the meningitis model, EC200(PP) administered 1 h or 7 h postinfection rescued 100% of the animals. The most delayed treatments were associated with the selection of phage-resistant S242 mutants. However, a representative mutant was highly sensitive to killing serum activity and avirulent in an animal model. EC200(PP) is a potential therapeutic agent for sepsis and meningitis caused by the widespread E. coli O25:H4-ST131 multidrug-resistant clone.

Genetically engineered virulent phage banks in the detection and control of emergent pathogenic bacteria.

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be used or engineered as agents of bioterrorism. From a biosecurity standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, for which there may be no effective antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this end, we developed technologies allowing us to (1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, (2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, (3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population, and (4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including phage host-range. Screening of such a bank should allow the rapid isolation of recombinant T4 particles capable of detecting (ie, diagnosing), infecting, and destroying hosts belonging to gram-negative bacterial species far removed from the original E. coli host.

Characterization of chromosomal regions conserved in Yersinia pseudotuberculosis and lost by Yersinia pestis.

The transformation of the enteropathogenic bacterium Yersinia pseudotuberculosis into the plague bacillus, Yersinia pestis, has been accompanied by extensive genetic loss. This study focused on chromosomal regions conserved in Y. pseudotuberculosis and lost during its transformation into Y. pestis. An extensive PCR screening of 78 strains of the two species identified five regions (R1 to R5) and four open reading frames (ORFs; orf1 to orf4) that were conserved in Y. pseudotuberculosis and absent from Y. pestis. Their conservation in Y. pseudotuberculosis suggests a positive selective pressure and a role during the life cycle of this species. Attempts to delete two ORFs (orf3 and orf4) from the chromosome of strain IP32953 were unsuccessful, indicating that they are essential for its viability. The seven remaining loci were individually deleted from the IP32953 chromosome, and the ability of each mutant to grow in vitro and to kill mice upon intragastric infection was evaluated. Four loci (orf1, R2, R4, and R5) were not required for optimal growth or virulence of Y. pseudotuberculosis. In contrast, orf2, encoding a putative pseudouridylate synthase involved in RNA stability, was necessary for the optimal growth of IP32953 at 37 degrees C in a chemically defined medium (M63S). Deletion of R1, a region predicted to encode the methionine salvage pathway, altered the mutant pathogenicity, suggesting that the availability of free methionine is severely restricted in vivo. R3, a region composed mostly of genes of unknown functions, was necessary for both optimal growth of Y. pseudotuberculosis at 37 degrees C in M63S and for virulence. Therefore, despite their loss in Y. pestis, five of the nine Y. pseudotuberculosis-specific chromosomal loci studied play a role in the survival, growth, or virulence of this species.

A putative DNA adenine methyltransferase is involved in Yersinia pseudotuberculosis pathogenicity.

Some adenine methyltransferases have been shown not only to protect specific DNA restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. This study focused on a type I restriction-modification system (designated yrmI) of Y. pseudotuberculosis. This system is composed of three adjacent genes which could potentially encode an N6-adenine DNA methylase (YamA), an enzyme involved in site-specific recognition (YrsA) and a restriction endonuclease (YreA). Screening of 85 isolates of Y. pestis and Y. pseudotuberculosis indicated that the yrmI system has been lost by Y. pestis and that yamA (but not yrsA or yreA) is present in all Y. pseudotuberculosis strains tested, suggesting that it may be important at some stages of the epidemiological cycle of this species. To further investigate the role of yamA in Y. pseudotuberculosis survival, multiplication or virulence, a DeltayamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that DeltayamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953DeltayamA mutant strain grew as well as the wild-type in a rich medium at both 28 degrees C and 37 degrees C. It also grew normally in a chemically defined medium at 28 degrees C, but exhibited a growth defect at 37 degrees C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the DeltayamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrmI locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.

A horizontally acquired filamentous phage contributes to the pathogenicity of the plague bacillus.

Yersinia pestis, the plague bacillus, has an exceptional pathogenicity but the factors responsible for its extreme virulence are still unknown. A genome comparison with its less virulent ancestor Yersinia pseudotuberculosis identified a few Y. pestis-specific regions acquired after their divergence. One of them potentially encodes a prophage (YpfPhi), similar to filamentous phages associated with virulence in other pathogens. We show here that YpfPhi forms filamentous phage particles infectious for other Y. pestis isolates. Although it was previously suggested that YpfPhi is restricted to the Orientalis branch, our results indicate that it was acquired by the Y. pestis ancestor. In Antiqua and Medievalis strains, YpfPhi genome forms an unstable episome whereas in Orientalis isolates it is stably integrated as tandem repeats. Deletion of the YpfPhi genome does not affect Y. pestis ability to colonize and block the flea proventriculus, but results in an alteration of Y. pestis pathogenicity in mice. Our results show that transformation of Y. pestis from a classical enteropathogen to the highly virulent plague bacillus was accompanied by the acquisition of an unstable filamentous phage. Continued maintenance of YpfPhi despite its high in vitro instability suggests that it confers selective advantages to Y. pestis under natural conditions.

Evaluation of O-antigen inactivation on Pla activity and virulence of Yersinia pseudotuberculosis harbouring the pPla plasmid.

Yersinia pestis is a species that emerged recently from Yersinia pseudotuberculosis and gained an exceptional pathogenicity potential. Among the major genetic differences between the plague bacillus and its ancestor is the acquisition of the pPla plasmid, which has been associated with the increased virulence of Y. pestis. In a previous study, introduction of pPla into Y. pseudotuberculosis did not lead to any modification of the virulence of the host bacterium. However, it was subsequently demonstrated that the presence of smooth lipopolysaccharide (LPS) inhibits the activity of Pla. In this study, pPla was introduced into a Y. pseudotuberculosis strain expressing smooth LPS, and into a variant in which a mutation that abrogates the formation of O-antigen (O-Ag) repeats (as in natural isolates of Y. pestis) was generated. It was found that in both strains, Pla was synthesized, exported to the bacterial membrane and processed as in Y. pestis. However, the ability of Pla to activate plasminogen was weak and observed only at 37 degrees C in the smooth strain, while this activity was similar to that of Y. pestis and expressed at both 28 and 37 degrees C in the O-Ag mutant strain. Similarly, Pla-mediated inactivation of the antiprotease alpha2-antiplasmin was not detected in the smooth Y. pseudotuberculosis strain grown at 28 degrees C, but was expressed at both temperatures in the O-Ag mutant strain. Despite the more efficient activity of Pla, the Y. pseudotuberculosis O-Ag mutant strain exhibited a lower pathogenicity upon subcutaneous infection of mice. The results thus indicate that, although abrogation of O side chain synthesis in a Y. pseudotuberculosis strain harbouring pPla potentiates the two proteolytic activities of Pla, this is not sufficient to confer to Y. pseudotuberculosis a higher pathogenicity potential. These results also suggest that acquisition of pPla may not have been sufficient to confer an immediate higher pathogenic potential to the ancestor Y. pestis strain.