Canonical germinant receptor is dispensable for spore germination in Clostridium botulinum group II strain NCTC 11219 (vol 7, 15426, 2017)

An amendment to this paper has been published and can be accessed via a link at the top of the paper.status: publishe

Completed Genomic Sequence ofBacillus thuringiensisHER1410 Reveals aCry-Containing Chromosome, Two Megaplasmids, and an Integrative Plasmidial Prophage

Bacillus thuringiensis is the most used biopesticide in agriculture. Its entomopathogenic capacity stems from the possession of plasmid-borne insecticidal crystal genes (cry), traditionally used as discriminant taxonomic feature for that species. As such, crystal and plasmid identification are key to the characterization of this species. To date, about 600 B. thuringiensis genomes have been reported, but less than 5% have been completed, while the other draft genomes are incomplete, hindering full plasmid delineation. Here we present the complete genome of Bacillus thuringiensis HER1410, a strain closely related to B. thuringiensis entomocidus and a known host for a variety of Bacillus phages. The combination of short and long-read techniques allowed fully resolving the genome and delineation of three plasmids. This enabled the accurate detection of an unusual location of a unique cry gene, cry1Ba4, located in a genomic island near the chromosome replication origin. Two megaplasmids, pLUSID1 and pLUSID2 could be delineated: pLUSID1 (368 kb), a likely conjugative plasmid involved in virulence, and pLUSID2 (156 kb) potentially related to the sporulation process. A smaller plasmidial prophage pLUSID3, with a dual lifestyle whose integration within the chromosome causes the disruption of a flagellar key component. Finally, phylogenetic analysis placed this strain within a clade comprising members from the B. thuringiensis serovar thuringiensis and other serovars and with B. cereus s. s in agreement with the intermingled taxonomy of B. cereus sensu lato group.status: publishe

Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a pseudomonas shirazica clinical strain

Horizontal transfer of plasmids plays a pivotal role in dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Plasmid sequencing is thus paramount for accurate epidemiological tracking in hospitals and routine surveillance. Combining Nanopore and Illumina sequencing allowed full assembly of a carbapenemase-encoding megaplasmid carried by multidrug-resistant clinical isolate FFUP_PS_41. Average nucleotide identity analyses revealed that FFUP_PS_41 belongs to the recently proposed new species Pseudomonas shirazica, related to the P. putida phylogenetic group. FFUP_PS_41 harbours a 498,516-bp megaplasmid (pJBCL41) with limited similarity to publicly-available plasmids. pJBCL41 contains genes predicted to encode replication, conjugation, partitioning and maintenance functions and heavy metal resistance. The |aacA7|blaVIM-2|aacA4| cassette array (resistance to carbapenems and aminoglycosides) is located within a class 1 integron that is a defective Tn402 derivative. This transposon lies within a 50,273-bp region bound by Tn3-family 38-bp inverted repeats and flanked by 5-bp direct repeats (DR) that composes additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach allowed full resolution of a carbapenemase-encoding megaplasmid from P. shirazica. Identification of novel megaplasmids sheds new light on the evolutionary effects of gene transfer and the selective forces driving antibiotic resistance.status: publishe

The ever-expanding Pseudomonas genus : description of 43 new species and partition of the Pseudomonas putida group

The genus Pseudomonas hosts an extensive genetic diversity and is one of the largest genera among Gram-negative bacteria. Type strains of Pseudomonas are well known to represent only a small fraction of this diversity and the number of available Pseudomonas genome sequences is increasing rapidly. Consequently, new Pseudomonas species are regularly reported and the number of species within the genus is constantly evolving. In this study, whole genome sequencing enabled us to define 43 new Pseudomonas species and provide an update of the Pseudomonas evolutionary and taxonomic relationships. Phylogenies based on the rpoD gene and whole genome sequences, including, respectively, 316 and 313 type strains of Pseudomonas, revealed sixteen groups of Pseudomonas and, together with the distribution of cyclic lipopeptide biosynthesis gene clusters, enabled the partitioning of the P. putida group into fifteen subgroups. Pairwise average nucleotide identities were calculated between type strains and a selection of 60 genomes of non-type strains of Pseudomonas. Forty-one strains were incorrectly assigned at the species level and among these, 19 strains were shown to represent an additional 13 new Pseudomonas species that remain to be formally classified. This work pinpoints the importance of correct taxonomic assignment and phylogenetic classification in order to perform integrative studies linking genetic diversity, lifestyle, and metabolic potential of Pseudomonas spp

Catabolism of the groundwater micropollutant 2,6-dichlorobenzamide beyond 2,6-dichlorobenzoate is plasmid encoded in Aminobacter sp MSH1

Aminobacter sp. MSH1 uses the groundwater micropollutant 2,6-dichlorobenzamide (BAM) as sole source of carbon and energy. In the first step, MSH1 converts BAM to 2,6-dichlorobenzoic acid (2,6-DCBA) by means of the BbdA amidase encoded on the IncP-1β plasmid pBAM1. Information about the genes and degradation steps involved in 2,6-DCBA metabolism in MSH1 or any other organism is currently lacking. Here, we show that the genes for 2,6-DCBA degradation in strain MSH1 reside on a second catabolic plasmid in MSH1, designated as pBAM2. The complete sequence of pBAM2 was determined revealing that it is a 53.9 kb repABC family plasmid. The 2,6-DCBA catabolic genes on pBAM2 are organized in two main clusters bordered by IS elements and integrase genes and encode putative functions like Rieske mono-/dioxygenase, meta-cleavage dioxygenase, and reductive dehalogenases. The putative mono-oxygenase encoded by the bbdD gene was shown to convert 2,6-DCBA to 3-hydroxy-2,6-dichlorobenzoate (3-OH-2,6-DCBA). 3-OH-DCBA was degraded by wild-type MSH1 and not by a pBAM2-free MSH1 variant indicating that it is a likely intermediate in the pBAM2-encoded DCBA catabolic pathway. Based on the activity of BbdD and the putative functions of the other catabolic genes on pBAM2, a metabolic pathway for BAM/2,6-DCBA in strain MSH1 was suggested.status: publishe

Integrative omics analysis of Pseudomonas aeruginosa virus PA5oct highlights the molecular complexity of jumbo phages

Pseudomonas virus vB_PaeM_PA5oct is proposed as a model jumbo bacteriophage to investigate phage-bacteria interactions and is a candidate for phage therapy applications. Combining hybrid sequencing, RNA-Seq and mass spectrometry allowed us to accurately annotate its 286,783 bp genome with 461 coding regions including four non-coding RNAs (ncRNAs) and 93 virion-associated proteins. PA5oct relies on the host RNA polymerase for the infection cycle and RNA-Seq revealed a gradual take-over of the total cell transcriptome from 21% in early infection to 93% in late infection. PA5oct is not organized into strictly contiguous regions of temporal transcription, but some genomic regions transcribed in early, middle and late phases of infection can be discriminated. Interestingly, we observe regions showing limited transcription activity throughout the infection cycle. We show that PA5oct upregulates specific bacterial operons during infection including operons pncA-pncB1-nadE involved in NAD biosynthesis, psl for exopolysaccharide biosynthesis and nap for periplasmic nitrate reductase production. We also observe a downregulation of T4P gene products suggesting mechanisms of superinfection exclusion. We used the proteome of PA5oct to position our isolate amongst other phages using a gene-sharing network. This integrative omics study illustrates the molecular diversity of jumbo viruses and raises new questions towards cellular regulation and phage-encoded hijacking mechanisms.status: publishe

Pseudomonas aeruginosa PA5oct Jumbo Phage Impacts Planktonic and Biofilm Population and Reduces Its Host Virulence

The emergence of phage-resistant mutants is a key aspect of lytic phages-bacteria interaction and the main driver for the co-evolution between both organisms. Here, we analyze the impact of PA5oct jumbo phage treatment on planktonic/cell line associated and sessile P. aeruginosa population. Besides its broad-spectrum activity and efficient bacteria reduction in both airway surface liquid (ASL) model, and biofilm matrix degradation, PA5oct appears to persist in most of phage-resistant clones. Indeed, a high percentage of resistance (20/30 clones) to PA5oct is accompanied by the presence of phage DNA within bacterial culture. Moreover, the maintenance of this phage in the bacterial population correlates with reduced P. aeruginosa virulence, coupled with a sensitization to innate immune mechanisms, and a significantly reduced growth rate. We observed rather unusual consequences of PA5oct infection causing an increased inflammatory response of monocytes to P. aeruginosa. This phenomenon, combined with the loss or modification of the phage receptor, makes most of the phage-resistant clones significantly less pathogenic in in vivo model. These findings provide new insights into the general knowledge of giant phages biology and the impact of their application in phage therapy.status: publishe

In Vitro Evaluation of the Therapeutic Potential of Phage VA7 against Enterotoxigenic Bacteroides fragilis Infection

Since the beginning of the 20th century, bacteriophages (phages), i.e., viruses that infect bacteria, have been used as antimicrobial agents for treating various infections. Phage preparations targeting a number of bacterial pathogens are still in use in the post-Soviet states and are experiencing a revival in the Western world. However, phages have never been used to treat diseases caused by Bacteroides fragilis, the leading agent cultured in anaerobic abscesses and postoperative peritonitis. Enterotoxin-producing strains of B. fragilis have been associated with the development of inflammatory diarrhea and colorectal carcinoma. In this study, we evaluated the molecular biosafety and antimicrobial properties of novel phage species vB_BfrS_VA7 (VA7) lysate, as well as its impact on cytokine IL-8 production in an enterotoxigenic B. fragilis (ETBF)-infected colonic epithelial cell (CEC) culture model. Compared to untreated infected cells, the addition of phage VA7 to ETBF-infected CECs led to significantly reduced bacterial counts and IL-8 levels. This in vitro study confirms the potential of phage VA7 as an antibacterial agent for use in prophylaxis or in the treatment of B. fragilis infections and associated colorectal carcinoma

New Bacteriophages against Emerging Lineages ST23 and ST258 of Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae

Klebsiella pneumoniae is a bacterial pathogen of high public health importance. Its polysaccharide capsule is highly variable but only a few capsular types are associated with emerging pathogenic sublineages. The aim of this work is to isolate and characterize new lytic bacteriophages and assess their potential to control infections by the ST23 and ST258 K. pneumoniae sublineages using a Galleria mellonella larvae model. Three selected bacteriophages, targeting lineages ST258 (bacteriophages vB_KpnP_KL106-ULIP47 and vB_KpnP_KL106-ULIP54) and ST23 (bacteriophage vB_KpnP_K1-ULIP33), display specificity for capsular types KL106 and K1, respectively. These podoviruses belong to the Autographivirinae subfamily and their genomes are devoid of lysogeny or toxin-associated genes. In a G. mellonella larvae model, a mortality rate of 70% was observed upon infection by K. pneumoniae ST258 and ST23. This number was reduced to 20% upon treatment with bacteriophages at a multiplicity of infection of 10. This work increases the number of characterized bacteriophages infecting K. pneumoniae and provides information regarding genome sequence and efficacy during preclinical phage therapy against two prominent sublineages of this bacterial species.status: publishe

Phage therapy to allow liver transplantation in a toddler infected by an extensively drug-resistant Pseudomonas aeruginosa

Brieuc Van Nieuwenhuyse1, Dimitri Van der Linden1,2, Olga Chatzis2, Cédric Lood3,4, Jeroen Wagemans3, Rob Lavigne4, Catherine de Magnée5, Étienne Sokal1,6, Hector Rodriguez-Villalobos7, Sarah Djebara8, Maya Merabishvili9, Patrick Soentjens8, Jean-Paul Pirnay9. 1Institute of Experimental and Clinical Research's Pediatric department, UCLouvain, Brussels, Belgium; 2Pediatric Infectious Diseases, General Pediatrics Department, Cliniques universitaires Saint-Luc, Brussels, Belgium; 3Department of Biosystems, Laboratory of Gene Technology, KULeuven, Leuven, Belgium; 4Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, KULeuven, Leuven, Belgium; 5Pediatric and Transplantation Surgery, Cliniques universitaires Saint-Luc, Brussels, Belgium; 6Pediatric Hepatology and Gastro-enterology, Cliniques universitaires Saint-Luc, Brussels, Belgium; 7Department of Microbiology, Cliniques universitaires Saint-Luc, Brussels, Belgium; 8Center for Infectious Diseases, Queen Astrid Military Hospital, Brussels, Belgium; 9Laboratory for Molecular and Cellular Technology, Queen Astrid Military Hospital, Brussels, Belgium A 14-month old boy undergoes a first liver transplantation (LT) (Day 0), from an ABO-incompatible living donor. On D+20, we detect a fecal carriage of an extensively drug-resistant (XDR) Pseudomonas aeruginosa (Pa) strain. Besides intermediate susceptibility to aztreonam and colistin and susceptibility to gentamycin, the strain is resistant to all other antibiotics. On D+53, the child enters a severe septic state due to a bacteremia with the same Pa strain. New antibiogram suggests a resistance to colistin. Liver bilomas' drainage material is cultured and grows the same Pa strain. Admission to the pediatric intensive care unit and adjunction of intravenous (IV) aztreonam, gentamycin, and colistin led to no improvement on the microbiological or clinical levels during the next four days. By collaborating with Queen Astrid Military Hospital (Brussels, Belgium), we initiated phage therapy (PT) on D+57 in accordance to the Article 37 of the Declaration of Helsinki and with the patient's parents' consent. PT is the use of lytic bacteriophage viruses to achieve antibacterial effect. Phage cocktail BFC1 contains two anti-Pa phages (PNM and 14/1) and one anti-Staphylococcus aureus phage (ISP). BFC1 was administered in situ by instillations through biliary catheter during six days, and in IV for 86 days (72 days until 2nd LT, 14 days afterwards), the longest described duration for IV PT in a child. Previous antibiotic therapy was pursued all along. Intraoperative PT was performed during 2nd LT by bathing the peritoneal cavity in phage solution during the anhepatic phase. To further our understanding of the case, seven Pa isolates, both bloodborne and liver-borne, were sequenced. Serum samples obtained before, during, and after phage therapy were analyzed through double agar overlay method to search for phage immune neutralization (PIN). Phage-induced virulence tradeoffs (PIVT) assays were performed in a Galleria mellonella model. In vitro phage-antibiotic interactions were evaluated with OmniLog® system. PT initiation was followed by immediate (<24 h) eradication of Pa from blood cultures. Reappearance of Pa in blood cultures after four days of PT led to a doubling of the PT dose, which was followed by eradication of Pa from bloodstream until 2nd LT. The child has known no further infectious episode since then. Sequencing confirmed the emergence of bacterial phage resistance (BPR) in four isolates. Such BPR did not lead to therapeutic failure, possibly thanks to PIVT. PIN against phage ISP was detected, but not against any anti-Pa phage. OmniLog® assays suggested synergistic properties between phage PNM and three antibiotics administered concomitantly to the patient. In conclusion, prolonged IV phage therapy combined with antibiotics led to the durable eradication of an XDR Pa sepsis in an immunosuppressed 14-month old boy, eventually allowing for 2nd LT. This possibly relied on synergy between phages and antibiotics. This combined therapy was safe