Salmonella enterica subsp. enterica virulence potential can be linked to higher survival within a dynamic in vitro human gastrointestinal model.

Salmonella enterica subsp. enterica is one of the leading causes of human foodborne infections and several outbreaks are now associated with the consumption of fresh fruit and vegetables. This study aims at evaluating whether Salmonella virulence can be linked to an enhanced ability to survive successive digestive environments. Thirteen S. enterica strains were selected according to high and low virulence phenotypes. Lettuce inoculated separately with each S. enterica strain was used as food matrix in the TNO gastrointestinal model (TIM-1) of the human upper gastrointestinal tract. During the passage in the stomach, counts determined using PMA-qPCR were 2-5 logs higher than the cultivable counts for all strains indicating the presence of viable but non-cultivable cells. Bacterial growth was observed in the duodenum compartment after 180 min for all but one strain and growth continued into the ileal compartment. After passage through the simulated gastrointestinal tract, both virulent and avirulent S. enterica strains survived but high virulence strains had a significantly (p = 0.004) better average survival rate (1003 %-3753 %) than low virulence strains (from 25 % to 3730%). The survival rates of S. enterica strains could be linked to the presence of genes associated with acid and bile resistance and their predicted products. The presence of single nucleotide polymorphisms may also impact the function of virulence associated genes and play a role in the resulting phenotype. These data provide an understanding of the relationship between measured virulence potential and survival of S. enterica during dynamic simulated gastrointestinal transit.

Model-Informed Drug Development in Pulmonary Delivery: Semimechanistic Pharmacokinetic-Pharmacodynamic Modeling for Evaluation of Treatments against Chronic Pseudomonas aeruginosa Lung Infections.

Antibiotic resistance is a major public health threat worldwide, and among others, about 80% of cystic fibrosis patients have chronic Pseudomonas aeruginosa (PA) lung infection resistant to many current antibiotics. Novel treatment strategies are therefore urgently needed. For lung infections, direct delivery of treatments to the site of action in the airway can achieve a higher local concentration with minimal systemic exposure and hence avoid risks of unwanted systemic adverse effects. Previously, a rat preclinical disease model for PA chronic lung infections has been reported. However, the role of this disease model in the development of new treatment has not been thoroughly evaluated. In this study, tobramycin (TOB) was used as a model antibiotic to evaluate the application of this preclinical disease model for PA treatments. The obtained data were used for pharmacokinetic-pharmacodynamic (PKPD) modeling. Plasma samples following pulmonary delivery of TOB via different dosing methods as well as growth and efficacy data from the chronic lung infection disease model following TOB treatments were collected for analysis and modeling. The developed PKPD model incorporates a semimechanistic description on biofilm development in chronic infections to allow the evaluation of drug action on bacteria in different states (i.e., planktonic, biofilm, and latent) and describes the available data from the efficacy study. The PKPD model can be used to support the application of the preclinical lung infection disease model by providing a quantitative description of the drug exposure-response relationship and a mechanistic platform to integrate all available PK and PKPD data with predictive capacity. With the support of appropriate experimental designs, the model can be further extended for other applications to, for instance, study the transition of bacteria between states and describe drug actions on biofilms.

Genes Required for Free Phage Production are Essential for Pseudomonas aeruginosa Chronic Lung Infections.

The opportunistic pathogen Pseudomonas aeruginosa causes chronic lung infection in patients with cystic fibrosis. The Liverpool Epidemic Strain LESB58 is highly resistant to antibiotics, transmissible, and associated with increased morbidity and mortality. Its genome contains 6 prophages and 5 genomic islands. We constructed a polymerase chain reaction (PCR)-based signature-tagged mutagenesis library of 9216 LESB58 mutants and screened the mutants in a rat model of chronic lung infection. A total of 162 mutants were identified as defective for in vivo maintenance, with 11 signature-tagged mutagenesis mutants having insertions in prophage and genomic island genes. Many of these mutants showed both diminished virulence and reduced phage production. Transcription profiling by quantitative PCR and RNA-Seq suggested that disruption of these prophages had a widespread trans-acting effect on the transcriptome. This study demonstrates that temperate phages play a pivotal role in the establishment of infection through modulation of bacterial host gene expression.

Comparative genomics and biological characterization of sequential Pseudomonas aeruginosa isolates from persistent airways infection.

BACKGROUND: Pseudomonas aeruginosa establishes life-long chronic airway infections in cystic fibrosis (CF) patients. As the disease progresses, P. aeruginosa pathoadaptive variants are distinguished from the initially acquired strain. However, the genetic basis and the biology of host-bacteria interactions leading to a persistent lifestyle of P. aeruginosa are not understood. As a model system to study long term and persistent CF infections, the P. aeruginosa RP73, isolated 16.9 years after the onset of airways colonization from a CF patient, was investigated. Comparisons with strains RP1, isolated at the onset of the colonization, and clonal RP45, isolated 7 years before RP73 were carried out to better characterize genomic evolution of P. aeruginosa in the context of CF pathogenicity. RESULTS: Virulence assessments in disease animal model, genome sequencing and comparative genomics analysis were performed for clinical RP73, RP45, RP1 and prototype strains. In murine model, RP73 showed lower lethality and a remarkable capability of long-term persistence in chronic airways infection when compared to other strains. Pathological analysis of murine lungs confirmed advanced chronic pulmonary disease, inflammation and mucus secretory cells hyperplasia. Genomic analysis predicted twelve genomic islands in the RP73 genome, some of which distinguished RP73 from other prototype strains and corresponded to regions of genome plasticity. Further, comparative genomic analyses with sequential RP isolates showed signatures of pathoadaptive mutations in virulence factors potentially linked to the development of chronic infections in CF. CONCLUSIONS: The genome plasticity of P. aeruginosa particularly in the RP73 strain strongly indicated that these alterations may form the genetic basis defining host-bacteria interactions leading to a persistent lifestyle in human lungs.

Requirement of the Pseudomonas aeruginosa CbrA sensor kinase for full virulence in a murine acute lung infection model.

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of respiratory tract and other nosocomial infections. The sensor kinase CbrA is a central regulator of carbon and nitrogen metabolism and in vitro also regulates virulence-related processes in P. aeruginosa. Here, we investigated the role of CbrA in two murine models of infection. In both peritoneal infections in leukopenic mice and lung infection models, the cbrA mutant was less virulent since substantially larger numbers of cbrA mutant bacteria were required to cause the same level of infection as wild-type or complemented bacteria. In contrast, in the chronic rat lung model the cbrA mutant grew and persisted as well as the wild type, indicating that the decrease of in vivo virulence of the cbrA mutant did not result from growth deficiencies on particular carbon substrates observed in vitro. In addition, a mutant in the cognate response regulator CbrB showed no defect in virulence in the peritoneal infection model, ruling out the involvement of certain alterations of virulence properties in the cbrA mutant including defective swarming motility, increased biofilm formation, and cytotoxicity, since these alterations are controlled through CbrB. Further investigations indicated that the mutant was more susceptible to uptake by phagocytes in vitro, resulting in greater overall bacterial killing. Consistent with the virulence defect, it took a smaller number of Dictyostelium discoideum amoebae to kill the cbrA mutant than to kill the wild type. Transcriptional analysis of the cbrA mutant during D. discoideum infection led to the conclusion that CbrA played an important role in the iron metabolism, protection of P. aeruginosa against oxidative stress, and the regulation of certain virulence factors.

Complete genome sequence of multidrug-resistant Enterobacter roggenkampii 0-E.

Here, we present the complete 4.77 Mb genome of Enterobacter roggenkampii 0-E assembled with Oxford Nanopore long reads. This genome harbors 19 antimicrobial resistance genes, including ramA and marA decreasing permeability to carbapenems. This genome adds novel knowledge on emerging multidrug resistance in the Enterobacter cloacae species complex.

Genomic and Phenotypic Analysis of Salmonella enterica Bacteriophages Identifies Two Novel Phage Species.

Bacteriophages (phages) are potential alternatives to chemical antimicrobials against pathogens of public health significance. Understanding the diversity and host specificity of phages is important for developing effective phage biocontrol approaches. Here, we assessed the host range, morphology, and genetic diversity of eight Salmonella enterica phages isolated from a wastewater treatment plant. The host range analysis revealed that six out of eight phages lysed more than 81% of the 43 Salmonella enterica isolates tested. The genomic sequences of all phages were determined. Whole-genome sequencing (WGS) data revealed that phage genome sizes ranged from 41 to 114 kb, with GC contents between 39.9 and 50.0%. Two of the phages SB13 and SB28 represent new species, Epseptimavirus SB13 and genera Macdonaldcampvirus, respectively, as designated by the International Committee for the Taxonomy of Viruses (ICTV) using genome-based taxonomic classification. One phage (SB18) belonged to the Myoviridae morphotype while the remaining phages belonged to the Siphoviridae morphotype. The gene content analyses showed that none of the phages possessed virulence, toxin, antibiotic resistance, type I-VI toxin-antitoxin modules, or lysogeny genes. Three (SB3, SB15, and SB18) out of the eight phages possessed tailspike proteins. Whole-genome-based phylogeny of the eight phages with their 113 homologs revealed three clusters A, B, and C and seven subclusters (A1, A2, A3, B1, B2, C1, and C2). While cluster C1 phages were predominantly isolated from animal sources, cluster B contained phages from both wastewater and animal sources. The broad host range of these phages highlights their potential use for controlling the presence of S. enterica in foods.

Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 µM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Evolution and host-specific adaptation of Pseudomonas aeruginosa.

The major human bacterial pathogen Pseudomonas aeruginosa causes multidrug-resistant infections in people with underlying immunodeficiencies or structural lung diseases such as cystic fibrosis (CF). We show that a few environmental isolates, driven by horizontal gene acquisition, have become dominant epidemic clones that have sequentially emerged and spread through global transmission networks over the past 200 years. These clones demonstrate varying intrinsic propensities for infecting CF or non-CF individuals (linked to specific transcriptional changes enabling survival within macrophages); have undergone multiple rounds of convergent, host-specific adaptation; and have eventually lost their ability to transmit between different patient groups. Our findings thus explain the pathogenic evolution of P. aeruginosa and highlight the importance of global surveillance and cross-infection prevention in averting the emergence of future epidemic clones.

Genetic diversity of Salmonella enterica isolated over 13 years from raw California almonds and from an almond orchard.

A comparative genomic analysis was conducted for 171 Salmonella isolates recovered from raw inshell almonds and raw almond kernels between 2001 and 2013 and for 30 Salmonella Enteritidis phage type (PT) 30 isolates recovered between 2001 and 2006 from a 2001 salmonellosis outbreak-associated almond orchard. Whole genome sequencing was used to measure the genetic distance among isolates by single nucleotide polymorphism (SNP) analyses and to predict the presence of plasmid DNA and of antimicrobial resistance (AMR) and virulence genes. Isolates were classified by serovars with Parsnp, a fast core-genome multi aligner, before being analyzed with the CFSAN SNP Pipeline (U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition). Genetically similar (≤18 SNPs) Salmonella isolates were identified among several serovars isolated years apart. Almond isolates of Salmonella Montevideo (2001 to 2013) and Salmonella Newport (2003 to 2010) differed by ≤9 SNPs. Salmonella Enteritidis PT 30 isolated between 2001 and 2013 from survey, orchard, outbreak, and clinical samples differed by ≤18 SNPs. One to seven plasmids were found in 106 (62%) of the Salmonella isolates. Of the 27 plasmid families that were identified, IncFII and IncFIB plasmids were the most predominant. AMR genes were identified in 16 (9%) of the survey isolates and were plasmid encoded in 11 of 16 cases; 12 isolates (7%) had putative resistance to at least one antibiotic in three or more drug classes. A total of 303 virulence genes were detected among the assembled genomes; a plasmid that harbored a combination of pef, rck, and spv virulence genes was identified in 23% of the isolates. These data provide evidence of long-term survival (years) of Salmonella in agricultural environments.

Draft genome sequence of Pseudomonas sp. ER28, a cyclohexane pentanoic acid degrader isolated from oil sands process-affected water from Alberta, Canada.

We report the draft genome sequence of Pseudomonas sp. ER28, capable of utilizing the model naphthenic acid, cyclohexane pentanoic acid, as its sole carbon source. It was recovered from oil sands process-affected water containing cyclic and acyclic naphthenic acids. The genome size is 5.7 Mbp, and the G + C content is 60%.

Draft genome sequences of six Pseudomonas spp. and one Rheinheimera sp. isolated from oil sands process-affected water from Alberta, Canada.

We report the draft genomes of seven bacterial strains (six Pseudomonas spp. and one Rheinheimera sp.) isolated from environmental water samples from oil sands tailings ponds that have accumulated a wide variety of organic compounds, salts and metals.

Complete genome sequences of agricultural azole-resistant Penicillium rubens encoding CYP51A and ERG11 paralogues.

Azoles are major antifungals in agriculture and medicine. However, the surge of intrinsic azole resistance is critical for public health. Here, we present the complete long-read sequencing of three azole-resistant Penicillium rubens from food crops. The presence of CYP51A and ERG11 paralogues was confirmed, as in other azole-resistant P. rubens.

Rapid on-site detection of harmful algal blooms: real-time cyanobacteria identification using Oxford Nanopore sequencing.

With the increasing occurrence and severity of cyanobacterial harmful algal blooms (cHAB) at the global scale, there is an urgent need for rapid, accurate, accessible, and cost-effective detection tools. Here, we detail the RosHAB workflow, an innovative, in-the-field applicable genomics approach for real-time, early detection of cHAB outbreaks. We present how the proposed workflow offers consistent taxonomic identification of water samples in comparison to traditional microscopic analyses in a few hours and discuss how the generated data can be used to deepen our understanding on cyanobacteria ecology and forecast HABs events. In parallel, processed water samples will be used to iteratively build the International cyanobacterial toxin database (ICYATOX; http://icyatox.ibis.ulaval.ca) containing the analysis of novel cyanobacterial genomes, including phenomics and genomics metadata. Ultimately, RosHAB will (1) improve the accuracy of on-site rapid diagnostics, (2) standardize genomic procedures in the field, (3) facilitate these genomics procedures for non-scientific personnel, and (4) identify prognostic markers for evidence-based decisions in HABs surveillance.

Complete genome sequences of three Pseudomonas aeruginosa isolates with phenotypes of polymyxin B adaptation and inducible resistance.

Clinical "superbug" isolates of Pseudomonas aeruginosa were previously observed to be resistant to several antibiotics, including polymyxin B, and/or to have a distinct, reproducible adaptive polymyxin resistance phenotype, identified by observing "skipped" wells (appearance of extra turbid wells) during broth microdilution testing. Here we report the complete assembled draft genome sequences of three such polymyxin resistant P. aeruginosa strains (9BR, 19BR, and 213BR).

Draft Genome Sequences of Two Clostridium botulinum Group II Strains Carrying Phage-Like Plasmids.

Clostridium botulinum is responsible for botulism, a potentially lethal foodborne intoxication. Here, we report the draft genome sequences of C. botulinum group II strains 202F (serotype F) and Hazen (serotype E). The genomes share many similarities, including multiple mobile genetic elements.

Complete Genome Sequence of a Pseudomonas Species Isolated from Tailings Pond Water in Alberta, Canada.

We report the complete genome sequence of strain OST1909, belonging to a Pseudomonas species. The genome size is 6,306,352 bp, with a G+C content of 59.6%. The isolate was recovered from oil sands process-affected water (OSPW), despite the numerous toxic compounds that accumulate in oil sands tailings ponds.

Design and Evaluation of New Quinazolin-4(3H)-one Derived PqsR Antagonists as Quorum Sensing Quenchers in Pseudomonas aeruginosa.

P. aeruginosa (PA) continues to pose a threat to global public health due to its high levels of antimicrobial resistance (AMR). The ongoing AMR crisis has led to an alarming shortage of effective treatments for resistant microbes, and hence there is a pressing demand for the development of novel antimicrobial interventions. The potential use of antivirulence therapeutics to tackle bacterial infections has attracted considerable attention over the past decades as they hamper the pathogenicity of target microbes with reduced selective pressure, minimizing the emergence of resistance. One such approach is to interfere with the PA pqs quorum sensing system which upon the interaction of PqsR, a Lys-R type transcriptional regulator, with its cognate signal molecules 4-hydroxy-2-heptylquinoline (HHQ) and 2-heptyl-3-hydroxy-4-quinolone (PQS), governs multiple virulence traits and host-microbe interactions. In this study, we report the hit identification and optimization of PqsR antagonists using virtual screening coupled with whole cell assay validation. The optimized hit compound 61 ((R)-2-(4-(3-(6-chloro-4-oxoquinazolin-3(4H)-yl)-2-hydroxypropoxy)phenyl)acetonitrile) was found to inhibit the expression of the PA PpqsA promoter controlled by PqsR with an IC50 of 1 µM. Using isothermal titration calorimetry, a Kd of 10 nM for the PqsR ligand binding domain (PqsRLBD) was determined for 61. Furthermore, the crystal structure of 61 with PqsRLBD was attained with a resolution of 2.65 Å. Compound 61 significantly reduced levels of pyocyanin, PQS, and HHQ in PAO1-L, PA14 lab strains and PAK6085 clinical isolate. Furthermore, this compound potentiated the effect of ciprofloxacin in early stages of biofilm treatment and in Galleria mellonella infected with PA. Altogether, this data shows 61 as a potent PqsR inhibitor with potential for hit to lead optimization toward the identification of a PA QS inhibitor which can be advanced into preclinical development.

Transmission, adaptation and geographical spread of the Pseudomonas aeruginosa Liverpool epidemic strain.

The Liverpool epidemic strain (LES) is an important transmissible clonal lineage of Pseudomonas aeruginosa that chronically infects the lungs of people with cystic fibrosis (CF). Previous studies have focused on the genomics of the LES in a limited number of isolates, mostly from one CF centre in the UK, and from studies highlighting identification of the LES in Canada. Here we significantly extend the current LES genome database by genome sequencing 91 isolates from multiple CF centres across the UK, and we describe the comparative genomics of this large collection of LES isolates from the UK and Canada. Phylogenetic analysis revealed that the 145 LES genomes analysed formed a distinct clonal lineage when compared with the wider P. aeruginosa population. Notably, the isolates formed two clades: one associated with isolates from Canada, and the other associated with UK isolates. Further analysis of the UK LES isolates revealed clustering by clinic geography. Where isolates clustered closely together, the association was often supported by clinical data linking isolates or patients. When compared with the earliest known isolate, LESB58 (from 1988), many UK LES isolates shared common loss-of-function mutations, such as in genes gltR and fleR. Other loss-of-function mutations identified in previous studies as common adaptations during CF chronic lung infections were also identified in multiple LES isolates. Analysis of the LES accessory genome (including genomic islands and prophages) revealed variations in the carriage of large genomic regions, with some evidence for shared genomic island/prophage complement according to clinic location. Our study reveals divergence and adaptation during the spread of the LES, within the UK and between continents.