99 results found
da Silva GC, Rosa J, de Castro A, et al., 2023, Identification of novel small RNAs in extracellular vesicles produced by Actinobacillus pleuropneumoniae, Frontiers in Microbiology, ISSN: 1664-302X
Abouelhadid S, Atkins ER, Kay EJ, et al., 2023, Development of a novel glycoengineering platform for the rapid production of conjugate vaccines, Microbial Cell Factories, Vol: 22, Pages: 1-13, ISSN: 1475-2859
Conjugate vaccines produced either by chemical or biologically conjugation have been demonstrated to be safe and efficacious in protection against several deadly bacterial diseases. However, conjugate vaccine assembly and production have several shortcomings which hinders their wider availability. Here, we developed a tool, Mobile-element Assisted Glycoconjugation by Insertion on Chromosome, MAGIC, a novel biotechnological platform that overcomes the limitations of the current conjugate vaccine design method(s). As a model, we focused our design on a leading bioconjugation method using N-oligosaccharyltransferase (OTase), PglB. The installation of MAGIC led to at least twofold increase in glycoconjugate yield via MAGIC when compared to conventional N-OTase based bioconjugation method(s). Then, we improved MAGIC to (a) allow rapid installation of glycoengineering component(s), (b) omit the usage of antibiotics, (c) reduce the dependence on protein induction agents. Furthermore, we show the modularity of the MAGIC platform in performing glycoengineering in bacterial species that are less genetically tractable than the commonly used Escherichia coli. The MAGIC system promises a rapid, robust and versatile method to develop vaccines against serious bacterial pathogens. We anticipate the utility of the MAGIC platform could enhance vaccines production due to its compatibility with virtually any bioconjugation method, thus expanding vaccine biopreparedness toolbox.
Nahar N, Tram G, Jen FE-C, et al., 2023, Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions, Nucleic Acids Research, Vol: 51, Pages: 3240-3260, ISSN: 0305-1048
Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.
Li Y, Bosse J, Stringer O, et al., 2023, Detection of novel Actinobacillus pleuropneumoniae serovars by multiplex PCR: a cautionary tale, Microbiology Spectrum, Vol: 11, Pages: 1-2, ISSN: 2165-0497
Di Blasio S, Clarke M, Hind CK, et al., 2023, Bolaamphiphile analogues of 12-bis-THA Cl2 are potent antimicrobial therapeutics with distinct mechanisms of action against bacterial, mycobacterial, and fungal pathogens., mSphere, Vol: 8, Pages: 1-20, ISSN: 2379-5042
12-Bis-THA Cl2 [12,12'-(dodecane-1,12-diyl)-bis-(9-amino-1,2,3,4-tetrahydroacridinium) chloride] is a cationic bolalipid adapted from dequalinium chloride (DQC), a bactericidal anti-infective indicated for bacterial vaginosis (BV). Here, we used a structure-activity-relationship study to show that the factors that determine effective killing of bacterial, fungal, and mycobacterial pathogens differ, to generate new analogues with a broader spectrum of activity, and to identify synergistic relationships, most notably with aminoglycosides against Acinetobacter baumannii and Pseudomonas aeruginosa, where the bactericidal killing rate was substantially increased. Like DQC, 12-bis-THA Cl2 and its analogues accumulate within bacteria and fungi. More hydrophobic analogues with larger headgroups show reduced potential for DNA binding but increased and broader spectrum antibacterial activity. In contrast, analogues with less bulky headgroups and stronger DNA binding affinity were more active against Candida spp. Shortening the interconnecting chain, from the most lipophilic twelve-carbon chain to six, improved the selectivity index against Mycobacterium tuberculosis in vitro, but only the longer chain analogue was therapeutic in a Galleria mellonella infection model, with the shorter chain analogue exacerbating the infection. In vivo therapy of Escherichia coli ATCC 25922 and epidemic methicillin-resistant Staphylococcus aureus 15 (EMRSA-15) infections in Galleria mellonella was also achieved with longer-chain analogues, as was therapy for an A. baumannii 17978 burn wound infection with a synergistic combination of bolaamphiphile and gentamicin. The present study shows how this class of bolalipids may be adapted further to enable a wider range of potential applications. IMPORTANCE While we face an acute threat from antibiotic resistant bacteria and a lack of new classes of antibiotic, there are many effective antimicrobials which have limited application due to concerns regar
Cunha da Silva G, Rossi CC, Nogueira Rosa J, et al., 2022, Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 13, ISSN: 1664-302X
The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.
Stringer OW, Li Y, Bosse JT, et al., 2022, Rapid detection of A. pleuropneumoniae from clinical samples using recombinase polymerase amplification, Frontiers in Veterinary Science, Vol: 9, ISSN: 2297-1769
Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine pleuropneumonia, resulting in high economic impact worldwide. There are currently 19 known serovars of APP, with different ones being predominant in specific geographic regions. Outbreaks of pleuropneumonia, characterized by sudden respiratory difficulties and high mortality, can occur when infected pigs are brought into naïve herds, or by those carrying different serovars. Good biosecurity measures include regular diagnostic testing for surveillance purposes. Current gold standard diagnostic techniques lacksensitivity (bacterial culture), require expensive thermocycling machinery (PCR) and are time consuming (culture and PCR). Here we describe the development of an isothermal point-of-care diagnostic test - utilizing recombinase polymerase amplification (RPA) for the detection of APP,targeting the species-specific apxIVA gene. Our APP-RPA diagnostic test achieved a sensitivity of 10 copies/µL using a strain of APP serovar 8, which is the most prevalent serovar in the UK. Additionally, our APP-RPA assay achieved a clinical sensitivity and specificity of 84.3% and 100%, respectively,across 61 extracted clinical samples obtained from farms located in England and Portugal. Using a small subset (n = 14) of the lung tissue samples, we achieved a clinical sensitivity and specificity of 76.9% and 100%, respectively) using lung imprints made on FTA cards tested directly in the APP- RPA reaction. Our results demonstrate that our APP-RPA assay enables a suitable rapid and sensitive screening tool for this important veterinary pathogen.
Stringer O, Li Y, Bosse J, et al., 2022, JMM Profile: Actinobacillus pleuropneumoniae: a major cause of lung disease in pigs but difficult to control and eradicate, Journal of Medical Microbiology, Vol: 71, ISSN: 0022-2615
The Gram-negative bacterium Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in pigs, its only known natural host. Typical symptoms of peracute disease include fever, apathy and anorexia, and time from infection to death may only be 6 h. Severe lung lesions result from presence of one or two of the ApxI-III toxins. Control is through good husbandry practice, vaccines and antibiotic use. Culture and presence of the species-specific apxIV gene by PCR confirms diagnosis, and identification of serovar, of which 19 are known, informs on appropriate vaccine use and epidemiology.
Srijuntongsiri G, Mhoowai A, Samngamnim S, et al., 2022, Novel DNA Markers for Identification of Actinobacillus pleuropneumoniae, Microbiology Spectrum, Vol: 10, ISSN: 2165-0497
Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-spe
da Silva G, Gonçalves O, Rosa J, et al., 2022, Mobile genetic elements drive antimicrobial resistance gene spread in Pasteurellaceae species, Frontiers in Microbiology, Vol: 12, Pages: 1-14, ISSN: 1664-302X
Mobile genetic elements (MGEs) and antimicrobial resistance (AMR) drive important ecological relationships in microbial communities and pathogen-host interaction. In this study, we investigated the resistome-associated mobilome in 345 publicly available Pasteurellaceae genomes, a large family of Gram-negative bacteria including major human and animal pathogens. We generated a comprehensive dataset of the mobilome integrated into genomes, including 10,820 insertion sequences, 2,939 prophages, and 43 integrative and conjugative elements. Also, we assessed plasmid sequences of Pasteurellaceae. Our findings greatly expand the diversity of MGEs for the family, including a description of novel elements. We discovered that MGEs are comparable and dispersed across species and that they also co-occur in genomes, contributing to the family's ecology via gene transfer. In addition, we investigated the impact of these elements in the dissemination and shaping of AMR genes. A total of 55 different AMR genes were mapped to 721 locations in the dataset. MGEs are linked with 77.6% of AMR genes discovered, indicating their important involvement in the acquisition and transmission of such genes. This study provides an uncharted view of the Pasteurellaceae by demonstrating the global distribution of resistance genes linked with MGEs.
Langford P, Stringer O, Li Y, et al., 2021, Application of the MISTEACHING(S) disease susceptibility framework to Actinobacillus pleuropneumoniae to identify research gaps: an exemplar of a veterinary pathogen, Animal Health Research Reviews, Vol: 22, Pages: 120-135, ISSN: 1466-2523
Historically, the MISTEACHING (microbiome, immunity, sex, temperature, environment, age, chance, history, inoculum, nutrition, genetics) framework to describe the outcome of host-pathogen interaction, has been applied to human pathogens. Here, weshow, using Actinobacillus pleuropneumoniaeas an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen enabling the identification of major research gaps, and the formulation of hypotheses whose study will lead to a greater understanding of pathogenic mechanisms, and/or improved prevention/therapeutic measures. We also suggest that the MISTEACHING framework should be extended with the inclusion of a “strain” category, to become MISTEACHINGS. We conclude that the MISTEACHINGS framework can be applied to veterinary pathogens, whether they be bacteria, viruses, or parasites, and hope to stimulate others to use it to identify research gaps and to formulate hypotheses worthy of study with their own pathogens.
Bossé JT, Li Y, Leanse LG, et al., 2021, Rationally designed mariner vectors for functional genomic analysis of Actinobacillus pleuropneumoniae and other Pasteurellaceae species by transposon-directed insertion-site sequencing (TraDIS), Animal Diseases, Vol: 1
Comprehensive identification of conditionally essential genes requires efficient tools for generating high-density transposon libraries that, ideally, can be analysed using next-generation sequencing methods such as Transposon Directed Insertion-site Sequencing (TraDIS). The Himar1 (mariner) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide, and this transposon has been used for mutagenesis of a wide variety of bacteria. However, plasmids for mariner delivery do not necessarily work well in all bacteria. In particular, there are limited tools for functional genomic analysis of Pasteurellaceae species of major veterinary importance, such as swine and cattle pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida, respectively. Here, we developed plasmids, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), that allow delivery of mariner into both these pathogens, but which should also be applicable to a wider range of bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating mariner mutant libraries in both A. pleuropneumoniae and P. multocida that showed a near random distribution of insertions around the respective chromosomes as detected by TraDIS. A preliminary screen of 5000 mutants each identified 8 and 14 genes, respectively, that are required for growth under anaerobic conditions. Future high-throughput screening of the generated libraries will facilitate identification of mutants required for growth under different conditions, including in vivo, highlighting key virulence factors and pathways that can be exploited for development of novel therapeutics and vaccines.
Bosse J, Li Y, Cohen LM, et al., 2021, Complete genome for Actinobacillus pleuropneumoniae serovar 8 reference strain 405: comparative analysis with draft genomes for different laboratory stock cultures indicates little genetic variation, Microbial Genomics, Vol: 7, ISSN: 2057-5858
We report here the complete genome sequence of the widely studied Actinobacillus pleuropneumoniae serovar 8 reference strain 405, generated using the Pacific Biosciences (PacBio) RS II platform. Furthermore, we compared draft sequences generated by Illumina sequencing of six stocks of this strain, including the same original stock used to generate the PacBio sequence, held in different countries and found little genetic variation, with only three SNPs identified, all within the degS gene. However, sequences of two small plasmids, pARD3079 and p405tetH, detected by Illumina sequencing of the draft genomes were not identified in the PacBio sequence of the reference strain.
Cohen LM, Bossé JT, Stegger M, et al., 2021, Comparative genome sequence analysis of actinobacillus pleuropneumoniae serovar 8 isolates from Norway, Denmark, and the United Kingdom indicates distinct phylogenetic lineages and differences in distribution of antimicrobial resistance genes, Frontiers in Microbiology, Vol: 12, Pages: 1-13, ISSN: 1664-302X
Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a disease of major impact on pig health, welfare, and productivity globally. Serovar 8 (APP) is the predominant clinical serovar in Norway and the United Kingdom (UK), and has been isolated from clinical cases in Denmark. The primary objective of this study was to characterize the genetic variability of isolates of A. pleuropneumoniae APP8 in the Norwegian population. The secondary objectives were to determine the within-host variability of APP8; to compare the APP8 bacterial populations in Norway, Denmark, and the UK, including antimicrobial resistance (AMR) gene profiles and to assess the effect of national differences in antimicrobial drug use and restricted animal movement on the occurrence of resistance. Isolates of APP8 from the UK (n=67), Denmark (n=22), and Norway (n=123) collected between 1983 and 2020 were compared using whole genome sequencing. To investigate genetic variability within individual hosts, an additional 104 APP8 isolates from the lungs of six Norwegian pigs were compared. Very low within-host variation was observed (≤ 2 single nucleotide polymorphisms). The phylogeny of 123 Norwegian APP8 isolates from 76 herds revealed some within-herd genetic variation, but substantial geographical clustering. When inferring the relatedness of the three international APP8 collections, the topology highlighted the existence of two distinct monophyletic branches characterized by the Norwegian and UK isolates, respectively. Three Danish isolates were scattered across the UK branch, whereas the remaining 19 Danish isolates clustered in two monophyletic groups nested in the Norwegian branch. Coalescence analysis, performed to estimate the divergences from a common ancestor, indicated a last common ancestor several centuries ago. The phylogenetic analyses also revealed striking differences in occurrence of AMR genes, as these were 23-times more prevalent among the UK isolates
Stringer O, Bosse J, Lacoutre S, et al., 2021, Rapid detection and typing of Actinobacillus pleuropneumoniae serovars directly from clinical samples: combining FTA® card technology with multiplex-PCR, Frontiers in Veterinary Science, Vol: 8, Pages: 1-9, ISSN: 2297-1769
Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is highly contagious and responsible for high morbidity, mortality and economic losses in the swine industry worldwide, but quick serotyping and diagnosis are still not widely available. In this study, we sought to validate the use of Whatman FTA® cards for collection and processing of A. pleuropneumoniae isolates, or porcine lung tissue samples, for direct use in diagnostic multiplex PCRs.We have optimized the processing of 3 mm discs punched from FTA® cards loaded with cultured A. pleuropneumoniae, or imprinted on lesioned regions of lung tissue, with only three distilled water washes before addition into our APP-mPCR assay for rapid, low-cost identification and serotyping. DNA captured on FTA® cards generated the same diagnostic PCR results as DNA extracted using commercial kits for 85 A. pleuropneumoniae clinical isolate cultures and 22 lung samples. Additionally, bacterial DNA bound to FTA® cards was detectable by PCR after six months of storage at 37°C.This study provides simple, efficient, rapid and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae.
Stringer O, Bosse J, Lacouture S, et al., 2021, Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars, Veterinary Microbiology, Vol: 255, ISSN: 0378-1135
Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-13 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13-14 and 16-19.
Gottschalk M, Lacouture S, Blackwell T, et al., 2021, Long-chain LPS-based enzyme-linked immunosorbent assay to detect swine herds infected by Actinobacillus pleuropneumoniae serotype 17, Canadian Veterinary Journal, Vol: 62, Pages: 62-65, ISSN: 0008-5286
Actinobacillus pleuropneumoniae serotype 17, one of the two most recent serotypes described, has been isolated from diseased pigs in North America. Yet, no serological test for surveillance has been developed so far. An enzyme-linked immunosorbent assay (ELISA) using the long-chain lipopolysaccharide antigen (LC-LPS) of this serotype is described. As predicted by previous genetic data on the O-antigen locus, cross reactions were observed between this serotype and serotypes 3, 6, 8, and 15. Although animals infected by serotype 17 would be detected using the current serotype 3 LC-LPS ELISA, better results may be obtained when plates are coated with the antigen purified from the homologous serotype.
Crispim JS, da Silva TF, Sanches NM, et al., 2020, Serovar-dependent differences in Hfq-regulated phenotypes in Actinobacillus pleuropneumoniae., Pathogens and Disease, Vol: 78, Pages: 1-12, ISSN: 2049-632X
The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions, and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen A. pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 hour time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes.
Gottschalk M, Lacouture S, Blackwell T, et al., 2020, Long-chain LPS-based ELISA to detect herds infected by Actinobacillus pleuropneumoniae serotype 17, Canadian Veterinary Journal, ISSN: 0008-5286
Hau SJ, Luan S-L, Loving CL, et al., 2020, Evaluation of the recombinant proteins RlpB and VacJ as a vaccine for protection against<i>Glaesserella parasuis</i>in pigs, BMC VETERINARY RESEARCH, Vol: 16
Eberle KC, Hau SJ, Luan S-L, et al., 2020, Generation and evaluation of a glaesserella (Haemophilus) parasuis capsular mutant, Infection and Immunity, Vol: 88, Pages: 1-11, ISSN: 0019-9567
Glaesserella (Haemophilus) parasuis is a commensal bacterium of the upper respiratory tract in pigs and also the causative agent of Glässer’s disease, which causes significant morbidity and mortality in pigs worldwide. Isolates are characterized into 15 serovars by their capsular polysaccharide, which has shown a correlation with isolate pathogenicity. To investigate the role the capsule plays in G. parasuis virulence and host interaction, a capsule mutant of the serovar 5 strain HS069 was generated (HS069Δcap) through allelic exchange following natural transformation. HS069Δcap was unable to cause signs of systemic disease during a pig challenge study and had increased sensitivity to complement killing and phagocytosis by alveolar macrophages. Compared with the parent strain, HS069Δcap produced more robust biofilm and adhered equivalently to 3D4/31 cells; however, it was unable to persistently colonize the nasal cavity of inoculated pigs, with all pigs clearing HS069Δcap by 5 days postchallenge. Our results indicate the importance of the capsular polysaccharide to G. parasuis virulence as well as nasal colonization in pigs.
Christensen H, Bosse J, Angen Ø, et al., 2020, Immunological and molecular techniques used for determination of serotypes in Pasteurellaceae, Methods in Microbiology, ISSN: 0580-9517
Wang S, Yin B, Yu L, et al., 2020, Overexpression of AmpC promotes bacteriophage lysis of ampicillin- resistant Escherichia coli, Frontiers in Microbiology, Vol: 10, ISSN: 1664-302X
Infections caused by antibiotic-resistant Escherichia coli are a threat to human and animal health globally. Phage therapy has made great progress for the treatment of drug-resistant infections, but it is still unclear whether E. coli resistance to antibiotics could change the lysis ability of phages. In this study, we demonstrate that over expression of AmpC, an important β-lactamase for ampicillin resistance, promotes lysis of E. coli by phage utilizing OmpA as a receptor. E. coli strains expressing more AmpC showed higher levels of OmpA, an E. coli outer membrane protein known to serve as a receptor for T-even phages, which resulted in increased adsorption and lysis by the phage tested in this study. These data demonstrate that increased ampicillin resistance can increase the sensitivity of E. coli to some lytic phage, which provides evidence for the feasibility of synergistic application of phage and antibiotics.
Bosse JT, Li Y, Fernandez Crespo R, et al., 2020, Draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319), two NAD-dependent bacterial species found in the respiratory tract of pigs, Microbiology Resource Announcements, Vol: 9, Pages: 1-3, ISSN: 2576-098X
We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2CT) and Actinobacillus porcinus (NM319T). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology.
Weinert LA, Chaudhuri RR, Wang J, et al., 2019, Publisher Correction: Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis (vol 6, 6740, 2015), Nature Communications, Vol: 10, ISSN: 2041-1723
Wileman TM, Weinert LA, Howell KJ, et al., 2019, Pathotyping the zoonotic pathogen streptococcus suis: Novel genetic markers to differentiate invasive disease-associated isolates from non-disease-associated isolates from England and Wales, Journal of Clinical Microbiology, Vol: 57, ISSN: 0095-1137
Streptococcus suis is one of the most important zoonotic bacterial pathogens of pigs, causing significant economic losses to the global swine industry. S. suis is also a very successful colonizer of mucosal surfaces, and commensal strains can be found in almost all pig populations worldwide, making detection of the S. suis species in asymptomatic carrier herds of little practical value in predicting the likelihood of future clinical relevance. The value of future molecular tools for surveillance and preventative health management lies in the detection of strains that genetically have increased potential to cause disease in presently healthy animals. Here we describe the use of genome-wide association studies to identify genetic markers associated with the observed clinical phenotypes (i) invasive disease and (ii) asymptomatic carriage on the palatine tonsils of pigs on UK farms. Subsequently, we designed a multiplex PCR to target three genetic markers that differentiated 115 S. suis isolates into disease-associated and non-disease-associated groups, that performed with a sensitivity of 0.91, a specificity of 0.79, a negative predictive value of 0.91, and a positive predictive value of 0.79 in comparison to observed clinical phenotypes. We describe evaluation of our pathotyping tool, using an out-of-sample collection of 50 previously uncharacterized S. suis isolates, in comparison to existing methods used to characterize and subtype S. suis isolates. In doing so, we show our pathotyping approach to be a competitive method to characterize S. suis isolates recovered from pigs on UK farms and one that can easily be updated to incorporate global strain collections.
Bosse J, Li Y, Stringer O, et al., 2019, Serotyping of Actinobacillus pleuropneumoniae field isolates by PCR, Publisher: Pig33 (www.pig333.com)
Li Y, da Silva GC, Li Y, et al., 2018, Evidence of illegitimate recombination between two Pasteurellaceae plasmids resulting in a novel multi-resistance replicon, pM3362MDR, in Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 9, ISSN: 1664-302X
Evidence of plasmids carrying the tetracycline resistance gene, tet(B), was found in the previously reported whole genome sequences of 14 United Kingdom, and 4 Brazilian, isolates of Actinobacillus pleuropneumoniae. Isolation and sequencing of selected plasmids, combined with comparative sequence analysis, indicated that the four Brazilian isolates all harbor plasmids that are nearly identical to pB1001, a plasmid previously found in Pasteurella multocida isolates from Spain. Of the United Kingdom isolates, 13/14 harbor plasmids that are (almost) identical to pTetHS016 from Haemophilus parasuis. The remaining United Kingdom isolate, MIDG3362, harbors a 12666 bp plasmid that shares extensive regions of similarity with pOV from P. multocida (which carries blaROB-1, sul2, and strAB genes), as well as with pTetHS016. The newly identified multi-resistance plasmid, pM3362MDR, appears to have arisen through illegitimate recombination of pTetHS016 into the stop codon of the truncated strB gene in a pOV-like plasmid. All of the tet(B)-carrying plasmids studied were capable of replicating in Escherichia coli, and predicted origins of replication were identified. A putative origin of transfer (oriT) sequence with similar secondary structure and a nic-site almost identical to that of RP4 was also identified in these plasmids, however, attempts to mobilize them from an RP4-encoding E. coli donor strain were not successful, indicating that specific conjugation machinery may be required.
Bossé JT, Li Y, Leanse LG, et al., 2018, Rationally designed<i>mariner</i>vectors to allow functional genomic analysis of<i>Actinobacillus pleuropneumoniae</i>and other bacteria by transposon-directed insertion-site sequencing (TraDIS)
<jats:title>Abstract</jats:title><jats:p>Transposon Directed Insertion Sequencing (TraDIS) is a high-throughput method for mapping insertion sites in large libraries of transposon mutants. The<jats:italic>Himar1</jats:italic>(<jats:italic>mariner</jats:italic>) transposon is ideal for generating near-saturating mutant libraries, especially in AT-rich chromosomes, as the requirement for integration is a TA dinucleotide. In this study, we generated two novel<jats:italic>mariner</jats:italic>vectors, pTsodCPC9 and pTlacPC9 (differing only in the promoter driving expression of the transposase gene), in order to facilitate TraDIS identification of conditionally essential genes in<jats:italic>Actinobacillus pleuropneumoniae</jats:italic>and other bacteria. Using the pTlacPC9 vector, we have generated, for the first time, saturating<jats:italic>mariner</jats:italic>mutant libraries in both<jats:italic>A. pleuropneumoniae</jats:italic>and<jats:italic>Pasteurella multocida</jats:italic>that showed a near random distribution of insertions around the respective chromosomes. A preliminary screen of 5000 mutants each identified 8 and 15 genes, respectively, that are required for growth under anaerobic conditions.</jats:p>
Li Y, Spiropoulos J, Cooley J, et al., 2018, Galleria mellonella - a novel infection model for the Mycobacterium tuberculosis complex, Virulence, Vol: 9, Pages: 1126-1137, ISSN: 2150-5594
Animal models have long been used in tuberculosis research to understand disease pathogenesis and to evaluate novel vaccine candidates and anti-mycobacterial drugs. However, all have limitations and there is no single animal model which mimics all the aspects of mycobacterial pathogenesis seen in humans. Importantly mice, the most commonly used model, do not normally form granulomas, the hallmark of tuberculosis infection. Thus there is an urgent need for the development of new alternative in vivo models. The insect larvae, Galleria mellonella has been increasingly used as a successful, simple, widely available and cost-effective model to study microbial infections. Here we report for the first time that G. mellonella can be used as an infection model for members of the M. tuberculosis complex. We demonstrate a dose-response for G. mellonella survival infected with different inocula of bioluminescent, Mycobacterium bovis BCG lux, and demonstrate suppression of mycobacterial luminesence over 14 days. Histopathology staining and transmission electron microscopy of infected G. mellonella phagocytic haemocytes show internalization and aggregation of M. bovis BCG lux in granuloma-like structures, and increasing accumulation of lipid bodies within M. bovis BCG lux over time, characteristic of latent tuberculosis infection. Our results demonstrate that G. mellonella can act as a surrogate host to study the pathogenesis of mycobacterial infection and shed light on host-mycobacteria interactions, including latent tuberculosis infection
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