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Bosse J, Li Y, Fernandez Crespo R, et al., 2018, Comparative sequence analysis of the capsular polysaccharide loci of Actinobacillus pleuropneumoniae serovars 1-18, and development of two multiplex PCRs for comprehensive capsule typing, Veterinary Microbiology, Vol: 220, Pages: 83-89, ISSN: 0378-1135
Problems with serological cross-reactivity have led to development of a number of PCRs (individual and multiplex) for molecular typing of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. Most of these assays were developed for detection of specific amplicons within capsule biosynthetic genes before the availability of complete sequences for the different serovars. Here we describe comparative analysis of the complete capsular loci for all 18 serovars of A. pleuropneumoniae, and development of two multiplex PCRs for comprehensive capsule typing of this important pig pathogen.
Michael G, Bosse J, Schwarz S, 2018, Antimicrobial resistance in pasteurellaceae of veterinary origin, Microbiology Spectrum, Vol: 6, Pages: 1-33, ISSN: 2165-0497
Members of the highly heterogeneous family Pasteurellaceae cause a wide variety of diseases in humans and animals. Antimicrobial agents are the most powerful tools to control such infections. However, the acquisition of resistance genes, as well as the development of resistance-mediating mutations, significantly reduces the efficacy of the antimicrobial agents. This article gives a brief description of the role of selected members of the family Pasteurellaceae in animal infections and of the most recent data on the susceptibility status of such members. Moreover, a review of the current knowledge of the genetic basis of resistance to antimicrobial agents is included, with particular reference to resistance to tetracyclines, β-lactam antibiotics, aminoglycosides/aminocyclitols, folate pathway inhibitors, macrolides, lincosamides, phenicols, and quinolones. This article focusses on the genera of veterinary importance for which sufficient data on antimicrobial susceptibility and the detection of resistance genes are currently available (Pasteurella, Mannheimia, Actinobacillus, Haemophilus, and Histophilus). Additionally, the role of plasmids, transposons, and integrative and conjugative elements in the spread of the resistance genes within and beyond the aforementioned genera is highlighted to provide insight into horizontal dissemination, coselection, and persistence of antimicrobial resistance genes. The article discusses the acquisition of diverse resistance genes by the selected Pasteurellaceae members from other Gram-negative or maybe even Gram-positive bacteria. Although the susceptibility status of these members still looks rather favorable, monitoring of their antimicrobial susceptibility is required for early detection of changes in the susceptibility status and the newly acquired/developed resistance mechanisms.
Sassu E, Bosse JT, Tobias TJ, et al., 2018, Update on Actinobacillus pleuropneumoniae ‐ knowledge, gaps andchallenges, Transboundary and Emerging Diseases, Vol: 65, Pages: 72-90, ISSN: 0931-184X
Porcine pleuropneumonia, caused by the bacterial porcine respiratory tract pathogen Actinobacillus pleuropneumoniae, leads to high economic losses in affected swine herds in most countries of the world. Pigs affected by peracute and acute disease suffer from severe respiratory distress with high lethality. The agent was first described in 1957 and, since then, knowledge about the pathogen itself, and its interactions with the host, has increased continuously. This is, in part, due to the fact that experimental infections can be studied in the natural host. However, the fact that most commercial pigs are colonized by this pathogen has hampered the applicability of knowledge gained under experimental conditions. In addition, several factors are involved in development of disease, and these have often been studied individually. In a DISCONTOOLS initiative, members from science, industry and clinics exchanged their expertise and empirical observations and identified the major gaps in knowledge. This review sums up published results and expert opinions, within the fields of pathogenesis, epidemiology, transmission, immune response to infection, as well as the main means of prevention, detection and control. The gaps that still remain to be filled are highlighted, and present as well as future challenges in the control of this disease are addressed.
Bosse JT, Li Y, Sárközi R, et al., 2018, Proposal of serovars 17 and 18 of Actinobacillus pleuropneumoniae based on serological and genotypic analysis, Veterinary Microbiology, Vol: 217, Pages: 1-6, ISSN: 0378-1135
The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as NT or as ‘K2:07’, which did not produce serovar-specific amplicons in PCR assays.We used whole genome sequencing to identify the capsule (CPS) loci of six previously designated biovar 1 non-typable (NT) and two biovar 1 ‘K2:O7’ isolates of A. pleuropneumoniae from Denmark, as well as a recent biovar 2 NT isolate from Canada. All of the NT isolates have the same six-gene type I CPS locus, sharing common cpsABC genes with serovars 2, 3, 6, 7, 8, 9, 11 and 13. The two ‘K2:O7’ isolates contain a unique three-gene type II CPS locus, having a cpsA gene similar to that of serovars 1, 4, 12, 14 and 15. The previously NT isolates share the same O-antigen genes, found between erpA and rpsU, as serovars 3, 6, 8, and 15. Whereas the ‘K2:O7’ isolates, have the same O-antigen genes as serovar 7, which likely contributed to their previous mis-identification. All of the NT and ‘K2:O7’ isolates have only the genes required for production of ApxII (apxIICA structural genes, and apxIBD export genes).Rabbit polyclonal antisera raised against representative isolates with these new CPS loci demonstrated distinct reactivity compared to the 16 known serovars. The serological and genomic results indicate that the isolates constitute new serovars 17 (previously NT) and 18 (previously ‘K2:O7’). Primers designed for amplification of specific serovar 17 and 18 sequences for molecular diagnostics will facilitate epidemiological tracking of these two new serovars of A. pleuropneumoniae.
Brockmeier SL, Loving CL, Nicholson TL, et al., 2018, Use of proteins identified through a functional genomic screen to develop a protein subunit vaccine that provides significant protection against virulent Streptococcus suis in pigs, Infection and Immunity, Vol: 86, ISSN: 0019-9567
Streptococcus suis is a bacterium commonly carried in the respiratory tract that is also one of the most important invasive pathogens of swine, commonly causing meningitis, arthritis, and septicemia. Due to the existence of many serotypes and a wide range of immune evasion capabilities efficacious vaccines are not readily available. The selection of S. suis protein candidates for inclusion in a vaccine was accomplished by identifying fitness genes through a functional genomics screen and selecting conserved predicted surface-associated proteins. Five candidate proteins were selected for evaluation in a vaccine trial and administered both intranasally and intramuscularly with one of two different adjuvant formulations. Clinical protection was evaluated by subsequent intranasal challenge with virulent S. suis While subunit vaccination with the S. suis proteins induced IgG antibody titers to each individual protein, a cellular immune response to the pool of proteins, and provided substantial protection from challenge with virulent S. suis, the immune response elicited and degree of protection were dependent on the parenteral adjuvant given. Subunit vaccination induced IgG reactive against different S. suis serotypes indicating a potential for cross-protection.
Michael GB, Bosse JT, Schwarz S, 2018, Antimicrobial Resistance in <i>Pasteurellaceae</i> of Veterinary Origin, ANTIMICROBIAL RESISTANCE IN BACTERIA FROM LIVESTOCK AND COMPANION ANIMALS, Editors: Schwarz, Cavaco, Shen, Publisher: AMER SOC MICROBIOLOGY, Pages: 331-363, ISBN: 978-1-55581-979-8
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Bosse JT, Langford PR, 2017, Regarding flagellar expression in clinical isolates of non-typeable Haemophilus influenzae, Journal of Medical Microbiology, Vol: 66, Pages: 1705-1705, ISSN: 1473-5644
Antenucci F, Fougeroux C, Bosse JT, et al., 2017, Identification and characterization of serovar-independent immunogens in Actinobacillus pleuropneumoniae, Veterinary Research, Vol: 48, ISSN: 0928-4249
Despite numerous actions to prevent disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) remains a majorcause of porcine pleuropneumonia, resulting in economic losses to the swine industry worldwide. In this paper, wedescribe the utilization of a reverse vaccinology approach for the selection and in vitro testing of serovar-independentA. pleuropneumoniae immunogens. Potential immunogens were identifed in the complete genomes of three A. pleuropneumoniaestrains belonging to diferent serovars using the following parameters: predicted outer-membrane subcellularlocalization; ≤ 1 trans-membrane helices; presence of a signal peptide in the protein sequence; presence in allknown A. pleuropneumoniae genomes; homology with other well characterized factors with relevant data regardingimmunogenicity/protective potential. Using this approach, we selected the proteins ApfA and VacJ to be expressedand further characterized, both in silico and in vitro. Additionally, we analysed outer membrane vesicles (OMVs) of A.pleuropneumoniae MIDG2331 as potential immunogens, and compared deletions in degS and nlpI for increasing yieldsof OMVs compared to the parental strain. Our results indicated that ApfA and VacJ are highly conserved proteins, naturallyexpressed during infection by all A. pleuropneumoniae serovars tested. Furthermore, OMVs, ApfA and VacJ wereshown to possess a high immunogenic potential in vitro. These fndings favour the immunogen selection protocolused, and suggest that OMVs, along with ApfA and VacJ, could represent efective immunogens for the prevention ofA. pleuropneumoniae infections in a serovar-independent manner. This hypothesis is nonetheless predictive in nature,and in vivo testing in a relevant animal model will be necessary to verify its validity.
Li Y, Li Y, Fernandez Crespo R, et al., 2017, Characterisation of the Actinobacillus pleuropneumoniae SXT-related Integrative and Conjugative Element ICEApl2, and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux, Journal of Antimicrobial Chemotherapy, Vol: 73, Pages: 57-65, ISSN: 0305-7453
ObjectivesTo characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR.MethodsICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli.ResultsICEApl2 is 92 660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol.ConclusionsOur results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae.
Howell KJ, Weinert LA, Peters SE, et al., 2017, "Pathotyping" Multiplex PCR Assay for haemophilus parasuis: a tool for prediction of virulence, Journal of Clinical Microbiology, Vol: 55, Pages: 2617-2628, ISSN: 0095-1137
Haemophilus parasuis is a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study of H. parasuis identified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates of H. parasuis based on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates of H. parasuis that had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases of H. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the “current standard” of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.
Hernandez-Garcia J, Wang J, Restif O, et al., 2017, Patterns of antimicrobial resistance in Streptococcus suis isolates from pigs with or without streptococcal disease in England between 2009 and 2014., Veterinary Microbiology, Vol: 207, Pages: 117-124, ISSN: 0378-1135
Antimicrobial resistance in Streptococcus suis, a global zoonotic pathogen of pigs, has been mostly studied only in diseased animals using surveys that have not evaluated changes over time. We compared patterns of resistance between S. suis isolates from clinical cases of disease (CC) and non-clinical case (NCC) pigs in England, collected over two discrete periods, 2009-2011 and 2013-2014. Minimum inhibitory concentrations (MIC) of 17 antimicrobials (nine classes) were determined on 405 S. suis isolates categorised by sampling period and disease association to assess changes in resistance over time and association with disease. First, isolates were characterized as resistant or susceptible using published clinical breakpoints. Second, epidemiological cut-offs (ECOFF) were derived from MIC values, and isolates classified as wild type (WT) below the ECOFF and non-wild type (NWT) above the ECOFF. Finally, isolate subsets were analysed for shifts in MIC distribution. NCC isolates were more resistant than CC isolates to cephalosporins, penams, pleuromutilins, potentiated sulphonamides and tetracyclines in both study periods. Resistance levels among CC isolates increased in 2013-2014 relative to 2009-2011 for antimicrobials including aminoglycosides, cephalosporins, fluoroquinolones, pleuromutilins, potentiated sulphonamides and tetracyclines. The prevalence of isolates categorised as NWT for five or more classes of antimicrobials was greater among NCC than CC isolates for both time periods, and increased with time. This study used standardised methods to identify significant shifts in antimicrobial resistance phenotypes of S. suis isolated from pigs in England, not only over time but also between isolates from known clinical cases or disease-free pigs.
Cunha da silva G, Rossi CC, Santana MF, et al., 2017, p518, a small floR plasmid from a South American isolate of Actinobacillus pleuropneumoniae, Veterinary Microbiology, Vol: 204, Pages: 129-132, ISSN: 1873-2542
A small (3.9 kb) plasmid (p518), conferring resistance to florfenicol (MIC >8 μg/mL) and chloramphenicol (MIC >8 μg/mL) was isolated from an Actinobacillus pleuropneumoniae clinical isolate from Southeastern Brazil. To date, this is the smallest florfenicol resistance plasmid isolated from a member of the Pasteurellaceae. The complete nucleotide of this plasmid revealed a unique gene arrangement compared to previously reported florfenicol resistance plasmids found in other members of the Pasteurellaceae. In addition to the floR gene and a lysR gene, common to various florfenicol resistance plasmids, p518 also encodes strA and a partial strB sequence. An origin of replication (oriV) similar to that in the broad host range plasmid, pLS88, was identified in p518, and transformation into Escherichia coli MFDpir confirmed the ability to replicate in other species. Mobilisation genes appear to have been lost, with only a partial mobC sequence remaining, and attempts to transfer p518 from a conjugal donor strain (E. coli MFDpir) were not successful, suggesting this plasmid is not mobilisable. Similarly, attempts to transfer p518 into a competent A. pleuropneumoniae strain, MIDG2331, by natural transformation were also not successful. These results suggest that p518 may be only transferred by vertical descent.
Bosse JT, Li Y, Rogers J, et al., 2017, Whole genome sequencing for surveillance of antimicrobial resistance in Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 8, ISSN: 1664-302X
The aim of this study was to evaluate the correlation between antimicrobial resistance (AMR) profiles of 96 clinical isolates of Actinobacillus pleuropneumoniae, an important porcine respiratory pathogen, and the identification of AMR genes in whole genome sequence (wgs) data. Susceptibility of the isolates to nine antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, sulfisoxazole, tetracycline, tilmicosin, trimethoprim, and tylosin) was determined by agar dilution susceptibility test. Except for the macrolides tested, elevated MICs were highly correlated to the presence of AMR genes identified in wgs data using ResFinder or BLASTn. Of the isolates tested, 57% were resistant to tetracycline [MIC ≥ 4 mg/L; 94.8% with either tet(B) or tet(H)]; 48% to sulfisoxazole (MIC ≥ 256 mg/L or DD = 6; 100% with sul2), 20% to ampicillin (MIC ≥ 4 mg/L; 100% with blaROB-1), 17% to trimethoprim (MIC ≥ 32 mg/L; 100% with dfrA14), and 6% to enrofloxacin (MIC ≥ 0.25 mg/L; 100% with GyrAS83F). Only 33% of the isolates did not have detectable AMR genes, and were sensitive by MICs for the antimicrobial agents tested. Although 23 isolates had MIC ≥ 32 mg/L for tylosin, all isolates had MIC ≤ 16 mg/L for both erythromycin and tilmicosin, and no macrolide resistance genes or known point mutations were detected. Other than the GyrAS83F mutation, the AMR genes detected were mapped to potential plasmids. In addition to presence on plasmid(s), the tet(B) gene was also found chromosomally either as part of a 56 kb integrative conjugative element (ICEApl1) in 21, or as part of a Tn7 insertion in 15 isolates. Our results indicate that, with the exception of macrolides, wgs data can be used to accurately predict resistance of A. pleuropneumoniae to the tested antimicrobial agents and provides added value for routine surveillance.
Cuccui J, Terra VS, Bossé JT, et al., 2017, The N-linking glycosylation system from Actinobacillus pleuropneumoniae is required for adhesion and has potential use in glycoengineering, Open Biology, Vol: 7, ISSN: 2046-2441
Actinobacillus pleuropneumoniae is a mucosal respiratory pathogen causing contagious porcine pleuropneumonia. Pathogenesis studies have demonstrated a major role for the capsule, exotoxins and outer membrane proteins. Actinobacillus pleuropneumoniae can also glycosylate proteins, using a cytoplasmic N-linked glycosylating enzyme designated NGT, but its transcriptional arrangement and role in virulence remains unknown. We investigated the NGT locus and demonstrated that the putative transcriptional unit consists of rimO, ngt and a glycosyltransferase termed agt. From this information we used the A. pleuropneumoniae glycosylation locus to decorate an acceptor protein, within Escherichia coli, with a hexose polymer that reacted with an anti-dextran antibody. Mass spectrometry analysis of a truncated protein revealed that this operon could add up to 29 repeat units to the appropriate sequon. We demonstrated the importance of NGT in virulence, by creating deletion mutants and testing them in a novel respiratory cell line adhesion model. This study demonstrates the importance of the NGT glycosylation system for pathogenesis and its potential biotechnological application for glycoengineering.
Bosse JT, Li Y, Sárközi R, et al., 2017, A unique capsule locus in the newly designated Actinobacillus pleuropneumoniae serovar 16 and development of a diagnostic PCR assay, Journal of Clinical Microbiology, Vol: 55, Pages: 902-907, ISSN: 1098-660X
Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant, lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal put forward that they comprised a new serovar 16. Here we used whole genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of A-85/14 and a PCR formulated that differentiated serovar 16 isolates from all 15 known serovars, and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data, show the existence of a sixteenth serovar – designated serovar 16 - of A. pleuropneumoniae.
Li Y, Bosse JT, Williamson SN, et al., 2016, Actinobacillus pleuropneumoniae serovar 8 predominates in England and Wales, Veterinary Record, Vol: 179, ISSN: 0042-4900
Rossi CC, Bosse JT, Li Y, et al., 2016, A computational strategy for the search of regulatory small RNAs inActinobacillus pleuropneumoniae, RNA, Vol: 22, Pages: 1373-1385, ISSN: 1355-8382
Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and arefrequently connected to the expression of virulence factors in diverse bacteria. Only a fewsRNAs have been described for Pasteurellaceae pathogens and no sRNA has yet beendescribed for Actinobacillus pleuropneumoniae, the causative agent of porcinepleuropneumonia, responsible for considerable losses in the swine industry. To search forsRNAs in A. pleuropneumoniae, we developed a pipeline for the computational analysis ofthe bacterial genome by using four algorithms with different approaches, followed byexperimental validation. The coding strand and expression of 17 out of 23 RNA candidateswere confirmed by Northern blotting, RT-PCR and RNA sequencing. Among them, two arelikely riboswitches, three are housekeeping regulatory RNAs, two are the widely studiedGcvB and 6S sRNAs, and ten are putative novel trans-acting sRNAs, never before describedfor any bacteria. The latter group has several potential mRNAs targets, many of which areinvolved with virulence, stress resistance or metabolism, and connect the sRNAs in acomplex gene regulatory network. The sRNAs identified are well conserved among thePasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the samehost. Our results show that the combination of newly developed computational programs canbe successfully utilized for the discovery of novel sRNAs and indicate an intricate system ofgene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thusproviding clues for novel aspects of virulence that will be explored in further studies.
Bosse JT, Li Y, Fernandez Crespo R, et al., 2016, ICEApl1, an integrative conjugative element related to ICEHin1056, identified in the pig pathogen Actinobacillus pleuropneumoniae, Frontiers in Microbiology, Vol: 7, ISSN: 1664-302X
ICEApl1 was identified in the whole genome sequence of MIDG2331, a tetracycline-resistant (MIC = 8 mg/L) serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia. PCR amplification of virB4, one of the core genes involved in conjugation, was used to identify other A. pleuropneumoniae isolates potentially carrying ICEApl1. MICs for tetracycline were determined for virB4 positive isolates, and shotgun whole genome sequence analysis was used to confirm presence of the complete ICEApl1. The sequence of ICEApl1 is 56083 bp long and contains 67 genes including a Tn10 element encoding tetracycline resistance. Comparative sequence analysis was performed with similar integrative conjugative elements (ICEs) found in other members of the Pasteurellaceae. ICEApl1 is most similar to the 59393 bp ICEHin1056, from Haemophilus influenzae strain 1056. Although initially identified only in serovar 8 isolates of A. pleuropneumoniae (31 from the UK and 1 from Cyprus), conjugal transfer of ICEApl1 to representative isolates of other serovars was confirmed. All isolates carrying ICEApl1 had a MIC for tetracycline of 8 mg/L. This is, to our knowledge, the first description of an ICE in A. pleuropneumoniae, and the first report of a member of the ICEHin1056 subfamily in a non-human pathogen. ICEApl1 confers resistance to tetracycline, currently one of the more commonly used antibiotics for treatment and control of porcine pleuropneumonia.
Bosse JT, Chaudhiri RR, Li Y, et al., 2016, The complete genome sequence of MIDG2331, a genetically tractable serovar 8 clinical isolate of Actinobacillus pleuropneumoniae, Genome Announcements, Vol: 4, ISSN: 2169-8287
We report here the complete annotated genome sequence of a clinical serovar 8 isolate Actinobacillus pleuropneumoniae MIDG2331. Unlike the serovar 8 reference strain 405, MIDG2331 is amenable to genetic manipulation via natural transformation as well as conjugation, making it ideal for studies of gene function.
Hathroubi S, Hancock MA, Bossé JT, et al., 2015, Surface polysaccharide mutants reveal that absence of O antigen reduces biofilm formation of Actinobacillus pleuropneumoniae., Infection and Immunity, Vol: 84, Pages: 127-137, ISSN: 1098-5522
Actinobacillus pleuropneumoniae is a Gram-negative bacterium belonging to the Pasteurellaceae family and the causative agent of porcine pleuropneumonia, a highly contagious lung disease causing important economic losses. Surface polysaccharides including lipopolysaccharides (LPS) and capsular polysaccharides (CPS) are implicated in the adhesion and virulence of A. pleuropneumoniae, but their role in biofilm formation is still unclear. In this study, we investigated the requirement of these surface polysaccharides in biofilm formation by A. pleuropneumoniae serotype 1. Well characterized mutants were used: an O-antigen LPS mutant, a truncated core-LPS mutant with an intact O-antigen, a capsule mutant and a poly-N-acetylglucosamine (PGA) mutant. We compared the amount of biofilm produced by the parental strain and the isogenic mutants using static and dynamic systems. Compared to the parental or other strains, the biofilm of the O-antigen and the PGA mutants was dramatically reduced and it had less cell-associated PGA. Real-time PCR analyses revealed a significant reduction in the level of pgaA, cpxR and cpxA mRNA in the biofilm cells of the O-antigen mutant compared to the parental strain. Specific binding between PGA and LPS was consistently detected by surface plasmon resonance but the lack of O-antigen did not abolish these interactions. In conclusion, the absence of the O-antigen reduces the ability of A. pleuropneumoniae to form a biofilm and this is associated with a reduction in the expression and the production of PGA.
Baig A, Weinert LA, Peters SE, et al., 2015, Whole genome investigation of a divergent clade of the pathogen Streptococcus suis, Frontiers in Microbiology, Vol: 6, ISSN: 1664-302X
Li G, Xie F, Zhang Y, et al., 2015, Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8., PLOS One, Vol: 10, ISSN: 1932-6203
Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae.
Howell KJ, Peters SE, Wang J, et al., 2015, Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis., Journal of Clinical Microbiology, Vol: 53, Pages: 3812-3821, ISSN: 1098-660X
Haemophilus parasuis causes Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus and in silico serovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars of H. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 10(5) ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensal Pasteurellaceae and other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequenced H. parasuis collection was used to validate the mPCR with 100% accuracy compared to the in silico results. In addition, the two in silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars of H. parasuis.
Bosse JT, Li Y, Atherton TG, et al., 2015, Corrigendum to 'Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae' (Veterinary Microbiology 178 (2015) 279-282), Veterinary Microbiology, Vol: 179, Pages: 341-341, ISSN: 1873-2542
Bosse JT, Li Y, Atherton TG, et al., 2015, Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae, Veterinary Microbiology, ISSN: 1873-2542
Bosse J, Li Y, Walker S, et al., 2015, Identification of dfrA14 in two distinct plasmids conferring trimethoprimresistance in Actinobacillus pleuropneumoniae, Journal of Antimicrobial Chemotherapy, Vol: 70, Pages: 2217-2222, ISSN: 1460-2091
Objectives The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England.Methods Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids.Results A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene.Conclusions This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial.
Weinert LA, Chaudhuri RR, Wang J, et al., 2015, Genomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis, Nature Communications, Vol: 6, ISSN: 2041-1723
Pereira MF, Rossi CC, de Queiroz MV, et al., 2015, Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection, Microbiology, Vol: 161, Pages: 387-400, ISSN: 1350-0872
Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 104 to 107 c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.
Howell KJ, Weinert LA, Chaudhuri RR, et al., 2014, The use of genome wide association methods to investigate pathogenicity, population structure and serovar in Haemophilus parasuis., BMC Genomics, Vol: 15, Pages: 1179-1179, ISSN: 1471-2164
BACKGROUND: Haemophilus parasuis is the etiologic agent of Glasser's disease in pigs and causes devastating losses to the farming industry. Whilst some hyper-virulent isolates have been described, the relationship between genetics and disease outcome has been only partially established. In particular, there is weak correlation between serovar and disease phenotype. We sequenced the genomes of 212 isolates of H. parasuis and have used this to describe the pan-genome and to correlate this with clinical and carrier status, as well as with serotype. RESULTS: Recombination and population structure analyses identified five groups with very high rates of recombination, separated into two clades of H. parasuis with no signs of recombination between them. We used genome-wide association methods including discriminant analysis of principal components (DAPC) and generalised linear modelling (glm) to look for genetic determinants of this population partition, serovar and pathogenicity. We were able to identify genes from the accessory genome that were significantly associated with phenotypes such as potential serovar specific genes including capsule genes, and 48 putative virulence factors that were significantly different between the clinical and non-clinical isolates. We also show that the presence of many previously suggested virulence factors is not an appropriate marker of virulence. CONCLUSIONS: These genes will inform the generation of new molecular diagnostics and vaccines, and refinement of existing typing schemes and show the importance of the accessory genome of a diverse species when investigating the relationship between genotypes and phenotypes.
Bosse JT, Soares-Bazzolli DM, Li Y, et al., 2014, The Generation of Successive Unmarked Mutations and Chromosomal Insertion of Heterologous Genes in <i>Actinobacillus pleuropneumoniae</i> Using Natural Transformation, PLOS ONE, Vol: 9, ISSN: 1932-6203
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