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Extracellular vesicle (EV) production by bacteria is an important mechanism for microbial communication and host-pathogen interaction. EVs of some bacterial species have been reported to contain nucleic acids. However, the role of small RNAs (sRNAs) packaged in EVs is poorly understood. Here, we report on the RNA cargo of EVs produced by the pig pathogen Actinobacillus pleuropneumoniae , the causal agent of porcine pleuropneumonia, a disease which causes substantial economic losses to the swine industry worldwide. The EVs produced by aerobically and anaerobically grown bacteria were only slightly different in size and distribution. Total cell and outer membrane protein profiles and lipid composition of A. pleuropneumoniae whole cell extracts and EVs were similar, although EVs contained rough lipopolysaccharide compared to the smooth form in whole cells. Approximately 50% of Galleria mellonella larvae died after the injection of EVs. RNAseq, RT-PCR, protection from nuclease degradation, and database searching identified previously described and 13 novel A. pleuropneumoniae sRNAs in EVs, some of which were enriched compared to whole cell content. We conclude that A. pleuropneumoniae EVs contain sRNAs, including those known to be involved in virulence, and some with homologs in other Pasteurellaceae and/or non- Pasteurellaceae . Further work will establish whether the novel sRNAs in A. pleuropneumoniae EVs play any role in pathogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 da Silva, Rosa, Fontes, de Castro, Barbosa, Clarindo, Mantovani, Li, Bossé, Langford and Bazzolli.)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 da Silva, Rossi, Rosa, Sanches, Cardoso, Li, Witney, Gould, Fontes, Callaghan, Bossé, Langford and Bazzolli.)

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 lack sensitivity (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., Competing Interests: TN, PM, EV, and PP are employed by Ceva Animal Health Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stringer, Li, Bossé, Forrest, Hernandez-Garcia, Tucker, Nunes, Costa, Mortensen, Velazquez, Penny, Rodriguez-Manzano, Georgiou and Langford.)

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.

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-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 da Silva, Gonçalves, Rosa, França, Bossé, Santana, Langford and Bazzolli.)

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, we show, using Actinobacillus pleuropneumoniae as an exemplar, that the MISTEACHING framework can be applied to a strict veterinary pathogen, enabling the identification of major research gaps, 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, fungi, 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.

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.

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 than among the Norwegian isolates. An increased understanding of the effects of population strategies is helpful in surveillance and control of infectious diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cohen, Bossé, Stegger, Li, Langford, Kielland, Klem, Gulliksen, Ranheim, Grøntvedt and Angen.)

Actinobacillus pleuropneumoniae (APP), 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-multiplex PCR (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 6 months of storage at 37°C. This study provides simple, efficient, rapid, and practical sample processing for detection and molecular serotyping of A. pleuropneumoniae ., Competing Interests: EV and PP are employed by Ceva Animal Health Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stringer, Bossé, Lacouture, Gottschalk, Fodor, Angen, Velazquez, Penny, Lei, Langford and Li.)

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 A08-013 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interestsAuthor Paul R. Langford was not involved in the journal’s review or decisions related to this manuscript. The authors declare no competing interests., (© The Author(s) 2021.)

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 Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 h 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., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

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., (Copyright © 2020 Wang, Yin, Yu, Dang, Guo, Yan, Hu, Gu, Du, Feng, Han, Adam, Sun, Bossé and Lei.)

We report here the draft genome sequences of the type strains of Actinobacillus indolicus (46K2C) and Actinobacillus porcinus (NM319). These NAD-dependent bacterial species are frequently found in the upper respiratory tract of pigs and are occasionally associated with lung pathology., (Copyright © 2020 Bossé et al.)

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 bla ROB-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.

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., (Copyright © 2018. Published by Elsevier B.V.)

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., (© 2017 Blackwell Verlag GmbH.)

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.

The aim of this study was to investigate isolates of Actinobacillus pleuropneumoniae previously designated serologically either as non-typable (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 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., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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 Mycobacterium 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.

Objectives: To 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., Methods: ICEApl2 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., Results: ICEApl2 is 92660 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., Conclusions: Our 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., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Despite numerous actions to prevent disease, Actinobacillus pleuropneumoniae (A. pleuropneumoniae) remains a major cause of porcine pleuropneumonia, resulting in economic losses to the swine industry worldwide. In this paper, we describe the utilization of a reverse vaccinology approach for the selection and in vitro testing of serovar-independent A. pleuropneumoniae immunogens. Potential immunogens were identified in the complete genomes of three A. pleuropneumoniae strains belonging to different serovars using the following parameters: predicted outer-membrane subcellular localization; ≤ 1 trans-membrane helices; presence of a signal peptide in the protein sequence; presence in all known A. pleuropneumoniae genomes; homology with other well characterized factors with relevant data regarding immunogenicity/protective potential. Using this approach, we selected the proteins ApfA and VacJ to be expressed and 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 yields of OMVs compared to the parental strain. Our results indicated that ApfA and VacJ are highly conserved proteins, naturally expressed during infection by all A. pleuropneumoniae serovars tested. Furthermore, OMVs, ApfA and VacJ were shown to possess a high immunogenic potential in vitro. These findings favour the immunogen selection protocol used, and suggest that OMVs, along with ApfA and VacJ, could represent effective immunogens for the prevention of A. 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.

A small (3.9kb) plasmid (p518), conferring resistance to florfenicol (MIC >8μg/mL) and chloramphenicol (MIC >8μg/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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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 bla ROB-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 (ICE Apl1 ) in 21, or as part of a Tn 7 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.

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 was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was 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 ., (Copyright © 2017 Bossé et al.)

Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies., (© 2016 Rossi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)