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The escalating threat of antimicrobial resistance has increased pressure to develop novel therapeutic strategies to tackle drug-resistant infections. Antimicrobial peptides have emerged as a promising class of therapeutics for various systemic and topical clinical applications. In this study, the de novo design of α-helical peptides with idealized facial amphiphilicities, based on an understanding of the pertinent features of protein secondary structures, is presented. Synthetic amphiphiles composed of the backbone sequence (X 1 Y 1 Y 2 X 2 ) n , where X 1 and X 2 are hydrophobic residues (Leu or Ile or Trp), Y 1 and Y 2 are cationic residues (Lys), and n is the number repeat units (2 or 2.5 or 3), demonstrated potent broad-spectrum antimicrobial activities against clinical isolates of drug-susceptible and multi-drug resistant bacteria. Live-cell imaging revealed that the most selective peptide, (LKKL) 3 , promoted rapid permeabilization of bacterial membranes. Importantly, (LKKL) 3 not only suppressed biofilm growth, but effectively disrupted mature biofilms after only 2 h of treatment. The peptides (LKKL) 3 and (WKKW) 3 suppressed the production of LPS-induced pro-inflammatory mediators to levels of unstimulated controls at low micromolar concentrations. Thus, the rational design strategies proposed herein can be implemented to develop potent, selective and multifunctional α-helical peptides to eradicate drug-resistant biofilm-associated infections. Statement of Significance Antimicrobial peptides (AMPs) are increasingly explored as therapeutics for drug-resistant and biofilm-related infections to help expand the size and quality of the current antibiotic pipeline in the face of mounting antimicrobial resistance. Here, synthetic peptides rationally designed based upon principles governing the folding of natural α-helical AMPs, comprising the backbone sequence (X 1 Y 1 Y 2 X 2 ) n , and which assemble into α-helical structures with idealized facial amphiphilicity, is presented. These multifunctional peptide amphiphiles demonstrate high bacterial selectivity, promote the disruption of pre-formed drug-resistant biofilms, and effectively neutralize endotoxins at low micromolar concentrations. Overall, the design strategies presented here could provide a useful tool for developing therapeutic peptides with broad-ranging clinical applications from the treatment and prevention of drug-resistant biofilms to the neutralization of bacterial endotoxins. [ABSTRACT FROM AUTHOR]

Background A recently-licensed 10-valent pneumococcal conjugate vaccine (PHiD-CV; Synflorix, GSK) uses Protein D from Haemophilus influenzae as a carrier protein. PHiD-CV therefore has the potential to provide additional protection against nontypeable H. influenzae (NTHi). NTHi frequently causes respiratory tract infections and is associated with significant morbidity and mortality worldwide and there is currently no vaccine. Methods We developed mouse models of NTHi infection and influenza/NTHi superinfection. Mice were immunized with PHiD-CV, heat-killed NTHi, or a 13-valent pneumococcal conjugate vaccine that did not contain Protein D (PCV13; Prevenar, Pfizer) and then infected intranasally with NTHi. Results Infection with NTHi resulted in weight loss, inflammation and airway neutrophilia. In a superinfection model, prior infection with pandemic H1N1 influenza virus (strain A/England/195/2009) augmented NTHi infection severity, even with a lower bacterial challenge dose. Immunization with PHiD-CV produced high levels of antibodies that were specific against Protein D, but not heat-killed NTHi. Immunization with PHiD-CV led to a slight reduction in bacterial load, but no change in disease outcome. Conclusions PHiD-CV induced high levels of Protein D-specific antibodies, but did not augment pulmonary clearance of NTHi. We found no evidence to suggest that PHiD-CV will offer added benefit by preventing NTHi lung infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Contagious porcine pleuropneumonia (CPP), caused by Actinobacillus pleuropneumoniae (APP), is a highly transmissible and fatal respiratory illness that causes tremendous economic losses for the pig breeding industry worldwide. Propionibacterium acnes (PA) has a strong cross-reaction with anti-APP1 and anti-APP5 serum and can efficiently prevent APP infection, which was fortuitously found in researching the differential gene between the different APP serotypes. There seems to be some natural cross-protection between PA and APP. To identify the common epitope, the phage display library of a PA whole genome was constructed, whose size is 10 5 . The DNA sequence of the positive clone was determined after three rounds of biopanning, and ten common protein types were identified and the epitope was predicted by computer software. Six peptide epitopes were selected and synthesized for further analysis. Among these epitopes, Ba1, Bb5 and C1 could bind to anti-PA serum and anti-APP1 serum and vice versa. Furthermore, the IgG and IL-4 levels and CD4 + / CD8 + T cell ratios in the Ba1, Bb5 and C1 groups were significantly higher than that in the control group, indicating that the epitopes could trigger an immune response, which was mainly humoral immunity. Moreover, Ba1 and Bb5 equally protected 80% of mice from a fatal dose of APP1 infection compared with the control group. Mice could resist APP1 and APP5 challenge after being treated with the combination of Ba1 and Bb5, with survival rates of 80% and 90%, respectively. These findings suggest that the PA epitope confers antigenicity and can heterologously resist to the APP infection. This finding provides a novel strategy for preventing APP infection. [ABSTRACT FROM AUTHOR]

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 MFD pir 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 MFD pir ) 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. [ABSTRACT FROM AUTHOR]

Abstract: The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic α-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti-mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK)2M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB. [Copyright &y& Elsevier]

Highlights: [•] P. acnes antigens protect against A. pleuropneumoniae infection. [•] Six P. acnes proteins were identified and produced as recombinant proteins. [•] PA-Ssb gave the best protection against A. pleuropneumoniae serotype 1 and 5 strains. [•] P. acnes induced immunity may be mediated by antibodies to ApxIV and ZnuA. [Copyright &y& Elsevier]

Abstract: APXIVA is an RTX toxin of Actinobacillus pleuropneumoniae that is a candidate antigen to differentiate infected from vaccinated animals (DIVA). Insertion of ISApl1 into the apxIVA gene is known to compromise an APXIVA-based DIVA approach, as is potentially a TGG to TGA mutation in the apxIVA gene. ISApl1 was found in 63/349 (18.1%) A. pleuropneumoniae isolates from England and Wales including serovars 2, 3, 6–8 and 12. No ISApl1 insertions into apxIVA were found. Only two serovar 3 isolates contained the TGG to TGA mutation. We conclude that an ApxIVA-based DIVA approach would potentially be viable in England and Wales. [Copyright &y& Elsevier]

Summary: Actinobacillus pleuropneumoniae is an important respiratory pathogen of swine, for which there is no highly effective vaccine. A phage expression library of the A. pleuropneumoniae genome was constructed and screened to identify potential vaccine components. Open reading frames within immuno-reactive phage were analyzed in silico to identify conserved outer membrane proteins. Four ORFs, named comL, lolB, lppC and ompA were chosen for further study. The four encoded proteins were shown experimentally to be antigenic, highly conserved, outer membrane, in vivo-expressed proteins. In pig protection studies, none of the proteins was individually capable of protecting pigs from colonization and infection with the homologous A. pleuropneumoniae strain, despite a detectable specific antibody response being induced. [Copyright &y& Elsevier]

Abstract: Several of the most virulent Salmonella enterica strains possess two genes encoding periplasmic Cu,Zn superoxide dismutase, sodC1 and sodC2, located on a lambdoid prophage and on the chromosome, respectively. These genes contribute to Salmonella virulence by protecting bacteria from superoxide generated by the host''s phagocytes. To investigate the respective contributions of sodC1 and sodC2 to the virulence of a clinical isolate of Salmonella enterica serovar Choleraesuis (S. choleraesuis), we have analyzed both the intracellular survival of wild type and sodC mutant strains within J774 macrophages and Caco-2 cells, and their ability to proliferate in intraperitoneally-infected mice in competition assays. In agreement with previous studies, mutant strains lacking one or both sodC genes were equally impaired in their ability to survive within activated macrophages. However, when macrophage killing experiments were carried out with non-opsonized bacteria, sodC2 contributed to intracellular survival more than sodC1, indicating that changes in the pathways of bacterial uptake can modify the relative role of the two sodC genes. More unexpectedly, we have found that the ability of S. choleraesuis to survive within Caco-2 cells was severely affected by inactivation of sodC genes, sodC2 being more important than sodC1. As Caco-2 cells actively produce superoxide, this suggests that oxygen radical production by colonic cells has a role in controlling proliferation of facultative intracellular bacteria. Mouse infection studies confirmed that, in the S. choleraesuis strain under investigation, both sodC genes are required to confer full virulence, sodC2 contributing slightly more than sodC1 to Salmonella pathogenesis. Our findings contrast with the results of other studies carried out in S. enterica serovar Typhimurium and suggest that the relative contributions of sodC1 and sodC2 to host-pathogen interactive biology may vary depending on the Salmonella serovar or strain. [Copyright &y& Elsevier]

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a highly contagious disease for which there is no effective vaccine. This review considers how adhesins, iron-acquisition factors, capsule and lipopolysaccharide, RTX cytotoxins and other potential future vaccine components contribute to colonisation, to avoidance of host clearance mechanisms and to damage of host tissues. [Copyright &y& Elsevier]

[Display omitted] • Identification of A. pleuropneumoniae serovar 19 type II capsule locus. • Serovar 19 isolates are biovar 1, encode ApxII and ApxIV toxins, and have either a serogroup 3/6/8/15 or 4/7 O-Ag locus. • Reformulation of two mPCRs for unequivocal capsule typing of all known A. pleuropneumoniae serovars. 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. [ABSTRACT FROM AUTHOR]

• The bacterium Actinobacillus pleuropneumoniae infects pig lungs. • Serum and broncheolavage fluid cytokine and chemokine levels did not correlate. • Blood measurements may not be representative of those found in the lung. • There are substantial differences in immune cells and cytokines in the lung and peripheral blood of APP infected piglets at same time points. Excessive cytokine production is an important component of the acute respiratory distress syndrome and multiple organ failure. Pneumonia can lead to an overexpression of cytokines, although comparatively little is known about the relevance and differences in cytokines between blood and lung. In this study, piglets were experimentally infected intranasally with Actinobacillus pleuropneumoniae (APP), and transcriptomes of lung tissue and peripheral blood mononuclear cells determined. In addition, the levels of 30 cytokines in broncheoalveolar lavage fluid (BALF) and sera were determined by ELISA. Post infection, there was an early increase in lung monocytes, and a later rise in inflammatory cytokines in BALF. Blood lymphocytes increased early in infection and there was a rise in inflammatory cytokines in the peripheral blood of infected piglets. Genes involved in cytokine production, leukocyte migration and differentiation, lymphocyte activation, and cytokine-mediated signaling pathways in the transcriptomes of lung tissue were significantly down-regulated early in infection. At this early phase of APP infection (0−6 h), the cytokines IL-1β, MCP-1, and IL-5 in sera increased rapidly and significantly, while many cytokines in BALF decreased. At 48 h post-infection, cytokines in sera were no longer significantly increased, although some were up-regulated in BALF, and there was aggravated pathological damage in the lungs at this time. The data indicate there are substantial differences between immune cells and cytokines in the lung and peripheral blood of APP infected piglets at equivalent time points. The results increase our understanding of pig-APP host interactive biology, and will be important in formulating future therapeutic and preventative strategies to prevent disease caused by APP. [ABSTRACT FROM AUTHOR]