Actinobacillus Pleuropneumoniae Isolates Research Articles

The Susceptibility Trends of Respiratory and Enteric Porcine Pathogens to Last-Resource Antimicrobials.

Monitoring the antimicrobial susceptibility of last-resource antimicrobials for veterinary pathogens is urgently needed from a one-health perspective. The objective of this study was to analyze the antimicrobial susceptibility trends of Spanish porcine bacteria to quinolones, cephalosporins, and polymyxins. Isolates of Actinobacillus pleuropneumoniae, Pasteurella multocida, and Escherichia coli were isolated from sick pigs from 2019 to 2022. An antimicrobial susceptibility test was determined based on the minimal inhibitory concentration (MIC) following an internationally accepted methodology. The MIC categorization was based on distributing the range of MIC values in four categories, with category one being the most susceptible (lowest MIC value) and category four the least susceptible (highest MIC value). Moreover, clinical susceptibility (susceptible/non-susceptible) was also determined according to the CLSI and EUCAST clinical breakpoints. A logistic and multinomial logistic regression model was used to analyze the susceptibility data for dichotomized and categorized MIC data, respectively, for any pair of antimicrobial/microorganism. In general terms, the antimicrobial susceptibility of pig bacteria to these antimicrobials remained stable or increased in the last four years in Spain. In the case of A. pleuropneumoniae and quinolones, a significant temporal trend was observed where isolates from 2020 had significantly increased odds of being more susceptible than isolates from 2019. In the case of E. coli and polymyxins, a significant temporal trend was observed where isolates from 2020 and 2021 had significantly increased odds of being more susceptible than isolates from 2019 and 2020, respectively. Finally, significant odds of being less susceptible were only observed for cephalosporins and E. coli for 2020 versus 2019, stagnating for the rest of study period. These results provide sound data on critically important antimicrobials in swine medicine.

Characterization of nonencapsulated Actinobacillus pleuropneumoniae serovar K12:O3 isolates.

Three Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia were positive by capsular serovar 12-specific PCR assay, but not reactive to antiserum prepared against serovar 12 using the rapid slide agglutination (RSA) test. The isolates were positive for apxIICA, apxIIICA, apxIBD, apxIIIBD, and apxIVA in the PCR toxin gene assay, which is the profile seen in serovars 2, 4, 6, 8, and 15, and reacted with antisera against serovars 3, 6, 8, 15, and 17. Nucleotide sequence analysis revealed that genes involved in the biosynthesis of capsular polysaccharide of the 3 isolates were identical or nearly identical to those of serovar 12. However, genes involved in the biosynthesis of O-polysaccharide of the 3 isolates were highly similar to those of reference strains of serovars 3, 6, 8, 15, 17, and 19. In agreement with results from the RSA test, transmission electron microscopic analysis confirmed the absence of detectable capsular material in the 3 isolates. The existence of nonencapsulated A. pleuropneumoniae serovar K12:O3 would hamper precise serodetection.

Óleos essenciais isolados de plantas medicinais e temperos populares como compostos antimicrobianos e antibiofilme alternativos contra o patógeno de suínosActinobacillus pleuropneumoniae

ABSTRACT: Actinobacilluspleuropneumoniae is the causative agent of swine pleuropneumonia, a contagious respiratory disease associated with high morbidity and economic losses. While antibiotic therapy helps to control the spreading of the pathogen on the farm, resistance to several classes of antibiotics were reported, and treatment can be impaired by the bacterial ability to form biofilms. This increases the need for alternative therapy approaches, including the use of natural compounds with antimicrobial and/or antibiofilm activities. In this research we analyzed, by the broth microdilution method, the inhibitory and bactericidal activities of the essential oils obtained from eighteen Brazilian popular medicinal plants or spices against clinical isolates of Actinobacilluspleuropneumoniae. After that, sub-inhibitory concentrations of active oils were tested for their antibiofilm effects, analyzed by the crystal violet method. Among the eighteen oils tested, eight (extracted from cinnamon, coriander, peppermint, spearmint, thyme, marjoram, eucalyptus, and laurel) presented bacteriostatic and bactericidal activities against all isolates, and subinhibitory concentrations of five of them disrupted up to 80% preformed biofilms, and significantly inhibited biofilm formation. The chemical composition of such oils was assessed by gas chromatography/mass spectrometry (GC/MS) and is presented, indicating that their bactericidal and antibiofilm properties were mostly associated with the presence of monoterpenes and phenylpropanoids. To our knowledge, this is the first report of essential oils with potential to control environmental contamination and animal infection with A. pleuropneumoniae, representing an alternative to increasing levels of antibiotic resistance.

Genetic Diversity of Actinobacillus pleuropneumoniae Serovars in Hungary.

Simple SummaryActinobacillus pleuropneumoniae causes severe pneumonia in pigs, resulting in high economic losses. A total of 114 isolates from pneumonia were characterized by the examination of biotype, serovar, antibiotic resistance genes, and genes of toxin production. Analyzing their genetic relationship, 16 groups of related isolates were found. The genetic diversity was different in the different groups, however. It was remarkably small in the case of serovar 13, which was unusually frequent in Hungary. Therefore, representative isolates of serovar 13 were subjected to whole-genome sequencing, confirming low diversity. Antibiotic resistance was frequently found in isolates of serovar 13 but was less frequent in other serovars. The unusually high frequency and low diversity of serovar 13 suggest a clonal spread in Hungary, which may have been facilitated by a high frequency of resistance to beta-lactams and tetracyclines.A total of 114 Actinobacillus pleuropneumoniae isolates from porcine hemorrhagic necrotic pleuropneumonia were characterized by the examination of biotype, serovar, antibiotic resistance genes, and genes of toxin production. Pulsed-field gel electrophoresis was used to analyze their genetic relationship, which identified 16 clusters. Serovar 2 (50 isolates), serovar 13 (25 isolates), serovar 9 (11 isolates), and serovar 16 (7 isolates) were the most frequent serovars. Serovar 2 formed nine distinguishable clusters; serovar 13 and serovar 16 were less diverse, exhibiting two potentially related subclusters; serovar 9 was represented by a single cluster. Remarkably small differences were seen in the core genome when nine representative isolates of serovar 13 were subjected to whole-genome sequencing. Tetracycline resistance was relatively frequent in the two clusters of serovar 13; one of them was also frequently resistant against beta-lactams. Resistance in other serovars was sporadic. All isolates carried the apxIV gene. The toxin profiles of serovar 2 were characterized by the production of ApxII and ApxIII toxins, except for a small cluster of three isolates: serovar 9 and serovar 16 isolates produced ApxI and ApxII toxins. Serovar 13 carried apxII and apxIBD genes, indicating the production of the ApxII toxin, but not of ApxI or ApxIII. The unusually high frequency and low diversity of serovar 13 are not explained by its virulence properties, but the high frequency of resistance to beta-lactams and tetracyclines may have played a role in its spread. The emergence of serovar 16 may be facilitated by its high virulence, also explaining its high clonality.

Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known 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.

Sero- and apx-typing of German Actinobacillus pleuropneumoniae field isolates from 2010 to 2019 reveals a predominance of serovar 2 with regular apx-profile

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae, the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx-toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx-profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the “typical” apx-gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates.

Two Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia in Japan were positive in the capsular serovar 15-specific PCR assay, but nontypeable (NT) in the agar gel precipitation (AGP) test. Nucleotide sequence analysis of gene clusters involved in the biosynthesis of capsular polysaccharide (CPS) and lipopolysaccharide O-polysaccharide (O-PS) revealed that both clusters contained transposable element ISApl1 of A. pleuropneumoniae belonging to the IS30 family. Immunoblot analysis revealed that these 2 isolates could not produce O-PS. We conclude that the ISApl1 of A. pleuropneumoniae can interfere in the biosynthesis of both CPS and O-PS.

Comparison of metabolic adaptation and biofilm formation of Actinobacillus pleuropneumoniae field isolates from the upper and lower respiratory tract of swine with respiratory disease

Most outbreaks of disease due to infection with Actinobacillus (A.) pleuropneumoniae are caused by pigs already pre-colonised in tonsillar tissue, where the pathogen is protected from exposure to antibiotic substances administered for treatment. As it has been shown recently under experimental conditions, A. pleuropneumoniae displays host tissue-specific metabolic adaptation. In this study, pairs of A. pleuropneumoniae field isolates were recovered from lung as well as from tonsillar and nasal tissue from 20 pigs suffering from acute clinical signs of pleuropneumonia and showing characteristic pathological lung alterations. Metabolic adaptation to the porcine lower and upper respiratory tract of 32 A. pleuropneumoniae serotype 2 field isolates was examined using Fourier transform infrared (FTIR) spectroscopy as a high resolution metabolic fingerprinting method. All strains showed metabolic adaptations to organ tissue reflected by hierarchical cluster analysis of FTIR spectra similar to those previously observed under experimental conditions. Notably, differences in antimicrobial resistance patterns and minimal inhibitory concentrations of isolates from different tissues in the same animal, but not in biofilm production capability in a microtiter plate assay were found. Overall, biofilm formation was observed for 71 % of the isolates, confirming that A. pleuropneumoniae field isolates are generally able to form biofilms, although rather in a serotype-specific than in an organ-specific manner. A. pleuropneumoniae serotype 6 isolates formed significantly more biofilm than the other serotypes. Furthermore, biofilm production was negatively correlated to the lung lesion scores and tonsillar isolates tended to be more susceptible to antimicrobial substances with high bioavailability than lung isolates.

Actinobacillus pleuropneumoniae serotypes in Hungary.

A total of 255 Actinobacillus pleuropneumoniae isolates were collected from 634 lung samples representing 70 swine herds in Hungary between January 2012 and June 2016. On the basis of the indirect haemagglutination test 77 independent strains were included in the evaluation after the elimination of duplicate or multiple serotypes from the same herd. In the case of 7 herds strains of two different serotypes were identified. Fourteen Hungarian A. pleuropneumoniae isolates from the culture collection of the Department of Microbiology and Infectious Diseases, isolated before 2012, were also included in the evaluation (one each from 12 herds and two each from two herds, where two serotypes occurred). Out of the altogether 91 A. pleuropneumoniae strains 72 strains belonged to biotype I and 19 strains could be allocated to biotype II. In Hungary, the most common serotypes were serotype 2 (39.5%), 13 (15.4%), 8 (8.8%) and 16 (8.8%), but serotypes 9 (5.5%), 11 (3.3%) and 12 (3.3%) were also isolated. Twelve strains (13.2%) were untypable.

Nucleotide sequence analysis of a DNA region involved in capsular polysaccharide biosynthesis reveals the molecular basis of the nontypeability of two Actinobacillus pleuropneumoniae isolates.

The aim of our study was to reveal the molecular basis of the serologic nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1-15. Strain FH24-5 showed positive results in 2 serovar 15-specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serologic and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serologic nontypeability in A. pleuropneumoniae.

Serotyping reanalysis of unserotypable Actinobacillus pleuropneumoniae isolates by agar gel diffusion test.

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

Sub-inhibitory concentrations of penicillin G induce biofilm formation by field isolates of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and causative agent of porcine pleuropneumonia. This is a highly contagious disease that causes important economic losses to the swine industry worldwide. Penicillins are extensively used in swine production and these antibiotics are associated with high systemic clearance and low oral bioavailability. This may expose A. pleuropneumoniae to sub-inhibitory concentrations of penicillin G when the antibiotic is administered orally. Our goal was to evaluate the effect of sub-minimum inhibitory concentration (MIC) of penicillin G on the biofilm formation of A. pleuropneumoniae. Biofilm production of 13 field isolates from serotypes 1, 5a, 7 and 15 was tested in the presence of sub-MIC of penicillin G using a polystyrene microtiter plate assay. Using microscopy techniques and enzymatic digestion, biofilm architecture and composition were also characterized after exposure to sub-MIC of penicillin G. Sub-MIC of penicillin G significantly induced biofilm formation of nine isolates. The penicillin G-induced biofilms contained more poly-N-acetyl-d-glucosamine (PGA), extracellular DNA and proteins when compared to control biofilms grown without penicillin G. Additionally, penicillin G-induced biofilms were sensitive to DNase which was not observed with the untreated controls. Furthermore, sub-MIC of penicillin G up-regulated the expression of pgaA, which encodes a protein involved in PGA synthesis, and the genes encoding the envelope-stress sensing two-component regulatory system CpxRA. In conclusion, sub-MICs of penicillin G significantly induce biofilm formation and this is likely the result of a cell envelope stress sensed by the CpxRA system resulting in an increased production of PGA and other matrix components.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

ObjectivesThe objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England.MethodsClinical 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.ResultsA 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.ConclusionsThis 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.

Multiplex PCR assay for unequivocal differentiation of Actinobacillus pleuropneumoniae serovars 1 to 3, 5 to 8, 10, and 12.

An improved multiplex PCR, using redesigned primers targeting the serovar 3 capsule locus, which differentiates serovars 3, 6, and 8 Actinobacillus pleuropneumoniae isolates, is described. The new primers eliminate an aberrant serovar 3-indicative amplicon found in some serovar 6 clinical isolates. Furthermore, we have developed a new multiplex PCR for the detection of serovars 1 to 3, 5 to 8, 10, and 12 along with apxIV, thus extending the utility of this diagnostic PCR to cover a broader range of isolates.

Serotypes and antimicrobial resistance patterns of the recent Korean Actinobacillus pleuropneumoniae isolates

In Korea, the addition of antibiotics to feed was banned in July 2011; since then, reports of outbreaks of porcine pleuropneumonia have increased. Therefore, knowledge of the characteristics of clinical...

Resistance to selected beta-lactam antibiotics

Susceptibility in vitro and trends in resistance to antimicrobials were determined by a dilution micromethod in a group of Actinobacillus pleuropneumoniae, Pasteurella multocida, Mannheimia haemolytica and Escherichia coli isolates from clinical cases of cattle and swine diseases in the Czech Republic from 2007 to 2011. A high susceptibility of pig and cattle respiratory pathogens to antimicrobials was found, with the exception of the moderate prevalence of M. haemolytica resistance to ampicillin. In contrast to respiratory pathogens, low susceptibility of E. coli of pig and cattle isolates to ampicillin and amoxicillin/clavulanic acid was noted. Regarding resistance trends, an increase in levels of resistance among E. coli isolates to ampicillin and amoxicillin/clavulanic acid was identified, but the resistance of respiratory isolates was low, with the exception of M. haemolytica.For the period of 2007–2011, there was a significant and almost continuous increase in sales (compared with population correction unit) of ceftiofur, cefquinome and other beta lactams for pigs. Consumption peaked in 2010. In the case of amoxicillin in combination with clavulanic acid, data showed a significant decrease in sales from 2007 to 2008, followed by a period of fluctuation. In cattle, within the groups of 3rd and 4th generation cephalosporins and for the whole group of other betalactams for the period of 2007–2011, there was a significant and almost continuous increase in sales (compared with population correction unit). Consumption peaked in 2010. In the case of ceftiofur, there was a huge increase noted from 2010. In the case of amoxicillin in combination with betalactamase inhibitor (clavulanic acid) data shows a significant decrease from 2007 to 2008, followed by a period of fluctuation in sales.

Comparative activities of selected fluoroquinolones against dynamic populations of Actinobacillus pleuropneumoniae in an in vitro model of time–kill continuous culture experiment

The aim of the current study was to demonstrate and compare the impact of different pharmacokinetics of marbofloxacin, enrofloxacin and difloxacin on their antimicrobial effects, their killing and re-growth kinetics, and the population dynamics of Actinobacillus pleuropneumoniae clinical isolates in an in vitro dynamic model. Selected clinical isolates of A. pleuropneumoniae and three fluoroquinolones at a range of simulated AUC24/MIC ratios of multiple doses were investigated. At the same simulated AUC24/MIC ratios of the three fluoroquinolones, the killing re-growth profile and IE values (intensity of the antimicrobial effect) revealed strain- and fluoroquinolone-specific effects. For example, a 31% lower IE of difloxacin was observed in AppK5 (biofilm-former) than in AppK2 (biofilm-non-former) at the same AUC24/MIC ratio of 120h. In addition, losses in A. pleuropneumoniae susceptibility of both strains by the three fluoroquinolones were observed. AUC24/MPC ratios of 20.89 and 39.81 for marbofloxacin, 17.32 and 19.49 for enrofloxacin and 31.62 and 60.25 for difloxacin were estimated to be protective against the selection of AppK2 and AppK5 strain mutants, respectively. Integration of these in vitro data with published pharmacokinetics revealed the inadequacy of the conventional clinical doses of the three drugs to attain the above protective values for minimum biofilm eradication concentration (MBEC) and concentration to prevent growth of 90% of the mutant subpopulation (MPC90). In conclusion, the results suggest optimising doses could suffice for resistant mutants control, while for biofilm-forming strains combination with biofilm-disrupting agents to reduce the MBEC to achieve AUC/MBEC ratios within the possible dosing regimens is desired.

Pharmacokinetics of tildipirosin in porcine plasma, lung tissue, and bronchial fluid and effects of test conditions on in vitro activity against reference strains and field isolates of Actinobacillus pleuropneumoniae

The pharmacokinetics of tildipirosin (Zuprevo(®) 40 mg/mL solution for injection for pigs), a novel 16-membered-ring macrolide for the treatment for swine respiratory disease (SRD), was investigated in studies collecting blood plasma and postmortem samples of lung tissue and bronchial fluid (BF) from swine. In view of factors influencing the in vitro activity of macrolides, and for the interpretation of tildipirosin pharmacokinetics in relation to minimum inhibitory concentrations (MIC), additional experiments were conducted to study the effects of pH, carbon dioxide-enriched atmosphere, buffers, and serum on tildipirosin MICs for various reference strains and Actinobacillus (A.) pleuropneumoniae field isolates. After single intramuscular (i.m.) injection at 4 mg/kg body weight, maximum plasma concentration (Cmax) was 0.9 μg/mL observed within 23 min (Tmax ). Mean residence time from the time of dosing to the time of last measurable concentration (MRTlast) and terminal half-life (T1/2) both were about 4 days. A dose-response relationship with no significant sex effect is observed for area under the plasma concentration-time curve from time 0 to the last sampling time with a quantifiable drug concentration (AUClast) over the range of doses up to 6 mg/kg. However, linear dose proportionality could not be proven with statistical methods. The time-concentration profile of tildipirosin in BF and lung far exceeded that in blood plasma. In lung, tildipirosin concentrations reached 3.1 μg/g at 2 h, peaked at 4.3 μg/g at day 1, and slowly declined to 0.8 μg/g at day 17. In BF, tildipirosin levels were 14.3, 7.0, and 6.5 μg/g at days 5, 10, and 14. T1/2 in lung was ∼7 days. Tildipirosin is rapidly and extensively distributed to the respiratory tract followed by slow elimination. Culture media pH and carbon dioxide-enriched atmosphere (CO2 -EA) had a marked impact on in vitro activity of tildipirosin in reference strains of various rapidly growing aerobic and fastidious bacteria including Histophilus (H.) somni ATCC 700025 and A. pleuropneumoniae ATCC 27090. For A. pleuropneumoniae ATCC 27090 testing conditions without CO2 -EA resulted in reduced acidification of culture media pH and a reduction in the minimum inhibitory concentrations compared to standard in vitro test conditions by 2 log2 dilution steps (4-fold) from 8 to 2 μg/mL. Supplementary buffering of standard culture media resulted in a reduction in the A. pleuropneumoniae (n = 8) MIC range by 4 log2 dilution steps (16-fold) from 8-16 to 0.5-1 μg/mL. Incremental supplementation of culture media with 50% serum resulted in noticeable shifts to lower minimum or maximum MICs by at least 2 log2 dilution steps (≥4-fold) in all aerobic and fastidious reference strains tested except for Pasteurella (P.) multocida. The MIC of A. pleuropneumoniae ATCC 27090 decreased by 2-4 log2 dilution steps (4 to 16-fold) from 8 to 0.5-2 μg/mL when 50% serum was added to the standard assay. Considering a higher presence of serum and the rather neutral pH conditions maintained in vivo, it is suggested to take the influence of these factors on in vitro activity into account when interpreting tildipirosin MICs for A. pleuropneumoniae in relation to pharmacokinetics.

Determination of minimum inhibitory and minimum bactericidal concentrations of tiamulin against field isolates of Actinobacillus pleuropneumoniae

Tiamulin activity was measured against 19 UK field isolates of Actinobacillus pleuropneumoniae collected between 2003 and 2009 and the type strain ATCC 27090 as a control, with the intention of comparing broth with serum as growth media. Broth microdilution MIC/MBC tests were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3, in ‘Veterinary Fastidious Medium’ (VFM) (supplemented Mueller–Hinton broth at pH 7.3) and in 100% swine serum. For improved precision, a modified, overlapping doubling-dilution series was used (tiamulin concentration range 0.3–72 μg/ml). The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial density in the sub-cultured well contents, relative to the starting inoculum. The mean MBC/MIC ratio for tiamulin against A. pleuropneumoniae in VFM was low (1.74:1), even though tiamulin is classed as a bacteriostatic drug. Only three of the 19 isolates and the reference strain grew in 100% serum and their MICs were higher than those determined in VFM. It is postulated that this difference was due to differences in pH of the matrices or binding of tiamulin to serum proteins or a combination of both factors.

Microarray-based comparative genomic profiling of reference strains and selected Canadian field isolates of Actinobacillus pleuropneumoniae

BackgroundActinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is a highly contagious respiratory pathogen that causes severe losses to the swine industry worldwide. Current commercially-available vaccines are of limited value because they do not induce cross-serovar immunity and do not prevent development of the carrier state. Microarray-based comparative genomic hybridizations (M-CGH) were used to estimate whole genomic diversity of representative Actinobacillus pleuropneumoniae strains. Our goal was to identify conserved genes, especially those predicted to encode outer membrane proteins and lipoproteins because of their potential for the development of more effective vaccines.ResultsUsing hierarchical clustering, our M-CGH results showed that the majority of the genes in the genome of the serovar 5 A. pleuropneumoniae L20 strain were conserved in the reference strains of all 15 serovars and in representative field isolates. Fifty-eight conserved genes predicted to encode for outer membrane proteins or lipoproteins were identified. As well, there were several clusters of diverged or absent genes including those associated with capsule biosynthesis, toxin production as well as genes typically associated with mobile elements.ConclusionAlthough A. pleuropneumoniae strains are essentially clonal, M-CGH analysis of the reference strains of the fifteen serovars and representative field isolates revealed several classes of genes that were divergent or absent. Not surprisingly, these included genes associated with capsule biosynthesis as the capsule is associated with sero-specificity. Several of the conserved genes were identified as candidates for vaccine development, and we conclude that M-CGH is a valuable tool for reverse vaccinology.