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Surveillance of Pasteurella multocida resistance in food-producing animals is essential to guide the first-line treatment of respiratory diseases and to limit economic losses. Since Pasteurella are the most common bacteria isolated from dog and cat bites, this surveillance is also needed to guide treatment in humans in case of bites. The aim of this study was to characterize the phenotypic resistance of P. multocida strains isolated from respiratory infections in animals, including both food-producing animals and pets. Data collected between 2012 and 2017 by the French national surveillance network for antimicrobial resistance referred to as RESAPATH were analyzed. The proportions of resistance to antimicrobials of relevance in veterinary and human medicines were estimated for each animal species. For cattle, resistance trends over the period were investigated using non-linear analysis applied to time-series. In total, 5356 P. multocida isolates were analyzed. Proportions of resistance of P. multocida were almost all below 20% over the period, and, more precisely, all resistance proportions were below 10% for rabbits, sheep and dogs. The highest resistance proportions to enrofloxacin were identified for cattle (4.5%) and dogs (5.2%). Despite its frequent use in livestock, resistance to florfenicol was less than 1% in P. multocida strains, regardless of the animal species considered. Time series analyses revealed continuous increases in resistance to tetracycline, tilmicosin, flumequine and fluoroquinolones in P. multocida strains isolated from cattle. These trends contrast with the decrease in use of antibiotics in cattle in France and with the decrease in resistance observed in E. coli isolated from diseased cattle., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pasteurella multocida causes single or complex respiratory disease in pigs. Although antimicrobial therapy is the most effective treatment for porcine respiratory disease, P. multocida shows increased antimicrobial resistance in Korea. Therefore, we aimed to investigate the phenotypic and genotypic characterization of tetracycline-resistant P. multocida. Thirty-seven of 454 P. multocida isolates from South Korea between 2010 and 2016 were selected. Four tet genes [tet(B) (78.4%), tet(H) (16.2%), tet(C) (5.4%), and tet(O) (2.7%)] were observed. This is the first report of tet(C) in P. multocida. Various virulence factors were observed in both tetracycline-resistant and -susceptible P. multocida isolates. Genes encoding pmHAS and pfhA were more prevalent in tetracycline-resistant than in tetracycline-susceptible isolates. Some virulence factors exhibited association with serogroups. tadD and sodA were common in serogroup A, while hsf-l was significantly associated with serogroup D (p < 0.01). Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) results showed the genetic diversity of tetracycline-resistant P. multocida. MLST showed six different sequence types (ST), with clonal complex 13 encompassing 56.8% of the strains. PFGE was more efficient in differentiating the isolates, and 29 PFGE patterns of the strains were observed. By combining these methods, identical STs and PFGE patterns were observed in isolates from different farms, suggesting that transmission of antimicrobial-resistant P. multocida strains between farms might occur in a geographically discrete population. In future, epidemiological approaches and development of effective vaccines should focus on the major clonal lineages carrying the important virulence factors and frequently observed resistance genes to prevent the transmission and control the disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pasteurella multocida is an important respiratory tract pathogen in intensive livestock farming, especially in pigs. Antimicrobial agents are frequently used to combat infections caused by this pathogen. In a study on antimicrobial resistance among respiratory tract pathogens of pigs from 30 German pig-producing farms, P. multocida isolates (n = 9) with high minimal inhibitory concentration (MIC) values of 16/304 mg/L (n = 2), 32/608 mg/L (n = 3) or ≥64/1216 mg/L (n = 4) for trimethoprim/sulfamethoxazole (1:19) and of ≥512 mg/L (n = 9) for trimethoprim (TMP) were detected in three of these farms. The genetic relatedness of the isolates was investigated via capsule-specific PCR and macrorestriction analyses with ApaI and SmaI. Pulsed-field gel electrophoresis revealed indistinguishable restriction patterns per farm, with slight differences between the three farms. All isolates represented capsular type A. Four representative isolates, that were subjected to whole genome sequencing, shared the multi-locus sequence type (ST) 3. Their plasmids were transformed into E. coli TOP10 with subsequent selection on TMP-containing agar plates. Antimicrobial susceptibility testing and plasmid analysis of the transformants confirmed that they were resistant to sulfonamides and trimethoprim and carried only a single small plasmid. This plasmid was completely sequenced and revealed a size of 6050 bp. Sequence analyses identified the presence of a resistance gene cluster comprising the genes sul2-ΔstrA-dfrA14-ΔstrA-ΔstrB. Further analysis identified a dfrA14 gene cassette being integrated into the strA reading frame. Neither the gene dfrA14 nor this gene cluster have been detected before in P. multocida., (Copyright © 2019. Published by Elsevier B.V.)

VetPath is an ongoing pan-European antibiotic susceptibility monitoring programme collecting pathogens from diseased antimicrobial non-treated cattle, pigs and poultry. In the current study, 1001 isolates from cattle and pig respiratory tract infections were tested for their antimicrobial susceptibilities. Non-replicate lung samples or nasopharyngeal/nasal swabs were collected from animals with acute clinical signs in 11 countries during 2002-2006. Pasteurella multocida and Mannheimia haemolytica from cattle and P. multocida, Actinobacillus pleuropneumoniae and Streptococcus suis from pigs were isolated by standard methods. S. suis was also isolated from meningitis cases. MICs of 16 antibiotics were assessed centrally by broth microdilution following CLSI recommendations. Results were interpreted using CLSI breakpoints where available. P. multocida (231) and M. haemolytica (138) isolates were all susceptible to amoxicillin/clavulanic acid, ceftiofur, enrofloxacin and trimethoprim/sulfamethoxazole. Resistance to florfenicol and spectinomycin was 0.4% and 3.5% in P. multocida, respectively, and absent in M. haemolytica isolates. Tetracycline resistance was 5.7% and 14.6% for P. multocida and M. haemolytica. In pigs, 230 P. multocida, 220 A. pleuropneumoniae and 182 S. suis isolates were recovered. Resistance to amoxicillin/clavulanic acid, ceftiofur, enrofloxacin, florfenicol, tiamulin and tilmicosin was absent or <1%. Trimethoprim/sulfamethoxazole resistance was 3-6% and tetracycline resistance varied from 14.7% in A. pleuropneumoniae to 81.8% in S. suis. In conclusion, low resistance to antibiotics with defined clinical breakpoints, except for tetracycline, was observed among the major respiratory tract pathogens recovered from cattle and pigs. Since for approximately half of the antibiotics in this panel no CLSI-defined breakpoints were available, setting of the missing veterinary breakpoints is important., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A total of 378 isolates of Pasteurella multocida from clinically healthy and diseased calves were characterised for their susceptibility to 9 antimicrobial agents and screened by PCR for the presence of antimicrobial resistance genes and 22 genes virulence-associated, including capsule biosynthesis genes. Of the 378 isolates, 102 (27.0%) were resistant to at least one of the 9 tested antimicrobial agents. Resistance to oxytetracycline (21.7%) was the most frequently observed phenotype among the isolates. The tet(H) gene were the primary determinant detected. The resistance rates for thiamphenicol, ampicillin, kanamycin and florfenicol were 13.2%, 5.8%, 9.0% and 0.5%, respectively. Cefazolin, ceftiofur, cefquinome and enrofloxacin were effective antimicrobial agents, with no resistant isolates emerging over the course of the investigation. Most isolates were identified as capsular type A, only 6.3% belonged to capsular type D and no other capsular type was identified. Four of the virulence-associated genes (pfhA, tadD, tbpA and HAS) exhibited associations to the capsular type, and three (pfhA, tbpA and hgbB) were associated with the disease status of the animals. These virulence genes have been considered as epidemiological markers and are hypothesised to have a strong positive association with the outcome of disease in cattle., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Bacterial pneumonia is a common and often life-threatening respiratory problem in both meat and dairy goats. Options for approved antibiotic therapy in goats to combat these bacterial infections are severely limited and frequently drugs must be used in an extra-label manner. Tulathromycin, a triamilide macrolide antimicrobial drug shown to be effective against swine and cattle respiratory bacterial agents, has been identified as a potentially useful drug in caprines. The present study was conducted to determine the susceptibility of recognized bacterial respiratory pathogens to commonly prescribed antimicrobials, with a particular emphasis on the efficacy of tulathromycin against these agents. Minimum inhibitory concentration (MIC) testing using microbroth dilution was performed on a collection of 45 Mannheimia haemolytica, 11 Pasteurella multocida, and 11 Bibersteinia trehalosi isolates from the lungs of goats with clinical pneumonia. To further characterize efficacy of tulathromycin against these pathogens, minimum bactericidal concentration (MBC) testing and kinetic killing assays were conducted. Most isolates were susceptible to the antimicrobials tested; however, increased resistance as demonstrated by higher MIC values was seen in all species to penicillin, in P. multocida to sulfadimethoxine, and in B. trehalosi to the tetracyclines. All isolates were susceptible to tulathromycin, which demonstrated a high killing efficiency in both bactericidal assays. Results of this study indicate that most goat pneumonic bacterial pathogens remain susceptible to commonly prescribed antibiotics, although some evidence of resistance was seen to certain drugs; and that tulathromycin is highly effective against goat respiratory pathogens which could make it a valuable medication in this species., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The early curative uses of antimicrobial drugs such as fluoroquinolones before the onset of symptoms in veterinary medicine may be regarded as irrational antibiotic consumption. However, it should be stressed that in early curative antimicrobial treatment as in metaphylaxis, the bacterial burden at the infection site is often very low, and so the rapid eradication of the bacterial population could result. We investigated the impact of early versus later curative administrations of 1 or 40 mg/kg of marbofloxacin on the survival of mice, the eradication of the targeted pathogen and the selection of resistant bacteria in a mouse lung infection with Pasteurella multocida. In this model, for a given marbofloxacin dose, the clinical and bacteriological outcomes were better, and the selection of resistance less frequent, for the early rather than for the late treatment. Moreover, the early administration of 1mg/kg led to better clinical and similar bacteriological (eradication and selection of resistance) outcomes than the late administration of 40 mg/kg marbofloxacin. Our results suggest that the optimal doses for the animals' cure could be lower when administered early during the time course of the infection than when administered after the disease outbreak. As the main argument against early treatments such as metaphylaxis is the possible enhancement of resistance at the gut level, further studies should assess if lower doses of antibiotic administered to all the animals of a herd could have less impact on the commensal digestive flora than higher doses only administered to animals showing clinical symptoms., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The usage of antimicrobials for treatment of mink and the occurrence of antimicrobial resistance among the most important bacterial pathogens in mink was investigated. The aim of the study was to provide data, which may serve as a basis for the formulation of recommendations for prudent use of antimicrobials for mink. A total of 164 haemolytic staphylococci, 49 haemolytic streptococci, 39 Pseudomonas aeruginosa, 13 Pasteurella multocida, and 1093 Escherichia coli isolates from Danish mink were included in the study. A high frequency of resistance among S. intermedius was found for tetracyclines (54.7%), followed by penicillin (21.7%), lincosamides (20.4%), macrolides (19.1%), and spectinomycin (18.5%). Very low frequencies of resistance were recorded for other antimicrobials. The highest frequency among the E. coli isolates was recorded for ampicillin, streptomycin, sulphonamides, and tetracyclines, whereas resistance to other antimicrobials was rare. All P. aeruginosa were sensitive to gentamicin and colistin and sensitive or intermediate to enrofloxacin, whereas most isolates were resistant to all other antimicrobials. All P. multocida and haemolytic streptococci were sensitive to penicillin. There was a steady increase in the use of antimicrobials during the period 2001-2006, the majority of the prescribed amount being extended spectrum penicillins followed by aminoglycosides, sulphonamides with trimethoprim, and macrolides.

The transcriptional responses of Pasteurella multocida to eight antibiotics with known mode of actions (MoAs) and one novel antibiotic compound with an unknown MoA were collected to create a compendium of transcriptional profiles for MoA studies. At minimal inhibitory concentration the three bactericidal compounds enrofloxacin, cefquinome and the novel compound had a minor impact on gene regulation with approximately 1% of the P. multocida genome affected, whilst the bacteriostatic compounds florfenicol, tilmicosin, rifampin, trimethoprim and brodimoprim regulated 20% of the genome. Novobiocin was special in that it regulated 40% of all P. multocida genes. Regulation of target genes was observed for novobiocin, rifampin, florfenicol and tilmicosin and signature genes were identified for most antibiotics. The transcriptional profile induced by the novel compound was unrelated to the compendium profiles suggesting a new MoA. The transcription of many P. multocida virulence factors, particularly genes involved in capsule synthesis and export, LPS synthesis, competence, adherence and iron transport were altered in the presence of antibiotics. Virulence gene transcription was mainly negatively affected, however the opposite effect was also observed in the case of rifampin where the up-regulation of the tad locus involved in tight adherence was seen. Novobiocin and trimethoprim caused a marked reduction in the transcription of capsule genes, which correlated with a concomitant reduction of the capsular layer on the surface of P. multocida. The broad negative impact on virulence gene transcription supports the notion that the therapeutic effect of some antibiotics could be a combination of growth and virulence inhibition.

In vitro susceptibility tests were conducted on bovine and porcine respiratory pathogens isolated from European countries during 2004-2006 for susceptibility to tulathromycin using the recommended methodologies for broth microdilution. The results were compared with data from a similar survey conducted prior to launch in 1998-2001 to monitor for any shift in susceptibility. The importance of maintaining the pH of the culture media within the range 7.2-7.4 was re-affirmed as a key factor in obtaining consistent minimum inhibitory concentration data. The use of recently established interpretative breakpoints would indicate that to date there has been no apparent decrease in susceptibility to tulathromycin since it became widely used across Europe.

Pasteurella multocida exhibits nonspecific susceptibility to nonpolar antimicrobial agents such as triclosan, despite possessing an ultrastructurally typical gram-negative cell envelope. Capsulated and noncapsulated cell surface variants were examined to investigate the role outer membrane permeability plays in triclosan susceptibility. Test strains were unable to initiate growth in the presence of bile salts and were susceptible to triclosan with minimal inhibitory concentrations (MICs) ranging from 0.06 to 0.25 microg/ml. Disk agar diffusion bioassays revealed triclosan susceptibility to be dose dependent and all strains were susceptible to the hydrophobic antibiotics novobiocin, rifamycin SV, and chloramphenicol. Triclosan minimal bactericidal concentrations were greater than MICs, thereby suggesting that dose dependency reflected both bacteriostatic and bactericidal effects. Total and viable cell density growth kinetic determinations revealed a triclosan concentration of 2.0 microg/ml resulted in loss of batch culture viability within 4-24 h. Concentrations of 0.02 and 0.2 microg/ml exerted either a bacteriostatic or bactericidal effect depending on the strain. Uptake of the hydrophobic probe 1-N-phenylnaphthylamine was greater in P. multocida strains than refractory control organisms Pseudomonas aeruginosa and Escherichia coli thereby suggesting the presence of phospholipid bilayer regions in the outer membrane. Because triclosan inhibits a conserved enoyl-ACP reductase necessary for bacterial fatty acid biosynthesis, these data support the notion that extreme susceptibility in P. multocida is due to the general inability of the outer membrane to exclude nonpolar compounds. Moreover, susceptibility is independent of the presence of capsular material and the biocide is bactericidal in a concentration dependent manner.

The antimicrobial activity of linear, cationic alpha-helical peptides from cattle (BMAP28), sheep (SMAP28 and SMAP29), and pigs (PMAP23) were assessed to determine if activity was selective for Pasteurella multocida from a particular animal species or broad-spectrum against all P. multocida tested. The antimicrobial activities of synthetic peptides were determined for P. multocida isolated from cattle (10 isolates), sheep (10 isolates), and pigs (10 isolates) in a broth microdilution assay. All thirty isolates of P. multocida were susceptible to BMAP28 (MICs and MBCs, 1.0-1.9 microM); SMAP28 and SMAP29 (MICs and MBCs, 0.2-0.7 microM); and PMAP23 (MICs and MBCs, 4.3 to > or = 6.8 microM). Overall, the results of this study suggest that synthesized cathelicidins from cattle, sheep, and pigs had broad-spectrum antimicrobial activity against all P. multocida.

For 8 months, 121 pet rabbits of more than 2 months old were included in an epidemiological study aimed at determining the nature, prevalence and bacteriological susceptibility of pathogenic bacteria responsible for upper respiratory tract disease ("snuffles"). All rabbits presented with nasal discharge and sneezing at inclusion and had not received any antibiotics in the 30 days prior to the study. Nasal samples were taken from all the rabbits before they received any treatment. Isolation of bacterial strains, susceptibility testing by disk diffusion for marbofloxacin, enrofloxacin, danofloxacin, gentamicin, oxytetracycline, doxycycline, cefalexin, trimethoprim-sulfamethoxazole, and marbofloxacin MIC determination for each pathogenic bacterium were also performed. The main bacterial strains isolated were Pasteurella multocida (54.8%), Bordetella bronchiseptica (52.2%), Pseudomonas spp. (27.9%) and Staphylococcus spp. (17.4%). Snuffles was mainly due to a polybacterial infection, and the most frequently found combination was P. multocida and B. bronchiseptica (28.9% of rabbits). Marbofloxacin was shown to be the most effective agent against all bacterial strains (between 87.8% and 100% susceptibility according to strain) except B. bronchiseptica, for which gentamicin was slightly more effective (96% versus 88.9%). Compared to other fluoroquinolones tested, marbofloxacin exhibited the highest level of activity. Marbofloxacin MIC(90) was equivalent to 1.320, 0.079, 1.741 and 0.490microg/ml for B. bronchiseptica, P. multocida, Pseudomonas spp. and Staphylococcus spp. strains, respectively. In this study, marbofloxacin was shown to be a potentially good treatment option for upper respiratory tract disease in pet rabbits.

The broad host-range cloning vectors, pJRD215 and pMMB67EH, were evaluated for stability and cloning efficiency in Pasteurella multocida. Transformation of P. multocida by electroporation was unreliable and poorly efficient regardless of whether the transforming DNA was isolated from E. coli or P. multocida. Both vectors contain a mob site that enabled transfer by conjugation from E. coli to P. multocida with high efficiency. Kanamycin, streptomycin, and ampicillin resistance encoded by the vectors were expressed in P. multocida. LacZ was cloned in pMMB67EH, an expression vector, and was transferred to P. multocida by conjugation. The transconjugants expressed a functional beta-galactosidase as determined by o-nitrophenyl-beta-D-galactopyranoside (ONPG) test. We propose the use of these cosmid and expression vectors as a shuttle vectors for cloning in P. multocida.

A collection of 45 strains of Pasteurella multocida was assembled. The strains had been isolated from cases of fowl cholera in eastern Australia over 8 years, and included mainly type A strains. All the strains were examined for plasmids and resistance to 10 antimicrobial agents and most of the strains were examined for restriction fragment length polymorphism. Nine strains were assayed for pathogenicity for mice. Twenty strains yielded no plasmid. Seven contained a single plasmid of 1.3 kbp and 18 contained 2 plasmids, of 2.4 and 7.5 kbp. All the strains were resistant to streptomycin, trimethoprim and lincomycin while one strain was resistant to tetracycline. There was no correlation between plasmid content and resistance to antimicrobial agents. Three strains that lacked plasmids were highly virulent for mice, 6 strains containing plasmids were not. Restriction fragment length polymorphism generated by HpaII allowed the 39 strains that were tested to be divided into 10 groups.

A colorimetric assay using sodium 3,3'-[1[(phenylamino)carbonyl]3,4- tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) was adapted to quantitate bactericidal activity of chicken macrophage HD 11 cell line against five Pasteurella multocida strains and an avirulent transposon insertion mutant. The strains used were virulent P1059, and D92, and four avirulent strains including a streptomycin resistant mutant of P1059 (P1059 SmR), two live vaccine strains namely, the Clemson University (CU) and M9, and a transposon insertion mutant PmTn-294. Percentage of bacteria killed by chicken macrophage (HD 11) cells was determined by extrapolation from a standard formazan curve derived by incubating XTT with known bacterial cell numbers of each strain. The amount of formazan as measured by absorption at 450 nm was directly related to the number of viable bacterial cells. The percentages of P1059 SmR, CU, M9 and PmTn-294 killed by HD 11 cells were approximately 50%, 61%, 25% and 34%, respectively. By contrast, the virulent P1059 and D92 strains were resistant to killing, and were able to replicate inside the HD 11 cells. Association of virulence with resistance to phagocytic killing by HD 11 cells as assessed by the colorimetric bactericidal assay, was validated with resistance to complement (C')-mediated killing and a turkey mortality test. Strains P1059 and D92 were resistant to C'-mediated killing, whereas strains P1059 SmR, CU, M9 and PmTn-294 strains were susceptible. All turkeys challenged with P1059 or D92 were dead within 18 hrs. Mortality did not occur in turkeys challenged with strains of P1059 SmR, M9 and PmTn-294. The mortality among CU challenged turkeys ranged from 0 to 40%. The results suggest that the colorimetric bactericidal assay using XTT can be used to quantitate chicken macrophage phagocytic killing of P. multocida strains, and may be a valuable assay to differentiate virulent from avirulent strains of avian P. multocida.