Pasteurella Multocida Isolates Research Articles

Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves

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.

Characterisation of avian Pasteurella multocida strains with PCR-RFLP analysis of the ompH gene

The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

Characterization of Pasteurella multocida strains isolated from geese

Phenotypic and genotypic diversity of forty-two Pasteurella multocida isolates from geese were characterized by analysis of their capsular type, Heddleston serotype, biotype, fimbrial gene allele type, comparative outer membrane protein (OMP) electrophoresis patterns, and were analyzed using PCR for the presence of virulence-associated genes (toxA, tbpA, pfhA, hgbA, hgbB, nanH, nanB, fimA, hsf-1, and pmHAS). A sequence comparison of the thdF and rpoB housekeeping genes of twenty representative P. multocida strains from three different OMP groups demonstrated that seventeen strains were closely related phylogenetically to previously published strains of P. multocida subsp. multocida and P. multocida subsp. gallicida, and only three strains from geese were grouped with previously published strains of P. multocida subsp. septica. This study is the first report regarding the characterization of phenotypic and genotypic features of different P. multocida field strains isolated from geese with the different clinical representation of pasteurellosis and provide our overview on the usefulness of these in vitro tests for primary characterization of P. multocida strains for the epidemiological studies of fowl cholera.

Bovine respiratory disease complex (BRDC): Viral and bacterial pathogens in Serbia

Pathogens causing BRDC in Serbia were investigated. Two herds of beef cattle with bovine respiratory disease were included, with twenty diseased calves (10 from each farm) were chosen for isolation of bacteria on artificial culture media and determination by aerobic cultivation. The most common bacterial pathogen was isolated was Pasteurella multocida. Diffusion method of sensitivity to antibiotics (antibiogram), revealed that Enrofloxacin and Floron were most efficient antibiotics against Pasteurella multocida isolates (100 % isolates sensitive on both antibiotics). From the all examined samples (n=20) using the method of Real Time PCR (RT-PCR and PCR) we determined the genome sequences of bovine respiratory syncytial virus (BRSV), but in none of the samples genome of bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BoHV-1).

Antibacterial activity of successive extracts of some medicinal plants against field isolates of Pasteurella multocida

The outbreak of duck pasteurellosis caused by avian strains of Pasteurella multocida, which occur during monsoon period, is a serious problem with high mortality and morbidity affecting younger age groups. Medicinal plants reported to have antibacterial action were tested for their efficacy against Pasteurella multocida by in vitro methods. Successive extraction of whole plant materials of Leucas aspera, Vernonia cineria and Ocimum sanctum, leaves of Syzigium cumini, and bark of Polyalthia longifolia were performed with petroleum benzine, choloroform, acetone, methanol and water, and were tested for their antibacterial effect using microtitre plate technique (to estimate the minimum inhibitory concentration, MIC) and disc diffusion method (to estimate the zone of inhibition). The results from the above in vitro assay revealed the efficacy of different plant successive extracts against duck pasteurellosis.

Molecular Epidemiologic Investigation of Polish Avian Pasteurella multocida Strains Isolated from Fowl Cholera Outbreaks Showing Restricted Geographical and Host-Specific Distribution

Molecular epidemiologic analyses of the 42 clinical isolates of Pasteurella multocida from various avian hosts (geese, ducks, turkeys, and laying hens) in Poland from 2001 to 2011, including a single reference strain, were performed by enterobacterial repetitive intergenic consensus (ERIC)-PCR, single primer PCR, and repetitive extragenic palindromic (REP)-PCR. Forty-two isolates were identified as P. multocida (serotype A). The majority of P. multocida strains were obtained from waterfowl clustered within one genotype, and they were not consistent with the genotypes obtained from the turkey strains. Pasteurella multocida showed genetic homogeneity between the host species, especially when isolated on the same farm. Some of the clones also were characteristic to the particular farm. The strains obtained in different regions represent distinct molecular patterns. The present findings demonstrate that some clones of P. multocida are restricted in geographical and host distribution. In addition, this study suggests that ERIC-PCR, single primer PCR, and REP-PCR are suitable techniques for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

A ten-year (2000–2009) study of antimicrobial susceptibility of bacteria that cause bovine respiratory disease complex—Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni—in the United States and Canada

Bovine isolates of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, collected from 2000 to 2009, were tested for in vitro susceptibility to ceftiofur, penicillin, danofloxacin, enrofloxacin, florfenicol, tetracycline, tilmicosin, and tulathromycin. Ceftiofur remained very active against all isolates. Penicillin retained good activity against P. multocida and H. somni isolates with no appreciable changes in susceptibility or minimal inhibitory concentration (MIC) distributions with time. While there was no obvious trend, the percent of M. haemolytica that were susceptible to penicillin ranged from 40.9% to 66.7%. Danofloxacin MIC(50) and MIC(90) values for M. haemolytica and P. multocida did not change beyond a single dilution over the 6 years it was included in the testing panel. The MIC(90) for H. somni increased beyond 1 dilution. Enrofloxacin MIC(50) values for the 3 pathogens also did not change over time, unlike the MIC(90) values, which increased by at least 4-doubling dilutions. Ninety percent or more of M. haemolytica and H. somni isolates were susceptible to florfenicol, while susceptibility among P. multocida was 79% or greater. Less than 50% of the isolates tested as susceptible to tetracycline in many of the years. All 3 organisms showed declines in tilmicosin and tulathromycin MIC(50) and MIC(90) values over the years in which they were tested.

Tetracycline resistance genes in Pasteurella multocida isolates from bovine, ovine, caprine and swine pneumonic lungs originated from different Greek prefecturesa

The aim of the present work was to analyze the antibiotic resistance of Pasteurella multocida isolates from sheep, goats, cattle and pigs originated from eight different Greek prefectures, to determine the class(es) of the tet genes, to investigate any possible association with plasmids or/and chromosomes and to study the geographical distribution of the resistant isolates. Eighteen out to one hundred isolates were resistant to tetracycline with a minimal inhibitory concentration (MIC) higher than 128 μg/ml. The tetH gene was found in thirteen (72.2%) and the tetB gene was found in four isolates (22.2%). After treatment with the restriction enzymes EcoRV, EcoRI, PstI and BspHI, the tetB gene showed a new polymorphism. Partial sequencing of the tetH and tetB genes revealed five polymorphisms in the tetB gene and two polymorphisms in the tetH gene; phylogenetic and molecular evolutionary analyses were also conducted. Both tetB and tetH genes revealed patterns clearly differentiated from the rest of Pasteurella species meaning thatthe genes could be transferred from a non Pasteurella species. The results presented provide further knowledge and evidence of the role played by commensals as of resistance determinants. Key words: Pasteurella multocida, tet genes, multiple sequence analysis.

Detection of Pasteurella multocida isolates from local pigs of India by polymerase chain reaction and their antibiogram

Pasteurella multocida has been recognized as an important veterinary pathogen for over a century. Conventional methods for diagnosis of pasteurellosis rely on the detection of the organism by microscopy and its isolation and identification. However, as far as pasteurellosis is concerned, it is not just sufficient to know the identity of the organism. To constitute effective control measures, it is important to know the serotype of the organism. A study was undertaken to characterize the Pasteurella isolates from local pigs in India with clinical respiratory disease by determination of their capsule types and presence or absence of toxin gene. Pasteurella could be isolated from 66.70% of pigs with clinical respiratory disease. All the isolates were confirmed through biochemical characterization and P. multocida-specific polymerase chain reaction. It has also been observed that all the isolates belonged to capsular type D. All the isolates were sensitive to chloramphenicol, chlortetracycline, doxycycline, and enrofloxacin, while the rest of the antibiotics were less effective. It has also been observed that all isolates were resistant to cephalexin, penicillin G, and sulphadiazine. The study revealed the detection of P. multocida serotype D from clinical respiratory diseases of local pigs of India, which could be one of the important respiratory tract pathogens responsible for mortality of local pigs in India.

ISOLATION, IDENTIFICATION AND ANTIBIOGRAM OF Pasteurella multocida ISOLATES OF RABBITS SUFFERING FROM PASTEURELLOSIS

Isolation of Pasteurella multocida was attempted from the heart blood, spleen, liver and lung collected from rabbits (4 No,s) suspected to have died of pasteurellosis. A total of four P. multocida isolates were isolated and identified on the basis of biochemical characteristics, pathogenicity studies in mice and PM-PCR. The In vitro antibiotic sensitivity test of the organisms was conducted which revealed that all the four isolates showed sensitivity to Gentamicin and Oflaxacin. Three of them were sensitive to Enrofloxacin and Ciprofloxacin. Two isolates showed sensitivity to Doxycycline and Chloramphenicol. Only one isolate was found sensitive to Nalidixic acid. All the isolates were resistant to Doxycycline, Erythromycin, Neomycin, Ampicillin, Penicillin, Cephalexin and Poymixin B. Based on the antibiotic sensitivity test, Gentamicin was recommended as a drug of choice for the treatment of Pasteurellosis in rabbits and the disease was controlled.

Survey of marbofloxacin susceptibility of bacteria isolated from cattle with respiratory disease and mastitis in Europe

A monitoring programme conducted in Europe since 1994 to survey the marbofloxacin susceptibility of bacterial pathogens isolated from cattle has established the susceptibility of bacterial strains isolated before any antibiotic...

ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: analysis of the regions that comprise 12 antimicrobial resistance genes

In recent years, multiresistant Pasteurella multocida isolates from bovine respiratory tract infections have been identified. These isolates have exhibited resistance to most classes of antimicrobial agents commonly used in veterinary medicine, the genetic basis of which, however, is largely unknown. Genomic DNA of a representative P. multocida isolate was subjected to whole genome sequencing. Genes have been predicted by the YACOP program, compared with the SWISSProt/EMBL databases and manually curated using the annotation software ERGO. Susceptibility testing was performed by broth microdilution according to CLSI recommendations. The analysis of one representative P. multocida isolate identified an 82 kb integrative and conjugative element (ICE) integrated into the chromosomal DNA. This ICE, designated ICEPmu1, harboured 11 resistance genes, which confer resistance to streptomycin/spectinomycin (aadA25), streptomycin (strA and strB), gentamicin (aadB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)], chloramphenicol/florfenicol (floR), sulphonamides (sul2), tilmicosin/clindamycin [erm(42)] or tilmicosin/tulathromycin [msr(E)-mph(E)]. In addition, a complete bla(OXA-2) gene was detected, which, however, appeared to be functionally inactive in P. multocida. These resistance genes were organized in two regions of approximately 15.7 and 9.8 kb. Based on the sequences obtained, it is likely that plasmids, gene cassettes and insertion sequences have played a role in the development of the two resistance gene regions within this ICE. The observation that 12 resistance genes, organized in two resistance gene regions, represent part of an ICE in P. multocida underlines the risk of simultaneous acquisition of multiple resistance genes via a single horizontal gene transfer event.

Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain

Two hundred and five isolates of Pasteurella multocida from pigs were phenotypically and genetically characterised by determining their biovar, capsular type, virulence-associated genes and pulsed-field gel electrophoresis (PFGE) profiles. All...

Development of rapid PCR method for simultaneous identification of species, specific capsular type, and toxigenicity of Pasteurella sp. isolates

Toxigenicity, species, and type species of Pasteurella multocida isolates cannot be differentiated by morphology or standard biochemical reactions. A more rapid method is needed for P. multocida detection from clinical cases. These findings provide rapid insights into the characteristics of P. multocida isolates and suggest that this method can identify toxigenic and specific capsular type P. multocida. A PCR assay has been developed for rapid detection of P. multocida and differentiation of capsular types A and D. In this rapid method, kmt1, capA and capD, and toxA genes were amplified and a reliable multiplex PCR method for the detection of P. multocida in sheep and goats in the south of Iran was designed. Twenty isolates were obtained, which evinced characteristic morphological and cultural properties. Ten samples were identified simultaneously through the presence of the kmt1 gene as P. multocida species, the hydD–hydC gene as type A capsule, and the toxA gene as dermonecrotic toxin by mPCR, but none of them belonged to type D.

STUDY OF PATHOGENICITY AND IMMUNOGENICITY OF PASTEURELLA MULTOCIDA RECENTLY ISOLATED FROM INFECTED RABBITS

In this study, 150 samples were collected from rabbits in infected farms which show high mortality suspected to be affected with Pasteurellosis. Thirty of Pasteurella multocida isolates were obtained which were belonged to serotypes A:3, A:3,5, A:12, B:2, D:6 and uncommon types as A:4 and D:12. The determination of pathogenicity and immunogenicity of these isolates (in mice) indicated that some of these isolates were highly pathogenic, others are of moderate or low pathogenicity. The protective value was good in serotypes A:3, A:3,5, A:12, B:2, D:6 which gave high protective value (more than 2 log), when vaccinated mice were challenged with homologous strains. Rabbits vaccinated with currently used vaccine and challenged with homologous strains and heterologous recently isolates gave good immune response except with field serotypes B:2 and D:6.

Pathology, Tissue Metalloproteinase Transcription and Haptoglobin Responses in Mice after Experimental Challenge with Different Isolates of Pasteurella multocida Obtained from Cases of Porcine Pneumonia

Pasteurella multocida is a major cause of porcine pneumonia, but the pathogenesis of the disease is poorly defined. The aim of this study was to further understand the host response to infection by use of a mouse model of P. multocida pneumonia. Twenty female mice were divided into four groups (n=5). Three groups were infected with one of three isolates of P. multocida isolated from clinical cases of chronic porcine pneumonia with necrotizing, suppurative and non-suppurative lesions, respectively. The fourth group served as uninfected controls. Mice were killed 24 h postinfection and samples were collected for bacteriology, histopathology and in-situ hybridization for detection of P. multocida. Measurements of expression of genes encoding matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1) in lung tissue and quantification of serum haptoglobin concentration were performed. P. multocida was found in the lung and spleen. Lung lesions were characterized by deposition of fibrin in alveoli and bronchioles, perivascular oedema, suppuration and necrosis. The cellular infiltration was mainly of neutrophils. Splenic neutrophilic infiltration was also evident. Minor differences in the severity and nature of lesions were seen according to the isolate of P. multocida used for infection. Intranasal infection of mice can therefore be used to evaluate the host response and lesions caused by P. multocida obtained from porcine pneumonic infections. The inflammatory response in this model is associated with increased tissue expression of genes encoding MMP9, TIMP1 and serum haptoglobin concentration.

Diversity of caprine and ovine Pasteurella multocida isolates based on 16S rRNA gene sequencing.

Summary In this study, to increase information about the r elationship between caprine and ovine isolates of Pasteurella multocida , 16S rRNA gene sequencing of 9 goats (5) and sheep (4) isolates were investigated. Also, capsular type and toxA gene presentation was studied in this paper. All i solates, except one, belong to capsular type A, and toxA + strain distributed among strains were isolated fr om both species. Comparison of the 16S rRNA gene sequence showed the role of goat as a reservoir for P. multocida to sheep independent of toxA genes transmission.

Contribution of ROB-1 and PBP3 mutations to the resistance phenotype of a β-lactamase-positive amoxicillin/clavulanic acid-resistant Haemophilus influenzae carrying plasmid pB1000 in Italy

plasmid pB1000 bearing bla(ROB-1) is responsible for high-level β-lactam resistance in Haemophilus influenzae as well as in Pasteurella multocida and Haemophilus parasuis isolates from Spain. Here, we explore the presence of ROB-1 in Italy and investigate the relative contribution of penicillin-binding protein 3 (PBP3) mutations and ROB-1 to the β-lactam resistance phenotype in H. influenzae. the collection of the Italian Reference Laboratory of H. influenzae was investigated for ROB-1-positive isolates between 2004 and 2009. H. influenzae Rd KW20 was used as recipient for pB1000 electroporation and for mutagenesis of the ftsI gene encoding PBP3. the presence of plasmid pB1000 in a non-typeable H. influenzae isolated in Italy, BB1059, is reported in this work. This strain is not genetically related to the H. influenzae clinical isolates bearing pB1000 described in Spain. The sequence of ftsI from BB1059 revealed several mutations in the predicted amino acid sequence of PBP3. To determine the relative contribution of pB1000 and PBP3 mutations to the β-lactam resistance phenotype of BB1059, H. influenzae Rd KW20 was transformed with ftsI and/or pB1000 from BB1059. β-Lactam resistance profiles revealed the additive effect of pB1000 and PBP3 mutations conferring resistance to β-lactams, including amoxicillin/clavulanic acid and third-generation cephalosporins. intra-European spread of plasmid pB1000 among H. influenzae has been shown. The coexistence of plasmid pB1000 and mutations in PBP3 produces an additive resistance phenotype in H. influenzae.

Development of a multi-locus sequence typing scheme for avian isolates of Pasteurella multocida

A total of 63 isolates of Pasteurella multocida from Australian poultry, all associated with fowl cholera outbreaks, and three international reference strains, representing the three subspecies within P. multocida were used to develop a multi-locus sequence typing scheme. Primers were designed for conserved regions of seven house-keeping enzymes – adk, est, gdh, mdh, pgi, pmi and zwf – and internal fragments of 570–784 bp were sequenced for all isolates and strains. The number of alleles at the different loci ranged from 11 to 20 and a total of 29 allelic profiles or sequence types were recognised amongst the 66 strains. There was a strong concordance between the MLST data and the existing multi-locus enzyme electrophoresis and ribotyping data. When used to study a sub-set of isolates with a known detailed epidemiological history, the MLST data matched the results given by restriction endonuclease analysis, pulsed-field gel electrophoresis, ribotyping and REP-PCR. The MLST scheme provides a high level of resolution and is an excellent tool for studying the population structure and epidemiology of P. multocida.

Frequency and antibiotic susceptibility of Pasteurella multocida and Mannheimia haemolytica isolates from nasal cavities of cattle

The aim of the present study was to determine the frequency of Pasteurella multocida and Mannheimia haemolytica from nasal cavities of cattle, and to find antibiotic susceptibility profiles of the isolates. Bilateral nasal swab samples were collected from 47 clinically healthy Holstein cattle, with no history of antimicrobial treatment prior to sampling. Respectively, 5 and 27 isolates were identified as M. haemolytica and P. multocida. Seventeen samples from cattle of 1 year or younger were found to have P. multocida. On the other hand, from 29 samples from cattle of 2 years or older, 10 P. multocida and 5 M. haemolytica strains were isolated. Each isolate was tested for susceptibility against florfenicol, sulfamethoxazole/trimethoprim, enrofloxacin, ampicillin, and erythromycin. All P. multocida and M. haemolytica isolates were found susceptible against florfenicol, and 27 P. multocida and 5 M. haemolytica isolates showed susceptibility against enrofloxacin, sulfamethoxazole/trimethoprim, erythromycin, and ampicillin at the rates of 85.1% and 80% ; 92.6% and 80% ; 44.5% and 60% ; 100% and 80% , respectively.