Comparative analysis of the pulmonary microbiome in healthy and diseased pigs.

The lungs possess an effective antimicrobial system and a strong ability to eliminate microorganisms in healthy organisms, and were once considered sterile. With the development of culture-independent sequencing technology, the richness and diversity of porcine lung microbiota have been gaining attention. In order to study the relationship between lung microbiota and porcine respiratory disease complex (PRDC), the lung microbiota in healthy and diseased swine bronchoalveolar lavage fluids were analyzed and compared using the Illumina MiSeq sequencing platform. The predominant microbial communities of healthy and diseased swine were similar at the phylum level, mainly composed of Proteobacteria, Firmicutes, Tenericutes, and Bacteroidetes. However, the bacterial taxonomic communities of healthy and diseased swine differed at the genus level. The higher relative abundances of Lactococcus, Enterococcus, Staphylococcus, and Lactobacillus genera in healthy swine might provide more benefits for lung health, while the enhanced richness of Streptococcus, Haemophilus, Pasteurella, and Bordetella genera in diseased swine might be closely related to pathogen invasion and the occurrence of respiratory disease. In conclusion, the observed differences in the richness and diversity of lung microbiota can provide novel insights into their relationship with PRDC. Analyses of swine lung microbiota communities might produce an effective strategy for the control and prevention of respiratory tract infections.

Pasteurella canis infective endocarditis in a dog.

Infective endocarditis, an infrequent clinical syndrome in dogs, is typically associated with nondescript clinical signs such as fever, malaise and loss of appetite. Although an uncommonly reported infection in dogs, Pasteurella canis is an emerging pathogen with increasing relevance in the human microbiology literature. The goal of this study is to detail the clinical presentation and microbiological findings associated with a novel causative agent of infective endocarditis in the dog. Diagnostic evaluation as well as conventional, automated and molecular microbiological methods are highlighted. The recent literature regarding P. canis and infective endocarditis in companion animals and humans is reviewed. Although an unusual etiologic agent of infective endocarditis, awareness of P. canis as a diagnostic possibility is crucial to accurate microbial surveillance.

Classification of genera of Pasteurellaceae using conserved predicted protein sequences.

The aim of the investigation was to investigate the phylogeny of the 49 type strains of species of Pasteurellaceae and three genomospecies, which are available with whole genomic sequences. The genomes were downloaded from National Center for Biotechnological Information and for three species of Avibacterium sequenced in the present investigation. From the predicted protein sequences of proteins, which were conserved in all genomes, 31 proteins were randomly selected for the study. The protein sequences were concatenated for each taxon, and a multiple alignment reconstructed for the 52 taxa. Phylogenetic analysis was performed by using the maximum-likelihood and neighbour-joining methods and confirmed the classification of the genera, which have been classified based on phylogenetic analysis of 16S rRNA gene sequences. The comparison linked [Haemophilus]parainfluezae and [Haemophilus] pittmania with Haemophilus influenzae (type species of genus) although at a much lower level than observed for Haemophilus aegyptius, H. influenzae and Haemophilus haemolyticus. The comparison documented that three, three and nine species of Actinobacillus, Pasteurella and Haemophilus, respectively, are not properly classified at genus level. Similar conclusions have been drawn by 16S rRNA gene sequence comparisons. The highest inter genus pairwise similarity was 88 % based on the comparison of the 31 concatenated protein sequences of the species included in the comparison. The level of intra genus pairwise similarity was also 88 %.

Characterization of the upper and lower respiratory tract microbiota in Piedmontese calves.

BACKGROUND: The microbiota of the bovine upper respiratory tract has been recently characterized, but no data for the lower respiratory tract are available. A major health problem in bovine medicine is infectious bronchopneumonia, the most common respiratory syndrome affecting cattle. With this study, we used 16S rRNA gene sequencing to characterize and compare the microbial community composition of the upper and lower respiratory tracts in calves. RESULTS: The microbiota of the upper (nasal swab [NS]) and the lower (trans-tracheal aspiration [TTA]) respiratory tracts of 19 post-weaned Piedmontese calves with (8/19) and without (11/19) clinical signs of respiratory disease, coming from six different farms, was characterized by 16S rRNA gene metabarcoding. A total of 29 phyla (29 in NS, 21 in TTA) and 305 genera (289 in NS, 182 in TTA) were identified. Mycoplasma (60.8%) was the most abundant genus identified in both the NS (27.3%) and TTA (76.7%) samples, followed by Moraxella (16.6%) in the NS and Pasteurella (7.3%) in the TTA samples. Pasteurella multocida (7.3% of total operational taxonomic units [OTUs]) was the most abundant species in the TTA and Psychrobacter sanguinis (1.1% of total OTUs) in the NS samples. Statistically significant differences between the NS and the TTA samples were found for both alpha (Shannon index, observed species, Chao1 index, and Simpson index; P = 0.001) and beta (Adonis; P = 0.001) diversity. Comparison of the NS and TTA samples by farm origin and clinical signs revealed no statistical difference (P > 0.05), except for farm origin for the NS samples when compared by the unweighted UniFrac metric (P = 0.05). CONCLUSIONS: Using 16S rRNA gene sequencing, we characterized the microbiota of the upper and lower respiratory tracts of calves, both healthy individuals and those with clinical signs of respiratory disease. Our results suggest that environmental factors may influence the composition of the upper airway microbiota in cattle. While the two microbial communities (upper and lower airways) differed in microbial composition, they shared several OTUs, suggesting that the lung microbiota may be a self-sustaining, more homogeneous ecosystem, influenced by the upper respiratory tract microbiota.

[Pasteurella canis hemorragic sepsis and empyema]. / Septicemia hemorrágica y empiema pleural por Pasteurella canis.

We report the case of a 56-year-old female patient, with a three-day history of hematemesis, melena, abdominal wall hematoma and epistaxis associated with thrombocytopenia and anemia. Idiopathic thrombocytopenic purpura was diagnosed and she was treated with dexamethasone for four days. The patient developed acute respiratory failure with signs of systemic inflammatory response. Blood and pleural fluid cultures grew Pasteurella canis. This is the first case, to our knowledge, of P. canis empyema associated with hemorrhagic septicemia without epidemiological background and the third case of septicemia caused by P. canis reported in the literature.

Septicemia hemorrágica y empiema pleural por Pasteurella canis / Pasteurella canis hemorragic sepsis and empyema

We report the case of a 56-year-old female patient, with a three-day history of hematemesis, melena, abdominal wall hematoma and epistaxis associated with thrombocytopenia and anemia. Idiopathic thrombocytopenic purpura was diagnosed and she was treated with dexamethasone for four days. The patient developed acute respiratory failure with signs of systemic inflammatory response. Blood and pleural fluid cultures grew Pasteurella canis. This is the first case, to our knowledge, of P. canis empyema associated with hemorrhagic septicemia without epidemiological background and the third case of septicemia caused by P. canis reported in the literature.

Comunicamos el caso de una mujer de 56 años de edad, con un cuadro clínico de tres días de evolución caracterizado por hematemesis, melena, hematoma en la pared abdominal y epistaxis, asociado a trombocitopenia y anemia. Con un probable diagnóstico de un púrpura trombocitopénico idiopático, se trató con dexametasona por cuatro días. Evolucionó con una insuficiencia respiratoria aguda con signos de respuesta inflamatoria sistémica, por un empiema pleural izquierdo con aislamiento de Pasteurella canis en hemocultivos y líquido pleural. Este es el primer caso, según nuestro conocimiento, de un empiema por P. canis asociado a una septicemia hemorrágica, sin antecedentes epidemiológicos; y tercero de una sepsis por P. canis publicado en el mundo.

American kestrel (Falco spaverius) fledgling with severe bilateral periorbital swelling and infection with Mycoplasma buteonis, Avibacterium (Pasteurella) gallinarum, and Staphylococcus pasteuri.

Abstract: A female American kestrel (Falco spaverius) fledgling was found on the ground with a suspected trauma to the right eye and open-mouth breathing. During the first 2 days of hospitalization, the bird developed severe bilateral periorbital cellulitis, blepharoedema, and sinusitis. The periocular tissues of the right globe were devitalized and communicated with a fistula at the commissure of the right side of the beak. The blepharoedema of the left eye was aspirated and yielded a dark colored malodorous fluid, which was submitted for aerobic bacterial and Mycoplasma cultures. Results showed a mixed infection with Mycoplasma buteonis, Avibacterium gallinarum, and Staphylococcus pasteuri, all of which are not commonly isolated from birds of prey. With antimicrobial therapy, supportive care, and surgical debridement of the right periocular necrotic tissues and adhesed phthisical globe, the kestrel recovered from this severe mixed upper respiratory infection.

Proposed reclassification of Pasteurella lymphangitidis Sneath & Stevens 1990 as Yersinia pseudotuberculosis.

The 16S rRNA gene sequences of Pasteurella lymphangitidis, Yersinia pseudotuberculosis and Yersinia pestis were found to be identical and multilocus sequence analysis could not discriminate between the three species. The susceptibility to a Y. pseudotuberculosis phage and the presence of the Y. pseudotuberculosis-specific invasin gene in P. lymphangitidis indicate that the latter should be reclassified as Y. pseudotuberculosis.

Identification of clinical Pasteurella isolates by MALDI-TOF -- a comparison with VITEK 2 and conventional microbiological methods.

The aim of this study was to compare the performance of four methods that are widely used in the clinical microbiology laboratory for identification of Pasteurella species. The 4 methods evaluated were VITEK2, VITEK MS (BioMerieux), and Bruker Biotyper MS (Bruker) as well as traditional biochemical tests. Sequencing of the sodA gene was used as the reference method. Sixty-five isolates of Pasteurella spp. from 65 patients were analyzed. One Pasteurella multocida isolate from American Type Culture Collection (Manassas, VA, USA) was used as a reference. Traditional biochemical tests accurately identified 62/66 (94%) isolates. Both Bruker and Vitek matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) identified 59/66 (89%) strains, but VITEK2 could only identify 32/66 (48.5%) isolates correctly. The mean time to identification using biochemical tests was 20 hours; VITEK2 took 6 hours and MALDI-TOF approximately 10 minutes. In conclusion, MALDI-TOF is a quick method, which accurately identified most isolates of Pasteurella to the species level. Thus, MALDI-TOF constitutes a valuable diagnostic tool in the clinical laboratory.

Renal abscess caused by a Providencia stuartii isolate biochemically misidentified as Pasteurella.

Providencia stuartii is associated with urinary tract infection (UTI) in catheterized patients. Here we report an abscess containing P. stuartii in a patient with a history of UTI, renal stones, and stent placement. This organism was identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing following biochemical identification as Pasteurella.

A quantitative PCR method for assessing the presence of Pasteurella testudinis DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Pasteurella testudinis has been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Our goal was to develop a sensitive and specific qPCR method for detecting DNA from P. testudinis in nasal lavage fluid collected from desert tortoises in the field. Probes for 16S ribosomal RNA and RNA polymerase ß-subunit (rpoB) genes were designed. A standard curve generated with DNA extracted from known numbers of bacterial cells determined by flow cytometry revealed a lower detection limit of 50 fg/ml (10 bacteria/ml). The nasal lavage fluid contained no interfering substances, and the qPCR method did not recognize normal flora DNA. The nasal lavage samples from 20 desert tortoises captured in Clark County, Nevada, USA in 2007 and housed at the Desert Tortoise Conservation Center, were all positive for P. testudinis DNA by qPCR. Another set of 19 lavage samples collected in 2010 from wild desert tortoises in the Mojave Desert were tested and 84% were positive for P. testudinis DNA. Fully validated, this qPCR method will provide a means of determining colonization rate. When used in conjunction with serological methods and clinical evaluations, both infection rate and disease rate can be determined for this potential URTD pathogen. This new assay provides an important tool for managing the threatened populations of the Mojave Desert tortoise.

Prosthetic valve endocarditis caused by a Pasteurella dagmatis-like isolate originating from a patient's cat.

Pasteurella species are part of the oral flora of cats and dogs. In humans, they are frequently found in infected animal bite wounds, but invasive infections are rare. This is the first report of prosthetic-valve endocarditis with a Pasteurella dagmatis-like species, which originated from the patient's cat.

Classification of Pasteurella species B as Pasteurella oralis sp. nov.

Pasteurella species B has so far only been reported from the oral cavity of dogs, cats and a ferret. In the present study, information from 15 recent isolates from different sources, including African hedgehogs (Atelerix albiventris), banded mongoose (Mungos mungo), Moholi bushbabies (Galago moholi) and pneumonia of a cat, were compared to five strains investigated previously from bite wounds in humans inflicted by a cat and dog and from gingiva of a cat. rpoB gene sequence comparison showed that 17 isolates, including the reference strain (CCUG 19794(T)), had identical sequences, whereas two were closely related and demonstrated 97.9 and 99.6 % similarity to strain CCUG 19794(T), respectively; the type strain of Pasteurella stomatis was the most closely related strain, with 92.3 % similarity. This is within the mean range (76-100 %) of rpoB gene sequence similarity between species of the same genus within the family Pasteurellaceae. 16S rRNA gene sequencing of four strains selected based on rpoB sequence comparison showed at least 99.7 % similarity between strains of Pasteurella species B, with 96.2 % similarity to the type strain of the closest related species (Pasteurella canis), indicating that Pasteurella species B should have separate species status. Separate species status was also documented when recN sequence comparisons were converted to a genome similarity of 93.7 % within Pasteurella species B and 59.0 % to the type strain of the closest related species (P. canis). Based on analysis of the phylogenetic and phenotypic data, and since most isolates originate from the oral cavities of a diverse group of animals, it is suggested that these bacteria be classified as Pasteurella oralis sp. nov.; the type strain is P683(T) ( = CCUG 19794(T) = CCM 7950(T) = strain 23193(T) = MCCM 00102(T)), obtained from a cat. Previous reports of the type strain have shown ubiquinone-8, demethylmenaquinone-8 and menaquinone-8 as the major quinones. Polyamines in the type strain were reported as diaminopropane, putrescine, cadaverine, sym-norspermidine, spermidine and spermine in a previous investigation, and the major fatty acids of the type strain were reported to be C(16:0), C(16:1)ω7c and C(14:0), with minor amounts of C(18:0) and C(18:1)ω9c. The DNA G+C content of the type strain has been reported to be 40.0 mol%.

A case of wound dual infection with Pasteurella dagmatis and Pasteurella canis resulting from a dog bite -- limitations of Vitek-2 system in exact identification of Pasteurella species.

BACKGROUND: Pasteurella species, widely known as indigenous organisms in the oral and gastrointestinal floras of many wild and domestic animals, are important pathogens in both animals and humans. Human infections due to Pasteurella species are in most cases associated with infected injuries following animal bites. We encountered a rare case of dual infections caused by different two Pasteurella species occurred in a previously healthy 25-year-old female sustaining injury by a dog-bite. METHODOLOGY: Exudates from the open wound of her dog-bite site, together with the saliva of the dog were submitted for bacteriological examination. Predominantly appearing grayish-white smooth colonies with almost the same colonial properties but slightly different glistening grown on chocolate and sheep blood agar plates were characterized morphologically by Gram's stain, biochemically by automated instrument using Vitek 2 system using GN cards together with commercially available kit system, ID-Test HN-20 rapid panels, and genetically by sequencing the 16S rRNA genes of the organism using a Taq DyeDeoxy Terminator Cycle Sequencing and a model 3100 DNA sequencer instrument. RESULTS: The causative isolates from the dog-bite site were finally identified as P. canis and P. dagmatis from the findings of the morphological, cultural, and biochemical properties together with the comparative sequences of the 16S rRNA genes. Both the isolates were highly susceptible to many antibiotics and the patient was successfully treated with the administration of so-called the first generation cephalosporin, cefazolin followed by so-called the third generation cephalosporin, cefcapene pivoxil. The isolate from the dog was subsequently identified as P. canis, the same species as the isolate from the patient. CONCLUSIONS: To the best of our knowledge, this was the second report of a dual infection with Pasteurella species consisting of P. dagmatis and P. canis resulting from a dog-bite, followed by the first report of dual infections due to P. dagmatis and P. multocida in 1988. Our isolate finally identified as P. dagmatis was misidentified as P. pneumotripica by means of the Vitek 2 system. The species name "P. dagmatis" was not included in the database of the system. It is also important for routine clinical microbiology laboratories to know the limitation of the automated Vitek 2 system for the accurate identification of Pasteurella species especially P. dagmatis. It should be emphasized that there still exists much room for improvement in Vitek 2 system. Significant improvement of Vitek 2 system especially in the identification of Pasteurella species is urgently desired.

Evaluation of the Biolog system for the identification of certain closely related Pasteurella species.

The zoonotic impact of Pasteurella species in human wounds caused by cats and dogs has increased recently. In this study, the effectiveness of the Biolog Microstation ID System (Biolog, Hayward, CA) for the identification of certain species of Pasteurella sensu stricto was analysed. Thirty-eight isolates originating from dogs and cats were studied by pheno- and genotypic methods. The classical biochemical tests identified these isolates as P. multocida, P. dagmatis, and P. canis, while the Biolog system distinguished only 2 categories: P. multocida and P. dagmatis. The sodA gene sequence-based phylogenetic analysis revealed that the isolates identified as P. dagmatis by the Biolog system were either P. dagmatis, P. canis, or P. dagmatis-like genomospecies. The low discrimination power of the Biolog system in the case of these closely related Pasteurella species draws attention to the need of continuously improving the database of automated microbial identification systems.