Emergence of a floR-carrying plasmid in extended spectrum ß-lactamase (ESBL) producing Pasteurella aerogenes, isolated from an avian species in China.

Identification and analysis of the antimicrobial resistance of Pasteurella aerogenes (P. aerogenes) isolated from poultry. For susceptibility testing in accordance with the CLSI, plasmids were extracted via alkaline lysis and transferred by CaCl2 treatment. Genomic DNA of a representative P. aerogenes isolate was subjected to whole genome sequencing. CCCP was utilized to determine whether SF190908 contains an efflux pump. The blaVEB gene was ligated with the pET-28 plasmid and transferred to Escherichia coli to verify it as an ESBL gene. SF190908 isolated from poultry was identified as P. aerogenes based upon biochemical and 16s rRNA results. The isolate showed high MIC values for eight antimicrobials. Sequencing results showed that the mobile element-mediated antimicrobial resistance gene cluster conferred antimicrobial resistance on the strain, and a single 5,105-bp plasmid, designated pRCAD0752PA-1, was isolated. Four antimicrobial resistance gene clusters were identified in the SF190908 chromosome; one antimicrobial resistance gene cluster carried the blaVEB gene, which was verified as ESBL according to the CLSI and was detected in Pasteurellaceae for the first time, to the best of our knowledge. The efflux pump may confer antimicrobial resistance to SF190908. P. aerogenes isolated from poultry showed resistance genes encoded in mobile elements that confer multi-antimicrobial resistance to SF190908. The antimicrobial-resistant plasmid pRCAD0752PA-1 was isolated in SF190908 and conferred resistance to florfenicol. This study indicates an urgent need to increase efforts to monitor the spread of P. aerogenes multi-antimicrobial-resistant strains and plasmids, especially in newly discovered at-risk species such as poultry.

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.

Synthetic Virus-like Particles for Glutathione Biosynthesis.

Protein-based nanocompartments found in nature have inspired the development of functional nanomaterials for a range of applications including delivery of catalytic activities with therapeutic effects. As glutathione (GSH) plays a vital role in metabolic adaptation and many diseases are associated with its deficiency, supplementation of GSH biosynthetic activity might be a potential therapeutic when delivered directly to the disease site. Here, we report the successful design and production of active nanoreactors capable of catalyzing the partial or complete pathway for GSH biosynthesis, which was realized by encapsulating essential enzymes of the pathway inside the virus-like particle (VLP) derived from the bacteriophage P22. These nanoreactors are the first examples of nanocages specifically designed for the biosynthesis of oligomeric biomolecules. A dense packing of enzymes is achieved within the cavities of the nanoreactors, which allows us to study enzyme behavior, in a crowded and confined environment, including enzymatic kinetics and protein stability. In addition, the biomedical utility of the nanoreactors in protection against oxidative stress was confirmed using an in vitro cell culture model. Given that P22 VLP capsid was suggested as a potential liver-tropic nanocarrier in vivo, it will be promising to test the efficacy of these GSH nanoreactors as a novel treatment for GSH-deficient hepatic diseases.

Genetic parameters of resistance to pasteurellosis using novel response traits in rabbits.

BACKGROUND: Pasteurellosis (Pasteurella infection) is one of the most common bacterial infections in rabbits on commercial farms and in laboratory facilities. Curative treatments using antibiotics are only partly efficient, with frequent relapses. Breeding rabbits for improved genetic resistance to pasteurellosis is a sustainable alternative approach. In this study, we infected 964 crossbred rabbits from six sire lines experimentally with Pasteurella multocida. After post-mortem examination and bacteriological analyses, abscess, bacteria, and resistance scores were derived for each rabbit based on the extent of lesions and bacterial dissemination in the body. This is the first study to use such an experimental design and response traits to measure resistance to pasteurellosis in a rabbit population. We investigated the genetic variation of these traits in order to identify potential selection criteria. We also estimated genetic correlations of resistance to pasteurellosis in the experimental population with traits that are under selection in the breeding populations (number of kits born alive and weaning weight). RESULTS: Heritability estimates for the novel response traits, abscess, bacteria, and resistance scores, ranged from 0.08 (± 0.05) to 0.16 (± 0.06). The resistance score showed very strong negative genetic correlation estimates with abscess (- 0.99 ± 0.05) and bacteria scores (- 0.98 ± 0.07). A very high positive genetic correlation of 0.99 ± 0.16 was estimated between abscess and bacteria scores. Estimates of genetic correlations of the resistance score with average daily gain traits for the first and second week after inoculation were 0.98 (± 0.06) and 0.70 (± 0.14), respectively. Estimates of genetic correlations of the disease-related traits with average daily gain pre-inoculation were favorable but with high standard errors. Estimates of genetic and phenotypic correlations of the disease-related traits with commercial selection traits were not significantly different from zero. CONCLUSIONS: Disease response traits are heritable and are highly correlated with each other, but do not show any significant genetic correlations with commercial selection traits. Thus, the prevalence of pasteurellosis could be decreased by selecting more resistant rabbits on any one of the disease response traits with a limited impact on the selection traits, which would allow implementation of a breeding program to improve resistance to pasteurellosis in rabbits.

Pathogenicity of Pasteurella sp. in lumpsuckers (Cyclopterus lumpus L.).

The incidence of disease caused by Pasteurella sp. in farmed lumpsuckers in Norway has been steadily increasing in recent years, causing significant economic losses and fish welfare issues. The disease affects all life stages, both in hatcheries and after release into salmon cages. Therefore, it is important to establish robust challenge models, to be used for vaccine development. Exposure experiments via intramuscular and intraperitoneal injection underlined the high virulence of the bacteria, whereas the cohabitation and bath models allowed the chronic symptoms of the disease to be studied more accurately. Skin lesions and haemorrhage at the base of fins were observed in the more acute cases of the disease. Symptoms including white spots over the skin, especially around the eyes, characterized the chronic cases. The latter were most prominent from the bath challenge model. Histopathology indicated a systemic pattern of disease, whereas qPCR analysis from head kidney showed that bacteria may be present in survivor fish at the end of the challenges. In all the challenge models investigated, Pasteurella sp. was re-isolated from the fish, thus fulfilling Koch's postulates. These findings highlight the importance of screening of lumpsuckers prior to transfer to minimize the risks of carrying over asymptomatic carriers.

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.

Small ruminant pasteurellosis in Tigray region, Ethiopia: marked serotype diversity may affect vaccine efficacy.

The aim of this study was to investigate the prevalent Bibersteinia, Mannheimia and Pasteurella serotypes, risk factors and degree of serotype co-infections in sheep and goats in the Tigray region of Ethiopia. Serum was collected from 384 sheep and goats from the Tanqua-Abergelle district of Tigray region using cross-sectional random sampling. An indirect haemagglutination test was used for serotyping. Risk factors for infections were evaluated by logistic regression. Potential clustering of multiple serotypes within individual animals due to common risk factors was evaluated by redundancy analysis. Eight serotypes were identified: all studied animals were serologically positive for at least one serotype. Overall, 355 (92·45%) of the animals were infected by four or more serotypes. Of the five risk factors studied, peasant association (PA), animal species, age (serotype A1), and bodyweight (serotype T15) were significantly associated with infection, but sex was not significant. Only PA explained a significant proportion of the variation (adjusted R 2 = 0·16) in the serological responses. After the effect of PA was accounted for, T3 and T4; A7 and Pasteurella multocida A; and A7 and T10 were positively correlated for co-infection, while T4 and T10 were less likely to be found within the same animal. Diverse serotypes were circulating in the Tigray region and could be a challenge in selecting serotypes for vaccine.

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.

Dietary L-glutamine supplementation increases Pasteurella multocida burden and the expression of its major virulence factors in mice.

This study was conducted to determine the effects of graded doses of L-glutamine supplementation on the replication and distribution of Pasteurella multocida, and the expression of its major virulence factors in mouse model. Mice were randomly assigned to the basal diet supplemented with 0, 0.5, 1.0 or 2.0 % glutamine. Pasteurella multocida burden was detected in the heart, liver, spleen, lung and kidney after 12 h of P. multocida infection. The expression of major virulence factors, toll-like receptors (TLRs), proinflammatory cytokines (interleukin-1 beta, interleukin-6, and tumor necrosis factor alpha) and anti-oxidative factors (GPX1 and CuZnSOD) was analyzed in the lung and spleen. Dietary 0.5 % glutamine supplementation has little significant effect on these parameters, compared to those with basal diet. However, results showed that a high dose of glutamine supplementation increased the P. multocida burden (P < 0.001) and the expression of its major virulence factors (P < 0.05) as compared to those with a lower dose of supplementation. In the lung, high dose of glutamine supplementation inhibited the proinflammatory responses (P < 0.05) and TLRs signaling (P < 0.05). In the spleen, the effect of glutamine supplementation on different components in TLR signaling depends on glutamine concentration, and high dose of glutamine supplementation activated the proinflammatory response. In conclusion, glutamine supplementation increased P. multocida burden and the expression of its major virulence factors, while affecting the functions of the lung and spleen.

Pasteurella infection after muraenidae bite in an HIV infected patient.

We report a case of Pasteurella dagmatis wound infection in an immunocompromised HIV infected patient after bite by a marine carnivore in Caribbean Sea (Dominican Republic), presumably a muraenidae. Identification of the Pasteurella species from wound sampling was obtained twice by mass spectrometry and confirmed by 16S RNA sequencing.

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%.

Genetic diversity of Pasteurella dagmatis as assessed by analysis of the 16S rRNA and rpoB gene sequences.

A total of 16 Pasteurella dagmatis strains, including 11 feline and 4 canine isolates as well as one strain isolated from a tiger, were analyzed using partial 16S rRNA and rpoB gene sequence comparison. Phylogenetic studies based on both genes revealed that the population of P. dagmatis recovered from cats in Poland differs markedly from canine strains, constituting a well-separated cluster within Pasteurella sensu stricto species group. The isolate from a tiger seems to represent yet another evolutionary lineage within P. dagmatis.

Genetic diversity of Pasteurella species isolated from European vespertilionid bats.

Pasteurella are an important cause of fatal infections in free-ranging bats, but the genetic diversity of bat-derived strains is unclear. In the current study, 81 Pasteurella strains associated with pneumonia, severe organ necroses and systemic infection in free-ranging European vespertilionid bats were characterized by biochemical and molecular typing methods. Genetic relationships and subspecies status of Pasteurella multocida strains were determined by comparative 16S rDNA and rpoB gene sequence analysis. In addition, 30 representatives of the bat-derived P. multocida strains were selected based on phenotypic and genotypic tests to be compared by pulsed-field gel electrophoresis using SmaI. Most (85%) of the Pasteurella strains obtained from free-ranging bats in this study represented P. multocida ssp. septica. P. multocida ssp. multocida and Pasteurella species B were also identified in a small number of isolates. PFGE analysis correlated well with the sequencing results and revealed a high genetic diversity among bat-derived strains of P. multocida ssp. septica. Strains sharing identical or closely related SmaI fragment patterns were cultured from bats of different species, geographic origins, and years of isolation. The presence of numerous different P. multocida strains allows the assumption that Pasteurella infections in vespertilionid bats are not solely based on intra- but also on inter-species transmission. And indeed, our results present evidence of P. multocida infections in bats following cat predation.