Virulence and Inoculation Route Influence the Consequences of Mycoplasma hyorhinis Infection in Bama Miniature Pigs.

Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. Although the majority of M. hyorhinis-colonized pigs have no apparent clinical disease, the pathogen can induce diseases such as polyserositis, arthritis, and eustachitis in some cases. To explore the mechanisms for the occurrence of these diseases, we challenged 4 groups of Bama miniature pigs with M. hyorhinis isolated from pigs without clinical symptoms (non-clinical origin [NCO] strain) or with typical clinical symptoms (clinical origin [CO] strain) and investigated the impacts of different strains and inoculation routes (intranasal [IN], intravenous [IV] + intraperitoneal [IP], and IV+IP+IN) on disease induction. Another group of pigs was set as a negative control. Pigs inoculated with the CO strain through a combined intravenous and intraperitoneal (IV+IP) route showed a significant decrease in average daily weight gain (ADWG), serious joint swelling, and lameness compared with the pigs in the negative-control group. Furthermore, this group developed moderate-to-severe pericarditis, pleuritis, peritonitis, and arthritis, as well as high levels of IgG and IgM antibodies. Pigs inoculated IV+IP with the NCO strain developed less marked clinical, pathological changes and a weaker specific antibody response compared with the pigs inoculated with the CO strain. The challenging results of the NCO strain via different routes (IV+IP, IV+IP+IN, and IN) indicated that the combined route (IV+IP) induced the most serious disease compared to the other inoculation routes. Intranasal inoculation induced a smaller decrease in ADWG without obvious polyserositis or arthritis. These data suggest that differences in both strain virulence and inoculation route affect the consequences of M. hyorhinis infection. IMPORTANCE Mycoplasma hyorhinis is a widespread pathogen in pig farms worldwide. The mechanisms or conditions that lead to the occurrence of disease in M. hyorhinis-infected pigs are still unknown. The objective of this study was to evaluate the impact of differences in the virulence of strain and the inoculation route on the consequences of M. hyorhinis infection.

A multifunctional enolase mediates cytoadhesion and interaction with host plasminogen and fibronectin in Mycoplasma hyorhinis.

Mycoplasma hyorhinis may cause systemic inflammation of pigs, typically polyserositis and arthritis, and is also associated with several types of human cancer. However, the pathogenesis of M. hyorhinis colonizing and breaching the respiratory barrier to establish systemic infection is poorly understood. Glycolytic enzymes are important moonlighting proteins and virulence-related factors in various bacteria. In this study, we investigated the functions of a glycolytic critical enzyme, enolase in the infection and systemic spread of M. hyorhinis. Bacterial surface localization of enolase was confirmed by flow cytometry and colony hybridization assay. Recombinant M. hyorhinis enolase (rEno) was found to adhere to pig kidney (PK-15) cells, and anti-rEno serum significantly decreased adherence. The enzyme was also found to bind host plasminogen and fibronectin, and interactions were specific and strong, with dissociation constant (KD) values of 1.4 nM and 14.3 nM, respectively, from surface plasmon resonance analysis. Activation of rEno-bound plasminogen was confirmed by its ability to hydrolyze plasmin-specific substrates and to degrade a reconstituted extracellular matrix. To explore key sites during these interactions, C-terminal lysine residues of enolase were replaced with leucine, and the resulting single-site and double-site mutants show significantly reduced interaction with plasminogen in far-Western blotting and surface plasmon resonance tests. The binding affinities of all mutants to fibronectin were reduced as well. Collectively, these results imply that enolase moonlights as an important adhesin of M. hyorhinis, and interacts with plasminogen and fibronectin. The two lysine residues in the C-terminus are important binding sites for its multiple binding activities.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) moonlights as an adhesin in Mycoplasma hyorhinis adhesion to epithelial cells as well as a plasminogen receptor mediating extracellular matrix degradation.

Mycoplasma hyorhinis infects pigs causing polyserositis and polyarthritis, and has also been reported in a variety of human tumor tissues. The occurrence of disease is often linked with the systemic invasion of the pathogen. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH), one of the key enzymes of glycolysis, was reported as a surface multifunctional molecule in several bacteria. Here, we investigated whether GAPDH could manifest binary functions; as an adhesin to promote colonization as well as a plasminogen receptor functioning in extracellular matrix (ECM) degradation to promote systemic invasion. The surface localization of GAPDH was observed in M. hyorhinis with flow cytometry and colony blot analysis. Recombinant GAPDH (rGAPDH) was found to be able to bind porcine-derived PK-15 and human-derived NCI-H292 cells. The incubation with anti-GAPDH antibody significantly decreased the adherence of M. hyorhinis to both cell lines. To investigate its function in recruiting plasminogen, firstly, the interaction between rGAPDH and plasminogen was demonstrated by ELISA and Far-Western blot assay. The activation of the rGAPDH-bound plasminogen into plasmin was proved by using a chromogenic substrate, and furtherly confirmed to degrade extracellular matrix by using a reconstituted ECM. Finally, the ability of rGAPDH to bind different ECM components was demonstrated, including fibronectin, laminin, collagen type IV and vitronectin. Collectively, our data imply GAPDH as an important adhesion factor of M. hyrohinis and a receptor for hijacking host plasminogen to degrade ECM. The multifunction of GAPDH to bind both plasminogen and ECM components is believed to increase the targeting of proteolysis and facilitate the dissemination of M. hyorhinis.

Phenol hydroxylase from Pseudomonas sp. KZNSA: Purification, characterization and prediction of three-dimensional structure.

A 61.3 kDa Phenol hydroxylase (PheA) was purified and characterized from Pseudomonas sp. KZNSA (PKZNSA). Cell free extract of the isolate grown in mineral salt medium supplemented with 600 ppm phenol showed 21.58 U/mL of PheA activity with a specific activity of 7.67 U/mg of protein. The enzyme was purified to 1.6-fold with a total yield of 33.6%. The purified PheA was optimally active at pH 8 and temperature 30 °C, with ≈95% stability at pH 7.5 and temperature 30 °C after 2 h. The Lineweaver-Burk plot showed the vmax and Km values of 4.04 µM/min and 4.03 µM, respectively, for the substrate phenol. The ES-MS data generated from the tryptic digested fragments of pure protein and PCR amplification of a ≈600 bp gene from genomic DNA of PKZNSA lead to the determination of complete amino acid and nucleotide sequence of PheA. Bioinformatics tools and homology modelling studies indicated that PheA from PKZNSA is likely a probable protein kinase UbiB (2-octaprenylphenol hydroxylase) involving Lys and Asp at positions 153 and 288 for binding and active site, respectively. Characterization and optimization of PheA activity may be useful for a better understanding of 2,4-dichlorophenol degradation by this organism and for potential industrial application of the enzyme.

Exploratory Study into the Microbiological Quality of Spinach and Cabbage Purchased from Street Vendors and Retailers in Johannesburg, South Africa.

Knowledge of the microbiological quality and prevalence of antibiotic resistance and virulence genes in bacterial isolates from leafy green vegetables supplied by formal suppliers (retailers) and informal suppliers (street vendors) in South Africa is limited. Because leafy vegetables have been implicated in foodborne disease outbreaks worldwide, 180 cabbage and spinach samples were collected from three major retailers and nine street vendors in Johannesburg, South Africa. Escherichia coli and coliforms were enumerated using Petrifilm plates. The prevalence of Listeria monocytogenes, Salmonella, and Shigella was determined using real-time PCR analysis. Identities of presumptive E. coli isolates from the fresh produce were confirmed using matrix-assisted laser desorption-ionization time of flight mass spectroscopy. Isolates were characterized using phenotypic (antibiotic resistance) and genotypic (phylogenetic and virulence gene) analysis. Hygiene indicator bacteria levels on spinach from formal and informal retailers exceeded the maximum level specified by the Department of Health guidelines for fresh fruit and vegetables. E. coli counts for street vendor spinach were higher (P < 0.0789) than those for retailer spinach. E. coli was present in only two cabbage samples, at 0.0035 CFU/g. L. monocytogenes and Salmonella were detected in 7.2 and 5% of the 180 samples, respectively, based on real-time PCR analysis; Shigella was not detected. Of the 29 spinach E. coli isolates, 37.9% were multidrug resistant. Virulence genes eae and stx1 were present in 14 and 3% of the spinach E. coli isolates, respectively; the stx2 gene was not detected. Eighty-six percent of these isolates belonged to phylogroup A, 3% belonged to group C, 7% belonged to group E, and 3% belonged to clade 1. The results from the current exploratory study on the microbiological quality of spinach bought from selected retailers highlight the need for continued surveillance on a larger scale, especially in the informal sector, to characterize the potential health risks to the consumer.

Bilateral blunt carotid artery injury: a case report and review of the literature.

A 22-year-old man sustained a strangulation-type injury to the neck, with bilateral blunt carotid artery injuries detected on computed tomography (CT) angiography. His Glasgow Coma Score was 15/15, and he was managed conservatively with therapeutic low-molecular-weight heparin and antiplatelet therapy. A repeat CT angiogram 6 weeks later showed complete resolution of an intimal flap, and he demonstrated no neurological deterioration. There are no definitive management guidelines regarding this type of injury, and our report emphasises the role of conservative anticoagulation therapy in the management of this rare condition.