Genotyping and biofilm formation of Mycoplasma hyopneumoniae and their association with virulence.

Mycoplasma hyopneumoniae, the causative agent of swine respiratory disease, demonstrates differences in virulence. However, factors associated with this variation remain unknown. We herein evaluated the association between differences in virulence and genotypes as well as phenotype (i.e., biofilm formation ability). Strains 168 L, RM48, XLW-2, and J show low virulence and strains 232, 7448, 7422, 168, NJ, and LH show high virulence, as determined through animal challenge experiments, complemented with in vitro tracheal mucosa infection tests. These 10 strains with known virulence were then subjected to classification via multilocus sequence typing (MLST) with three housekeeping genes, P146-based genotyping, and multilocus variable-number tandem-repeat analysis (MLVA) of 13 loci. MLST and P146-based genotyping identified 168, 168 L, NJ, and RM48 as the same type and clustered them in a single branch. MLVA assigned a different sequence type to each strain. Simpson's index of diversity indicates a higher discriminatory ability for MLVA. However, no statistically significant correlation was found between genotypes and virulence. Furthermore, we investigated the correlation between virulence and biofilm formation ability. The strains showing high virulence demonstrate strong biofilm formation ability, while attenuated strains show low biofilm formation ability. Pearson correlation analysis revealed a significant positive correlation between biofilm formation ability and virulence. To conclude, there was no association between virulence and our genotyping data, but virulence was found to be significantly associated with the biofilm formation ability of M. hyopneumoniae.

Sesquiterpene lactones from Polydora serratuloides and their quorum sensing inhibitory activity.

The leaves of Polydora serratuloides, with the synonym Vernonia perrottetii are widely used as purgative agents for gastrointestinal problems, and other members of Vernonieae have been used in African traditional medicine for decades. A new sesquiterpene lactone of the keto-hirsutinolide type, 13-acetoxy-1(4ß),5(6)ß-diepoxy-8α-(senecioyloxy)-3-oxo-1,7(11)-germacradiene-12,6-olide 1, was isolated from the hexane extract of its leaves, in addition to the known 13-acetoxy-1,4ß-epoxy-8α-(senecioyloxy)-3-oxo-1,5,7(11)-germacratriene-12,6-olide 2. Three common flavonoids (apigenin 3, luteolin 4 and velutin 5) were also isolated. The antibacterial and quorum sensing inhibitory activities of compounds 1 and 2 and crudes extracts showed limited activity on Bacillus subtilis and Staphylococcus aureus, with no activity on Gram negative bacteria. However, quorum sensing (QSI) experiments indicated that 1 and 2, and the four crude extracts had interesting inhibitory activity on the biosensor organism, Chromobacterium violaceum ATCC 12472 in the range of 0.33-5.25 mg mL-1, with compound 1 being the most effective at 0.33 mg mL-1.

Biochar-Mediated Control of Phytophthora Blight of Pepper Is Closely Related to the Improvement of the Rhizosphere Fungal Community.

Biochar is a new eco-material with the potential to control soilborne diseases. This study explored the relationship between the rhizosphere fungal community and the suppression of Phytophthora blight of pepper in the context of time after biochar application. A pot experiment was conducted and rhizosphere soils were sampled to determine the biochar-induced soil chemical properties, fungal community composition, and abundance of biocontrol fungi. The biochar-enriched fungal strains were screened by the selective isolation method, and their control effects against Phytophthora blight of pepper were determined using a pot experiment. Biochar treatments effectively inhibited pathogen growth and controlled the disease, with biochar applied immediately before planting (BC0) having greater effects than that applied 20 days before planting (BC20). Compared to the control, biochar-amended rhizosphere soils had a higher pH, available nutrient content, and fungal richness and diversity. Moreover, biochar treatments significantly increased the abundance of potential biocontrol fungi. The proliferation in BC0 was stronger as compared to that in BC20. Several strains belonging to Aspergillus, Chaetomium, and Trichoderma, which were enriched by biochar amendment, demonstrated effective control of Phytophthora blight of pepper. Canonical correspondence and Pearson's correlation analysis showed that a high content of soil-available nutrients in biochar treatments was favorable to the proliferation of beneficial fungi, which was negatively correlated with both the abundance of Phytophthora capsici and disease severity. In conclusion, biochar-mediated improvement in the fungal community suppressed the Phytophthora blight of pepper. The biochar application time had a great impact on the control effect, possibly due to the short-term proliferative effect of the biochar on biocontrol fungi.

Emergence and Spread of Extended Spectrum ß-Lactamase Producing Enterobacteriaceae (ESBL-PE) in Pigs and Exposed Workers: A Multicentre Comparative Study between Cameroon and South Africa.

Extended spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) represent a significant public health concern globally and are recognized by the World Health Organization as pathogens of critical priority. However, the prevalence of ESBL-PE in food animals and humans across the farm-to-plate continuum is yet to be elucidated in Sub-Saharan countries including Cameroon and South Africa. This work sought to determine the risk factors, carriage, antimicrobial resistance profiles and genetic relatedness of extended spectrum ß-lactamase producing Enterobacteriaceae (ESBL-PE) amid pigs and abattoir workers in Cameroon and South Africa. ESBL-PE from pooled samples of 432 pigs and nasal and hand swabs of 82 humans were confirmed with VITEK 2 system. Genomic fingerprinting was performed by ERIC-PCR. Logistic regression (univariate and multivariate) analyses were carried out to identify risk factors for human ESBL-PE carriage using a questionnaire survey amongst abattoir workers. ESBL-PE prevalence in animal samples from Cameroon were higher than for South Africa and ESBL-PE carriage was observed in Cameroonian workers only. Nasal ESBL-PE colonization was statistically significantly associated with hand ESBL-PE (21.95% vs. 91.67%; p = 0.000; OR = 39.11; 95% CI 2.02⁻755.72; p = 0.015). Low level of education, lesser monthly income, previous hospitalization, recent antibiotic use, inadequate handwashing, lack of training and contact with poultry were the risk factors identified. The study highlights the threat posed by ESBL-PE in the food chain and recommends the implementation of effective strategies for antibiotic resistance containment in both countries.

Prevalence and characterization of plasmid-mediated quinolone resistance genes in Aeromonas spp. isolated from South African freshwater fish.

An increasing incidence of multidrug-resistant Aeromonas spp., which are both fish and emerging opportunistic human pathogens, has been observed worldwide. Quinolone-resistant Aeromonas spp. isolates are increasingly being observed in clinical and environmental settings, and this has been attributed primarily to target gene alterations, efflux, and transferable quinolone resistance. Thirty-four Aeromonas spp., obtained from freshwater aquaculture systems, were screened for the presence of GyrA and ParC substitutions, efflux activity and the prevalence of plasmid-mediated quinolone resistance genes, qnr and aac-6'-Ib-cr. Although 44% of isolates were resistant to nalidixic acid, the majority were susceptible to ciprofloxacin and ofloxacin. The predominant GyrA substitution was Ser-83→Val among Aeromonas veronii isolates whilst Aeromonas hydrophila isolates displayed a Ser-83→Ile substitution, and Ser-80→Ile substitutions were observed in ParC. Minimum inhibitory concentrations of fluoro(quinolones) were determined in the presence and absence of the efflux pump inhibitor, phenylalanine-arginine ß-naphthylamide (PAßN). Addition of PAßN had no effect on the levels of fluoro(quinolone) resistance observed for these isolates. Although no aac-6'-Ib-cr variant genes were identified, qnrB and qnrS were detected for 41% and 24% of isolates, respectively, by Southern hybridization and confirmed by PCR and sequencing. Quinolone resistance in these fish-associated Aeromonas isolates was related to mutations in the quinolone resistance determining regions of GyrA and ParC and presence of qnrB and qnrS. The presence of qnr alleles in Aeromonas spp. isolates may facilitate high-level fluoroquinolone resistance and potentially serve as reservoirs for the dissemination of qnr genes to other aquatic microbes.

Quorum sensing inhibitory potential and molecular docking studies of sesquiterpene lactones from Vernonia blumeoides.

The increasing incidence of multidrug-resistant Gram-negative bacterial pathogens has focused research on the suppression of bacterial virulence via quorum sensing inhibition strategies, rather than the conventional antimicrobial approach. The anti-virulence potential of eudesmanolide sesquiterpene lactones previously isolated from Vernonia blumeoides was assessed by inhibition of quorum sensing and in silico molecular docking. Inhibition of quorum sensing-controlled violacein production in Chromobacterium violaceum was quantified using violacein inhibition assays. Qualitative modulation of quorum sensing activity and signal synthesis was investigated using agar diffusion double ring assays and C. violaceum and Agrobacterium tumefaciens biosensor systems. Inhibition of violacein production was concentration-dependent, with ⩾90% inhibition being obtained with ⩾2.4 mg ml(-1) of crude extracts. Violacein inhibition was significant for the ethyl acetate extract with decreasing inhibition being observed with dichloromethane, hexane and methanol extracts. Violacein inhibition ⩾80% was obtained with 0.071 mg ml(-1) of blumeoidolide B in comparison with ⩾3.6 mg ml(-1) of blumeoidolide A. Agar diffusion double ring assays indicated that only the activity of the LuxI synthase homologue, CviI, was modulated by blumeoidolides A and B, and V. blumeoides crude extracts, suggesting that quorum sensing signal synthesis was down-regulated or competitively inhibited. Finally, molecular docking was conducted to explore the binding conformations of sesquiterpene lactones into the binding sites of quorum sensing regulator proteins, CviR and CviR'. The computed binding energy data suggested that the blumeoidolides have a tendency to inhibit both CviR and CviR' with varying binding affinities. Vernonia eudesmanolide sesquiterpene lactones have the potential to be novel therapeutic agents, which might be important in reducing virulence and pathogenicity of drug-resistant bacteria in vivo.