In vitro susceptibility of Brachyspira hyodysenteriae to a commercial citrus fruit extract
Introduction
Brachyspira hyodysenteriae is a Gram negative, motile, spiral and anaerobic bacterium of the Brachyspiraceae Family, Phylum Spirochaetes. It is the main causative agent of swine dysentery (SD), a disease characterized by mucohaemorrhagic diarrhoea which causes significant losses in pig production (Alvarez-Ordóñez et al., 2013c, Paster and Dewhirst, 2000). Since there is no commercially available vaccine, control of SD involves the use of antibiotics together with other biosecurity measures. However, the number of effective antibiotics is limited and the emergence of strains with reduced susceptibility to these molecules greatly complicates the treatment and control of SD (Hidalgo et al., 2011, Rugna et al., 2015).
Natural plant-derived products used as additives in animal feeding are also called phytobiotics, botanicals or phytogenic feed additives (Windisch et al., 2008). Besides their nutritional effects, they may contain secondary metabolites with antimicrobial properties, which are being evaluated as an alternative to the use of antibiotics in animal feeding (Benchaar et al., 2008, Brenes and Roura, 2010, Frankič et al., 2009, Hashemi and Davoodi, 2010, Michiels et al., 2009, Zeng et al., 2015).
BIOCITRO is a citrus extract manufactured by QUINABRA (Química Natural Brasileira, Ltda.) and commercialized by PROBENA S.L. in the European Union. It is extracted from four types of organic cultures of citrus fruits: grapefruit (Citrus paradisi), tangerine (Citrus reticulata blanco), bergamot (Citrus aurantium ss. bergamia) and sweet orange (Citrus sinensis). The main compounds of the extract are ascorbic acid (Vitamin C) and its salts, with high level of bioavailability and linked with the citrus bioflavonoids naringin, hesperidin, quercetin and rutin, with organic acids on an inert carrier, silica dioxide or vegetable glycerine for powder or liquid presentations of BIOCITRO respectively (http://www.probena.e.telefonica.net/biocitroi.htm).
The aim of the research was to evaluate the antimicrobial activity of the liquid presentation of BIOCITRO against B. hyodysenteriae and to investigate its main mechanisms of action by flow cytometry (FC), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).
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Brachyspira hyodysenteriae isolates and culture conditions
Two strains from the American Type Culture Collection (ATCC) and eight field strains from the Infectious Diseases Unit Collection of the University of León in Spain (IDUC), isolated from SD outbreaks in Spanish swine finishing farms between 2007 and 2016 (Table 1), were selected for the assessment based on their different antibiotic susceptibility.
Cultures were grown in an anaerobic chamber (80% N2, 10% H2 and 10% CO2) or in a jar with a pack of AnaeroGenTM (OXOID) at 42 °C. Columbia agar with
MICs and MBCs
The broth microdilution method showed no differences of more than one dilution step between MIC and MBC replicates. In nine isolates the inhibitory concentrations were also bactericidal, whereas only one showed the MBC as twice the MIC (Table 1). Both values, MIC and MBC, ranged from 32 to 128 ppm, with a MIC50 of 64 ppm, whereas the MIC90, MBC50 and MBC90 were 128 ppm.
Membrane integrity test
Membrane integrity test performed by FC revealed a similar behaviour for the two ATCC strains tested (Fig. 1). A slight damage due
Discussion
The assessment of the antimicrobial effect of BIOCITRO against B. hyodysenteriae revealed a relevant inhibitory and bactericidal activity against all isolates tested, as previously described for other citrus fruit extracts against different bacterial groups (Alvarez-Ordóñez et al., 2013b, Mandalari et al., 2007, Negi and Jayaprakasha, 2001, Torres-Alvarez et al., 2017). For most of the tested isolates the MIC was the same as the MBC, with a MIC90, MBC50 and MBC90 of 128 ppm. This activity may be
Conclusions
Our results evidenced the great activity of the citrus fruit extract BIOCITRO against B. hyodysenteriae, with MIC and MBC values ≤ 128 ppm. Cell damages were demonstrated by FC and SEM, as well as broad changes in the composition and the structure of the cells by FTIR. Considering these findings, BIOCITRO is shown as a possible alternative to antibiotics in the control of SD. However, further research in field conditions is necessary to evaluate this antagonistic effect in vivo.
Conflict of interest statement
The research was partly financed by PROBENA (2015/00119/001), the company commercializing BIOCITRO in the EU, but with having no influence over the experimental procedures neither results. None of the authors has any other financial or personal relationships that could inappropriately influence or bias the content of the paper.
Acknowledgements
We would like to thank the excellent technical assistance provided by G.F. Bayón, S. Costillas and A. Sánchez and the information provided by L. Álvarez from Aquilón CyL S.L. This research did not receive any specific grant from funding agencies in the public or not-for-profit sectors apart from the above mentioned of PROBENA.
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