Genetic diversity and antimicrobial resistance profiles of Campylobacter coli and Campylobacter jejuni isolated from broiler chicken in farms and at time of slaughter in central Italy.

AIMS: Genetic diversity and antimicrobial resistance of Campylobacter coli and Campylobacter jejuni were investigated along the broiler chicken production chain in central Italy. METHODS AND RESULTS: Campylobacter sp. isolated from cloacal swabs in farms (n = 116) and from the neck skin of chilled and eviscerated carcasses at slaughter (n = 24) were identified as C. coli (n = 99) and C. jejuni (n = 41) by multiplex PCR. Characterization by single amplified fragment length polymorphism (s-AFLP) revealed a specific genotype of Campylobacter for each farm. Minimal inhibitory concentration showed high prevalence of fluoroquinolones (70%), tetracycline (70%) and erythromycin (30%) resistance among C. coli isolates. Campylobacter jejuni isolates showed lower prevalence of fluoroquinolone (39%) and tetracycline (10%) resistance, and all isolates were susceptible to erythromycin. The S-AFLP types of the C. coli and C. jejuni isolates were associated with their antimicrobial resistance profiles (P < 0·001). CONCLUSIONS: The genetic diversity detected in Campylobacter isolates suggested that a specific genotype was harboured in each farm. A considerable number of C. coli isolates were resistant to erythromycin. SIGNIFICANCE AND IMPACT OF THE STUDY: Campylobacter coli was detected more frequently than C. jejuni in contrast to common findings for poultry. The high prevalence of 30% resistance to erythromycin in C. coli strains isolated from poultry is worrisome, as this is the first antibiotic of choice to treat human campylobacteriosis.

Evaluation of damage induced by Kwkt and Pikt zymocins against Brettanomyces/Dekkera spoilage yeast, as compared to sulphur dioxide.

AIMS: Over the last few decades, the use of zymocins as biological tools to counteract contamination by spoilage yeast in beverages and food has been widely studied. This study examined the damage induced by the Kwkt and Pikt, two zymocins produced by Kluyeromyces wickerhamii and Wickerhanomyces anomalus, respectively, with antimicrobial activity against Brettanomyces/Dekkera wine-spoilage yeast. METHODS AND RESULTS: The physiological and biochemical characterization of both of these proteins revealed that only Pikt showed a strict relationship between ß-glucosidase activity and killer activity. The minimum inhibitory concentrations and minimum fungicidal concentrations of Kwkt and Pikt showed inhibitory activities against Brettanomyces/Dekkera yeast. Cytofluorimetric evaluation of cell death was based on both cell membrane permeability and cell metabolism, using fluorescence techniques under increasing zymocin levels over different incubation times. The antimicrobial actions of Kwkt and Pikt were also compared with the mode of action of sulphur dioxide. In this last case, the induction of the viable but noncultivable (VBNC) state was confirmed, with the consequent recovery of Brettanomyces yeast after medium replacement. In contrast, Kwkt and Pikt caused irreversible death of these yeast, without recovery of sensitive cells. CONCLUSIONS: Kwkt and Pikt could be proposed as fungistatic or fungicide biocontrol agents in winemaking to control the colonization and development of Brettanomyces/Dekkera yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: These data support the potential use of zymocins to reduce wine contamination as an alternative to sulphur dioxide that act on sensitive cells. Differently from sulphur dioxide, that could induce a reversible VBNC state, Kwkt and Pikt determine the irreversible damage on sensitive yeasts, ensuring the complete control of spoilage Brettanomyces yeast.

Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts.

AIMS: In the present study, it was investigated the antagonistic behaviour of Metschnikowia pulcherrima, as biocontrol agent, against the main wine yeast species involved in the winemaking process. METHODS AND RESULTS: Seven strains of M. pulcherrima were evaluated for the antimicrobial activity against 114 yeast strains belonging to Pichia, Candida, Hanseniaspora, Kluyveromyces, Saccharomycodes, Torulaspora, Brettanomyces and Saccharomyces genera. Results showed both different inter-generic and intra-generic responses to the antimicrobial action of M. pulcherrima strains. Interestingly, the antimicrobial activity of M. pulcherrima did not have any influence on the growth of Saccharomyces cerevisiae. Instead, M. pulcherrima displayed a broad and effective antimicrobial action on undesired wild spoilage yeasts, such as Brettanomyces/Dekkera, Hanseniaspora and Pichia genera. Fermentation trials carried out in synthetic grape must confirmed the antimicrobial activity of M. pulcherrima, determining the early death of the non-Saccharomyces co-inoculated cultures. CONCLUSIONS: The antimicrobial activity of M. pulcherrima does not seem due to proteinaceous compounds such as killer phenomenon, but to the pulcherriminic acid (the precursor of pulcherrimin pigment) that depletes iron present in the medium, making it not available to the other yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: These data agree with and further support the potential use of selected M. pulcherrima strains in controlled multistarter fermentations with S. cerevisiae starter cultures.

The zymocidial activity of Tetrapisispora phaffii in the control of Hanseniaspora uvarum during the early stages of winemaking.

AIMS: The yeast strain Tetrapisispora phaffii DBVPG 6706 (formerly Kluyveromyces phaffii) secretes a killer toxin (Kpkt) that has antimicrobial activity against apiculate yeasts. The aim of this study was to evaluate the killer activity of Kpkt towards Hanseniaspora uvarum under winemaking conditions. METHODS AND RESULTS: The zymocidial activity of Kpkt on H. uvarum was assayed in microfermentation trials inoculated with free and immobilized T. phaffii cells. The microbial evolution and fermentation profiles of the wines were evaluated to determine the effects of Kpkt on apiculate yeasts, in comparison with SO(2). The results indicate that the fungicidal activity of Kpkt against H. uvarum is stable for at least 14 days in wine, and the zymocin can control the proliferation of apiculate yeasts. The analytical composition of wines with the inoculum of T. phaffii immobilized cells did not differ from the wines with SO(2). In contrast to wines without this control of apiculate yeasts, an increase in ethyl acetate was seen. CONCLUSIONS: Tetrapisispora phaffii is an excellent candidate for the biological control of undesired proliferation of apiculate yeasts during the first steps of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Tetrapisispora phaffii cells in an immobilized form can be used as a biocontrol agent to reduce the need for SO(2) addition.

Effect of Phanerochaete chrysosporium inoculation during maturation of co-composted agricultural wastes mixed with olive mill wastewater.

The co-composting of olive mill wastewater with a variety of agricultural wastes was investigated. To reduce the toxicity of the phenolic fraction and to improve the degree of maturity of the compost, inoculation with the white-rot fungus Phanerochaete chrysosporium was carried out during the maturation phase. The results showed that agricultural wastes that contain high levels of lignin-related compounds, such as the residue from trimmings, improved the microbial activity and thus reduced the soluble phenols residue. The inoculation of P. chrysosporium during compost maturation reduced and modified the phenolic fraction, allowing a reduction in the time to reach compost maturity with the improvement in the germination index of 100% after 36 days in two of three trials performed.

Survival of inoculated Saccharomyces cerevisiae strain on wine grapes during two vintages.

AIMS: To investigate the influence of a specific ecological niche, the wine grape, on the survival and development of Saccharomyces cerevisiae. METHODS AND RESULTS: A strain with a rare phenotype was sprayed onto the grape surfaces and monitored through two vintages using a specific indicative medium and analysing the internal transcribed spacer regions in the 5.8S rDNA. During the ripening process, there was a progressive colonization of the surface of the undamaged and damaged grapes by epiphytic yeasts, up to the time of harvest. The damaged wine grapes showed a much greater epiphytic yeast population. However, the inoculated S. cerevisiae strain showed a scarce persistence on both undamaged and damaged wine grapes, and the damaged grapes did not appear to improve the grape surface colonization of this strain. CONCLUSIONS: Results indicated that wine grape is not a favourable ecological niche for the development and colonization of S. cerevisiae species. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this work are further evidence that S. cerevisiae is not specifically associated with natural environments such as damaged and undamaged wine grapes.

Interactions between Saccharomyces cerevisiae and malolactic bacteria: preliminary characterization of a yeast proteinaceous compound(s) active against Oenococcus oeni.

AIMS: To investigate the occurrence and extent of Saccharomyces cerevisiae and Oenococcus oeni interactions. METHODS AND RESULTS: Interactions between S. cerevisiae and O. oeni were investigated by double-layer and well-plate assays showing the occurrence of specific interactions for each yeast-malolactic bacteria (MLB) coupling. Heat and protease treatments of synthetic grape juice fermented by the S. cerevisiae strain F63 indicated that the inhibitory activity exerted by this yeast on O. oeni is due to a proteinaceous factor(s) which exerts either bacteriostatic or bactericidal effect depending on concentration and affects malolactic fermentation in natural grape juice and wine. CONCLUSIONS: A proteinaceous factor(s) produced by a S. cerevisiae wine strain able to inhibit O. oeni growth and malic acid fermentation was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The individuation, characterization and exploitation of yeast proteinaceous factor(s) exerting inhibitory activity on MLB may offer new opportunities for the management of malolactic fermentation.