Celia J. Hugo

A polyphasic taxonomic study of Chryseobacterium strains isolated from dairy sources.

A polyphasic taxonomic study, employing protein electrophoresis (SDS-PAGE), gas chromatographic analysis of cellular fatty acids (FAME), mol% G+C determination and DNA-DNA hybridizations, was undertaken on 103 dairy isolates shown to belong to Chryseobacterium. Reference strains of the Chryseobacterium species, CDC group IIb and Embedobacter brevis were included. SDS-PAGE analysis yielded good differentiation between the investigated species. About half of the strains could be clustered into nine major groups while the other half occupied a separate position. With FAME analysis no clear differentiation of the Chryseobacterium species (except C. meningosepticum) and SDS-PAGE groups could be achieved. FAME analysis, however, gave good differentiation between the Chryseobacterium and Empedobacter strains. The mol% G+C of the isolates tested, ranged between 36.4 and 39.0. The combination of SDS-PAGE and DNA-DNA hybridization identified a large group of dairy isolates as C. indologenes, one isolate as C. gleum and two new genotypic groups, comprising five and 15 dairy isolates respectively, emerged from the polyphasic study. Another large part of strains have a separate or uncertain position in Chryseobacterium and remained classified as Chryseobacterium species CDC group IIb.

Effect of alternative preservatives on the microbial quality, lipid stability and sensory evaluation of boerewors

Boerewors is a South African fresh sausage preserved with 450mg/kg sulphur dioxide (SO(2)). The preservative effects of rosemary (Ros; 260mg/kg) and chitosan (Chi; 10g/kg) were compared to SO(2). Eight boerewors models were formulated. Microbial, colour, lipid and sensory characteristics were evaluated. Chi and Chi in combination with other preservatives had a significant effect on reducing total bacterial, coliform and Enterobacteriaceae counts, comparable to SO(2). Chi, however, had a better effect on decreasing yeasts and mould counts than SO(2). Chi showed good colour properties comparable to SO(2). Ros showed comparable lipid stability to SO(2) but better when compared to Chi. Ros had a better effect on the sensory taste when compared to Chi, but SO(2) was still preferred. Reduced levels of 100mg/kg SO(2) showed good antimicrobial and colour effects in combination with Chi and in combination with Ros as antioxidant and improving the sensory properties. Alternative preservatives can be used to reduce the SO(2) content of boerewors.

Characterization of bacterial pathogens in rural and urban irrigation water

The study aimed to compare the bacteriological quality of an urban and rural irrigation water source. Bacterial counts, characterization, identification and diversity of aerobic bacteria were determined. Escherichia coli isolated from both sites was subjected to antibiotic susceptibility testing, virulence gene (Stx1/Stx2 and eae) determination and (GTG)5 Rep-PCR fingerprinting. Low mean monthly counts for aerobic spore formers, anaerobic spore formers and Staphylococcus aureus were noted although occasional spikes were observed. The most prevalent bacterial species at both sites were Bacillus spp., E. coli and Enterobacter spp. In addition, E. coli and Bacillus spp. were most prevalent in winter and summer respectively. Resistance to at least one antibiotic was 84% (rural) and 83% (urban). Highest resistance at both sites was to cephalothin and ampicillin. Prevalence of E. coli possessing at least one virulence gene (Stx1/Stx2 and eae) was 15% (rural) and 42% (urban). All (rural) and 80% (urban) of E. coli possessing virulence genes showed antibiotic resistance. Complete genetic relatedness (100%) was shown by 47% of rural and 67% of urban E. coli isolates. Results from this study show that surface irrigation water sources regardless of geographical location and surrounding land-use practices can be reservoirs of similar bacterial pathogens.

Chryseobacterium carnipullorum sp. nov., isolated from raw chicken

Three Gram-staining-negative, rod-shaped, non-spore-forming, non-motile, oxidase-positive, yellow pigmented and aerobic bacterial isolates designated 8_R23573, 9_R23581(T) and 10_R23577 were isolated from raw chicken at a broiler processing plant in Bloemfontein, South Africa. A polyphasic taxonomic approach was used to determine their exact taxonomic identities. Phylogenetic analysis of the 16S rRNA gene sequences showed that the three strains belonged to the genus Chryseobacterium, exhibiting the highest similarities to Chryseobacterium shigense DSM 17126(T) (98.6-99.2%) and Chryseobacterium luteum DSM 18605(T) (98.3-98.7%). The most abundant quinone was menaquinone MK-6 and the predominant cellular fatty acids were iso-15:0, iso-17:1ω9c, iso-17:0 3-OH and summed feature 3 (iso-16:1ω7c and/or iso-15:0 2-OH), which supported the affiliation of the strains to the genus Chryseobacterium. The DNA G+C contents of the strains were 36.9, 36.7 and 36.6 mol% respectively. The DNA-DNA hybridization results gave relatedness values ranging from 78.8 to 87.2% among the three strains and 23.4 to 56.1% to the two nearest phylogenetic neighbours C. shigense DSM 17126(T) and C. luteum LMG 23785(T). On the basis of the data from this polyphasic study, the three strains are concluded to represent a novel species of the genus Chryseobacterium for which the name Chryseobacterium carnipullorum sp. nov. is proposed. The type strain is 9_R23581(T) ( = LMG 26732(T) =DSM 25581(T)).

The growth, survival and thermal inactivation of Escherichia coli O157:H7 in a traditional South African sausage

This study investigated the growth and survival of Escherichia coli O157:H7 inoculated into boerewors models with (B+P) and without (B-P) sulphur dioxide preservative at a low (L) and high (H) inoculum followed by storage at 0, 4 and 10 degrees C for 10 days. The pathogens thermal inactivation at 50, 60, 65 and 70 degrees C was also evaluated in B+P. The B-P at both low and high inocula had significantly higher recoveries at all temperatures compared to B+P. The BL+P and BH+P had significant reductions in recoveries at 0 degrees C, declining to below detectable limits at days 8 and 10, respectively. At 4 degrees C, the BL+P and BH+P recoveries declined significantly at day 10. At 10 degrees C, significant increases were observed from days 0 to day 10 in both models and at low and high inocula. At 0 degrees C, the BL-P and BH-P treatments had significant declines in recoveries. The combination of sulphur dioxide preservative and low temperature demonstrated the best efficacy against E. coli O157:H7 survival. Thermal inactivation of E. coli O157:H7 was 60 min at 60 degrees C, 80 s at 65 degrees C and 60s at 70 degrees C. This study demonstrated that E. coli O157:H7 can survive in boerewors with and without preservative and is more sensitive to heat treatment at 70 degrees C.

Pathogenic Gram-positive cocci in South African rainbow trout, Oncorhynchus mykiss (Walbaum)

Fish cultured in aquaculture systems are continuously exposed to pathogens present in the water, soil, air or in the fish (Rottmann, Francis-Floyd & Durborow 1992). The fish are weakened by stress conditions including increased fish density and poor water quality, injury during handling, inadequate nutrition, poor sanitation and increased water temperatures. During the last decade, Gram-positive cocci have become important pathogens causing disease in fish (Eldar & Ghittino 1999). Diseases have been reported from Japan (Hoshina, Sanu & Marimoto 1958), Singapore (Foo, Ho & Lam 1985), Australia (Carson, Gudkovs & Austin 1993), Israel (Eldar, Frealier, Asanta, Varner, Lawhon & Bercovier 1995), Italy (Ghittino & Praero 1992), Spain (Toranzo, Curtin, Romalde, Nunez & Barja 1995; Domenech, Fernandez-Garayzabal, Pasqual, Garcia, Cutuli, Moreno, Collins & Dominguez 1996), France (Michel, Nougayrède, Eldar, Sochon & De Kinkelin 1997), South Africa (Bragg & Broere 1986) and the United States (Perera, Johnson, Collins & Lewis 1994). This includes species from streptococci, lactococci and vagococci (Eldar & Ghittino 1999). In South Africa, a Streptococcus infection caused large numbers of deaths in rainbow trout, Oncorhynchus mykiss (Walbaum), (Bragg & Broere 1986) in the former Eastern Transvaal area. Clinical signs seen in the fish included extreme exophthalmus, often with haemorrhages in the eye chamber, and often resulting in the rupture of one or both eyes. Darkening of the skin pigmentation also occurred. Internal examination showed enlargement of the spleen, and a haemorrhagic intestine filled with a yellowish fluid, while both the stomach and intestine contained food. Only adult fish were affected and the largest, fast growing fish showed the disease first in most cases. These Streptococcus isolates could, however, not be classified (Bragg & Broere 1986). A number of the isolates from the study done by Bragg & Broere (1986) which were preserved by freeze drying were used in the current study. The aims were to identify some of these isolates using phenotypic tests as well as 16S rRNA sequencing. The twelve isolates used in this study are given in Table 1. The freeze-dried isolates were revived in brain heart infusion (BHI) broth (Biolab C114) and incubated under anaerobic conditions at 25 C for 3 days using an anaerobic jar with a gasgenerating kit (Oxoid BR0038). Anaerobic conditions enhanced the growth of the organisms. The purity of the cultures was confirmed by Gram staining. For the phenotypic tests, growth from a 24-h BHI agar slant culture was standardized in 5 mL sterile 1 N phosphate buffer to a density comparable to a McFarland 2 standard (Difco 0691326). Journal of Fish Diseases 2011, 34, 483–487 doi:10.1111/j.1365-2761.2011.01259.x

Chapter 21 Culture media for genera in the family flavobacteriaceae

The taxonomy of the flavobacteria has undergone many changes over the past 10 years. This fact may lead to confusion when seeking culture media for this group of bacteria. This chapter is organised in such a way that it orientates the reader in terms of the taxonomy of the family and the deals with the isolation, cultivation and maintenance media that can be used for members of the Flavobacteriaceae family that are of significance for the food microbiologist.

Intermediate added salt levels as sodium reduction strategy: Effects on chemical, microbial, textural and sensory quality of polony

The purpose of this study was to evaluate the use of intermediate added salt levels (1.33% w/w and 1.84% w/w) as a strategy for reducing the total sodium content of polony, an emulsified, heat-treated meat product, which is reliant on the various functions of salt normally included at a 2.5% (w/w) level. Chemical, microbial, and textural stability over 180days as well as sensory quality were evaluated. At 1.84% (w/w) added salt content, the product was indistinguishable from the positive control except for a slightly wetter cutting surface. A level of 1.33% (w/w) added salt showed similar results, except for a slight variation in initial moisture content and a much wetter cutting surface. The hardest texture was achieved at 1.33% (w/w) added salt up to 90days of shelf-life. Consumers had a slight preference for a lower salt content. From this research it can be recommended that salt reduction as sole approach in sodium reduction should be evaluated for meat products in limiting the possibly costly and negatively perceived use of sodium replacers.

International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Aerobic Bacteroidetes (formerly Flavobacterium and Cytophaga-like bacteria). Minutes of the meeting, 30 July 2014, Montréal, Canada

The current voting membership of the Subcommittee is as follows: J.-F. Bernardet (Chairman; Jouy-en-Josas, France); J. P. Bowman (Secretary; Hobart, Tasmania, Australia); J. Chun (Seoul, Korea); E. Frandsen Lau (Åarhus, Denmark); B. Holmes (London, UK); C. Hugo (Bloemfontein, South Africa); Y. Nakagawa (Kisarazu, Japan); and O. Nedashkovskaya (Vladivostok, Russia). Members are listed on the Subcommittee website, which includes reference to current affiliation and contact information (www.the-icsp.org/subcoms/Flavobacterium_Cytophaga.htm).

Comparison of the growth kinetics and proteolytic activities of Chryseobacterium species and Pseudomonas fluorescens

The effect of temperature on the growth kinetics and proteolytic activity of Chryseobacterium joostei and Chryseobacterium bovis was determined during this study. The results were compared with the activities of Pseudomonas fluorescens, which is regarded to be a major food spoilage psychrotolerant microorganism. For the growth studies, cultures were incubated in nutrient broth in a temperature gradient incubator (from 9 to 50 °C) and separately at 4 °C, and the optical density was measured at different time intervals. Growth temperature profiles for each organism were constructed. For determination of proteolytic activity, the cultures were incubated in fat-free ultra-high temperature processed milk in the temperature gradient incubator for 72 h (temperature range as above). Cell-free extracts were used to determine the proteolytic activity using the azocasein method. Results of the growth studies showed that C. joostei had the ability to grow over a wider temperature range than C. bovis and P. fluorescens without being affected by changes in the temperature. For the proteolytic activity, C. joostei had significantly (p < 0.001) higher activity per milligram of protein at 15.5 °C, followed by C. bovis and P. fluorescens. The results showed that C. joostei potentially has an even greater spoilage capacity in milk on the basis of growth rate and proteolytic activity than did P. fluorescens.