Phylogenetic analysis of Cronobacter isolates based on the rpoA and 16S rRNA genes.

The reclassification of the genus Cronobacter (previously known as Enterobacter sakazakii) was based on a polyphasic analysis that led to the description of five species. These bacteria are opportunistic pathogens that can cause neonatal meningitis and other infections in immuno-compromised individuals. Cronobacter species have been reported to show differences in sensitivity to antibiotics, heat and chemicals, as well as differences in virulence. The objective of this study was to classify Cronobacter isolates from infant formula milk, the food processing environment and fresh produce in South Africa and to evaluate the phylogenetic placement of these isolates based on the rpoA and 16S ribosomal RNA (rRNA) gene sequences. All the South African strains were identified as Cronobacter sakazakii despite the wide variety of isolation sources. No relation between the phylogenetic placement and strain origin could be determined. Strains of C. sakazakii, Cronobacter dublinensis, Cronobacter turicensis and Cronobacter muytjensii could be differentiated from each other, but it was not possible to differentiate between C. sakazakii and Cronobacter malonaticus based on the rpoA and 16S rRNA gene sequences alone. However, sequence data of these two genes can be used to differentiate between C. sakazakii and C. malonaticus when used in combination with biochemical analysis based on the utilisation of malonate.

Alicyclobacillus spoilage and isolation--a review.

Until recently, acidic products such as fruit juice and fruit based products were generally thought to be susceptible to spoilage by yeasts, mycelia fungi and lactic acid bacteria, as the low pH of these products acts as natural control measures against spoilage by most bacteria. Alicyclobacillus seem to be prevalent in fruit based products as they survive the acidic fruit juice environment, even when they are exposed to pasteurisation temperatures during production. In this review the historical background of the discovery of these bacteria is summarised. The bacterial characteristics and the reported spoilage incidences caused by members of this genus are discussed. As the isolation methods for these bacteria are controversial, this review includes a discussion of the various media that have been reported in the literature for the use in the isolation and enumeration of members of the genus Alicyclobacillus.

Isolation, identification and typification of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius strains from orchard soil and the fruit processing environment in South Africa.

Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius are thermo-acidophilic, non-pathogenic, spore-forming bacteria that can survive the typical heat processing of fruit juices and concentrates. Bacterial endospores then germinate, grow and cause spoilage of acid food products. Species of Alicyclobacillus were isolated from orchard soil and a fruit concentrate production factory in South Africa. Preliminary identification of the isolates was based on morphological, biochemical and physiological properties. Identification at species level was done by PCR amplification using genus-specific primers and 16S ribosomal RNA (rRNA) gene sequencing. The majority of isolates belonged to the species A. acidoterrestris, but A. acidocaldarius was also isolated and identified. As far as we could determine, this is the first report of the isolation of A. acidoterrestris from wash water and soil outside a fruit processing plant, as well as the isolation of A. acidocaldarius from vinigar flies. The genotypic relatedness between strains of A. acidoterrestris and between strains of A. acidocaldarius was determined by RAPD-PCR. Sixteen isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Three isolateT:/PGN/ELSEVIER/YFMIC/web/00001155/s identified as A. acidocaldarius gave three unique banding patterns.

Evaluation of different methods for the detection and identification of Enterobacter sakazakii isolated from South African infant formula milks and the processing environment.

Enterobacter sakazakii is an emerging pathogen associated with life-threatening neonatal infections resulting from the consumption of contaminated powdered infant formula milk (IFM). Recent taxonomic analyses have determined that E. sakazakii comprises a number of genomospecies, and it has been proposed that E. sakazakii be reclassified as a novel genus, "Cronobacter". Accurate methods are required for the rapid detection and identification of this group of micro-organisms, since even low cell numbers have been reported to cause disease. The aim of this study was to evaluate various E. sakazakii detection methods in order to ascertain the most suitable method for detection and identification of these pathogenic agents. Samples from IFM and the environment were evaluated for the presence of E. sakazakii using the isolation steps (pre-enrichment, enrichment and selection) described in the Food and Drug Administration (FDA) method for E. sakazakii detection. Sixty-four isolates (50 from IFM and 14 from the environment) were selected from tryptone soy agar (TSA), regardless of colony appearance, and these isolates were identified by 16S ribosomal DNA (rDNA) sequencing. Thereafter, different culture-dependent and culture-independent methods were evaluated to accurately detect and identify the E. sakazakii isolates. These methods included the assessment of yellow pigment production on TSA, typical colonies on chromogenic Druggan-Forsythe-Iversen (DFI) and Chromocult Enterobacter sakazakii (CES) media and polymerase chain reaction (PCR) using six different species-specific primer pairs described in the literature. Identification of E. sakazakii using yellow pigment production was demonstrated to have a low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively), which lowers the suitability of the FDA method. Chromogenic DFI and CES media were sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications ranged from 8% to 92%, emphasising the need for rigorous primer testing against closely related species. Of the primer pairs evaluated, Esakf/Esakr were the most suitable for E. sakazakii detection and identification. The detection limit of Esakf/Esakr was found to be 10(4) CFU/ml. This study demonstrated that no single method was capable of unambiguously confirming the presence and identity of E. sakazakii isolates, that each method had inherent advantages and disadvantages, and that in most cases several methods were required for accurate detection and identification. Further, it was demonstrated that the current FDA method for E. sakazakii detection should be revised in the light of the availability of more sensitive, specific and accurate detection methods.

Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa.

The development of DNA-based methods for the identification of fish species is important for fisheries research and control, as well as for the detection of unintentional or fraudulent species substitutions in the marketplace. The aim of this study was to generate a comprehensive reference database of DNA sequences from the mitochondrial 16S and 12S ribosomal RNA (rRNA) genes for 53 commercial fish species in South Africa and to evaluate the applicability of these genetic markers for the identification of fish at the species level. The DNA extracted from all target species was readily amplified using universal primers targeting both rRNA gene regions. Sequences from the 16S and 12S rRNA genes were submitted to GenBank for the first time for 34% and 53% of the fish species, respectively. Cumulative analysis of the 16S rRNA gene sequences revealed mean conspecific, congeneric and confamilial Kimura two parameter (K2P) distances of 0.03%, 0.70% and 5.10% and the corresponding values at the 12S level were 0.03%, 1.00% and 5.57%. K2P neighbour-joining trees based on both sequence datasets generally clustered species in accordance with their taxonomic classifications. The nucleotide variation in both the 16S and 12S sequences was suitable for identifying the large majority of the examined fish specimens to at least the level of genus, but was found to be less useful for the explicit differentiation of certain congeneric fish species. It is recommended that one or more faster-evolving DNA regions be analysed to confirm the identities of closely-related fish species in South Africa.

Guaiacol production from ferulic acid, vanillin and vanillic acid by Alicyclobacillus acidoterrestris.

Alicyclobacilli are thermophilic, acidophilic bacteria (TAB) that spoil fruit juice products by producing guaiacol. It is currently believed that guaiacol is formed by Alicyclobacillus in fruit juices as a product of ferulic acid metabolism. The aim of this study was to identify the precursors that can be metabolised by Alicyclobacillus acidoterrestris to produce guaiacol and to evaluate the pathway of guaiacol production. A. acidoterrestris FB2 was incubated at 45°C for 7days in Bacillus acidoterrestris (BAT) broth supplemented with ferulic acid, vanillin or vanillic acid, respectively. The samples were analysed every day to determine the cell concentration, the supplement concentration using high performance liquid chromatography with UV-diode array detection (HPLC-DAD) and the guaiacol concentration, using both the peroxidase enzyme colourimetric assay (PECA) and HPLC-DAD. The cell concentration of A. acidoterrestris FB2 during the 7days in all samples were above the critical cell concentration of 10(5)cfu/mL reportedly required for guaiacol production. The guaiacol produced by A. acidoterrestris FB2 increased with an increase in vanillin or vanillic acid concentration and a metabolic pathway of A. acidoterrestris FB2 directly from vanillin to guaiacol was established. The high concentration of vanillic acid (1000mg/L) resulted in an initial inhibitory effect on the cells, but the cell concentration increased after day 2. Guaiacol production did not occur in the absence of either a precursor or A. acidoterrestris FB2 and guaiacol was not produced by A. acidoterrestris FB2 in the samples supplemented with ferulic acid. The presence of Alicyclobacillus spp. that has the ability to produce guaiacol, as well as the substrates vanillin or vanillic acid is prerequisite for production of guaiacol.

Occurrence of Alicyclobacillus in the fruit processing environment--a review.

Concentrated fruit products have a significant place in modern consumption markets and are valuable semi-prepared food components to the bakery, dairy, confectionary, canning, baby food, frozen food, distilling and beverage industries. There is continuous pressure on the beverage industry to improve the quality of concentrated fruit products in order for reconstituted fruit beverages to compete with beverages that are made from fresh fruits. In recent years, Alicyclobacillus spp. have become a major concern to the beverage industry worldwide as many high-acid, concentrated fruit products have been found to be contaminated with these spoilage microbes. The thermo-acidophilic nature of alicyclobacilli and highly resistant endospores allows for their survival during the production of concentrated fruit products. Under favourable conditions, endospores can germinate and multiply to numbers high enough to cause spoilage and product deterioration through the production of chemical taint compounds. It is imperative to understand the nature of Alicyclobacillus within the fruit concentrate processing environment so as to develop effective control strategies and to prevent spoilage in juice and beverage products that are reconstituted from fruit concentrates. This paper reviews the occurrence of alicyclobacilli in the fruit processing environment, control measures, as well as detection, identification and standardised test methods that are currently used for Alicyclobacillus in concentrated fruit products.

Establishment of a mitochondrial DNA sequence database for the identification of fish species commercially available in South Africa.

The limitations intrinsic to morphology-based identification systems have created an urgent need for reliable genetic methods that enable the unequivocal recognition of fish species, particularly those that are prone to overexploitation and/or market substitution. The aim of this study was to develop a comprehensive reference library of DNA sequence data to allow the explicit identification of 53 commercially available fish species in South Africa, most of which were locally caught marine species. Sequences of approximately 655 base pairs were generated for all species from the cytochrome c oxidase I (COI) gene, the region widely adopted for DNA barcoding. Specimens of the genus Thunnus were examined in further detail, employing additional mitochondrial DNA control region sequencing. Cumulative analysis of the sequences from the COI region revealed mean conspecific, congeneric and confamilial Kimura 2-parameter distances of 0.10%, 4.58% and 15.43%, respectively. The results showed that the vast majority (98%) of fish species examined could be readily differentiated by their COI barcodes, but that supplementary control region sequencing was more useful for the discrimination of three Thunnus species. Additionally, the analysis of COI data raised the prospect that Thyrsites atun (snoek) could constitute a species pair. The present study has established the necessary genetic information to permit the unambiguous identification of 53 commonly marketed fish species in South Africa, the applications of which hold a plethora of benefits relating to ecology research, fisheries management and control of commercial practices.

Isolation of Alicyclobacillus and the influence of different growth parameters.

Alicyclobacillus species are thermo-acidophilic, endospore-forming bacteria that are able to survive pasteurisation and have been implicated in a number of spoilage incidents involving acidic foods and beverages. The aim of this study was to compare three isolation methods used for the detection of Alicyclobacillus acidoterrestris and to investigate the influence of incubation temperature on the growth of A. acidoterrestris and A. acidocaldarius. Peach juice samples inoculated with A. acidoterrestris K47 were analysed using either the International Federation of Fruit Juice Producers (IFU) Method No. 12 (Method A), which involved spread plating onto Bacillus acidoterrestris (BAT) agar at pH 4.0; Method B, which involved pour plating using potato dextrose agar (PDA) at pH 3.7; or Method C, which made use of membrane filtration followed by incubation on K agar at pH 3.7. The performance of the three methods differed significantly, with the IFU Method No. 12 recovering the highest percentage of cells at 75.97%, followed by Method B at 66.79% and Method C at 3.43%. These findings strengthen the proposal of the IFU for the use of the IFU Method No. 12 as a standard international method for the detection of Alicyclobacillus. To investigate the effect on growth of different incubation temperatures A. acidoterrestris (three strains) and A. acidocaldarius (two strains) were incubated at either 45 °C or 25 °C. Growth at 25°C was slower and maximum cell concentrations were lower (1 x 105-106 cfu/mL compared to 1 x 107-108 cfu/mL) than at 45 °C for A. acidoterrestris. A. acidocaldarius was unable to grow at 25°C and cell concentrations decreased by 1-2 logs. Since a growth temperature of 25 °C could not inhibit growth of A. acidoterrestris, cooling to room temperature (20°-25 °C) is not an effective control measure for A. acidoterrestris inhibition.

Phenylethanoid glycosides as major antioxidants in Lippia multiflora herbal infusion and their stability during steam pasteurisation of plant material.

Lippia multiflora, a perennial, aromatic shrub commonly known as bush tea has recently been identified as an African plant with high commercial potential due to its medicinal properties. The plant material was subjected to steam pasteurisation to improve its microbial quality. The major compounds of L. multiflora herbal infusion, i.e. the phenylethanoid glycosides (PhGs), verbascoside, isoverbascoside, nuomioside A and isonuomioside A, and the flavone, luteolin-7-O-glucuronide were quantified by HPLC. Verbascoside was the most abundant PhG. The PhGs are of interest due to their pharmacological properties. Liquid chromatography tandem-mass spectrometry (LC-MS(2)) was used to tentatively identify the compounds. The on-line DPPH() (2,2'-diphenyl-1-picrylhydrazyl radical) scavenging assay (reaction time=0.45s) applied to the infusion in "quantitative" mode, showed the relative order of activity: isonuomioside A>isoverbascoside>verbascoside>nuomioside A. In the microplate assay (reaction time=2h), isoverbascoside and verbascoside had similar activity. Both compounds were less active in the latter assay than the well-known flavan-3-ol antioxidant, (-)-epigallocatechin gallate, but more active than caffeic acid and an ester, rosmarinic acid. Steam pasteurisation of L. multiflora leaves at maximum exposure (150s at ca 99°C) for improved microbial quality did not decrease the soluble solids content, phenolic content and antioxidant activities of the infusion compared to the untreated control (p<0.05). The PhG content of the water soluble solids was as high as 15%, underscoring the potential of L. multiflora extract as functional ingredient.

Isolation and identification of species of Alicyclobacillus from orchard soil in the Western Cape, South Africa.

Alicyclobacilli were isolated from orchard soil collected from an apple and pear farm in Elgin, Western Cape, South Africa. Morphological, biochemical and physiological characteristics of the isolates were used to presumptively classify them as belonging to the genus Alicyclobacillus. Strains were identified to species level by polymerase chain reaction (PCR) with genus-specific primers, and 16S ribosomal RNA (rRNA) gene sequencing. To our knowledge this is the first report on the isolation of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius from orchard soil. The presence of these organisms in the soil suggests a possible source of contamination for the final fruit juice, concentrate or pulp.

Evaluation of different preservation techniques on the storage potential of Kefir grains.

Kefir is an acidic, mildly alcoholic dairy beverage produced by the fermentation of milk with a grain-like starter culture (Koroleva, 1988). These grains usually contain a relatively stable and specific balance of microbes that exist in a complex symbiotic relationship (Obermann & Libudzisz, 1998; Witthuhn et al. 2004). The different groups of microbes present in the grains are active at different stages of the fermentation (Koroleva, 1982). The lactococci, including Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Lc. lactis subsp. diacetilactis provide rapid acid development during the first hours of the fermentation (Litopoulou-Tzanetaki & Tzanetakis, 2000). As the acidity of the milk increases it provides favourable conditions for the growth of the lactobacilli (Rea et al. 1996). The yeasts, acetic acid bacteria and the aroma-producing microbes, mainly leuconostocs, have a much slower growth rate than the lactic acid producers, resulting in the slow production of the aroma compounds and the gradual increase in the concentration of these substances in the later stages of the fermentation (Koroleva, 1982). In the past the preservation of the microbial populations present in the traditional Kefir grains was achieved by methods including freezing (Garrote et al. 1997), lyophilisation (Oberman & Libudzisz, 1998), air-drying (Kroger, 1993) and refrigeration (Marshall, 1993). Research has shown that traditional Kefir grains preserved by air-drying and lyophilisation retain their activity for up to 12-18 months (Oberman & Libudzisz, 1998). Frozen grains stored at -20 degrees C were found to maintain the microbial activity for up to 7-8 months, whereas grains stored at refrigerated temperatures showed a decreased activity after about 10 d (Oberman & Libudzisz, 1998). The aim of this study was to evaluate the impact of four different preservation techniques on the activity of mass cultured Kefir grains (Schoevers & Britz, 2003). The activity of the grains was evaluated at different time intervals using four activity measurements, including changes in substrate pH, titratable acidity (TA), lactose and lactic acid levels of the final Kefir beverage.