Arthrospira (Spirulina’) strains from four continents are resolved into only two clusters, based on amplified ribosomal DNA restriction analysis of the internally transcribed spacer. FEMS

Abstract We present the results of a phylogenetic study, based on amplified ribosomal DNA restriction analysis of the rDNA operon, of 37 Arthrospira (`Spirulina') cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 51 tested cultures. The strain Spirulina laxissima SAG 256.80 was included as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by amplified ribosomal DNA restriction analysis, and thus the internally transcribed spacer was selected as molecular taxonomic marker. The internally transcribed spacer sequences situated between the 16S and the 23S rRNA were amplified by polymerase chain reaction and yielded amplicons of about 540 bp. Direct use of cells for polymerase chain reaction seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of bovine serum albumin in the polymerase chain reaction mix.The amplicons were digested with four restriction enzymes (EcoRV, HhaI, HinfI, MseI) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology.

Emended descriptions of Bacillus sporothermodurans and Bacillus oleronius with the inclusion of dairy farm isolates of both species

Bacillus sporothermodurans is an industrially important micro-organism because of its ability to produce endospores which resist ultra high temperature (UHT) and industrial sterilization processes. It was described by Pettersson et al. (1996) based on seven genetically homogeneous isolates all from UHT-milk. Bacillus oleronius, the closest phylogenetic neighbor of B. sporothermodurans, was described by Kuhnigk et al. (1995), based on a single strain, isolated from the hindgut of the termite Reticulitermes santonensis. A polyphasic study of a heterogeneous collection of B. sporothermodurans and B. oleronius strains isolated from various sources and geographic origins led to an emended description of both species. Additional data presented are the results of fatty acids, quinones and/or cell wall analysis (polar lipids). DNA-DNA hybridizations confirmed 3 subgroups of strains obtained after SDS-PAGE analysis of cellular proteins as B. sporothermodurans. One named B. sporothermodurans strain (R-7489) was reclassified as a Bacillus fordii strain. The phenotypic profiles of both species were rather heterogeneous, sometimes different from the original descriptions and did not differ in a large number of characters, although B. oleronius generally gave stronger reactions in its positive tests than did B. sporothermodurans; the variable and weak reactions for both organisms with some substrates blurred the distinction between both. However, differences in polar lipid, SDS-PAGE and menaquinone profiles clearly allow distinction between the two species

Efficacy of electrolyzed oxidizing water and lactic acid on the reduction of Campylobacter on naturally contaminated broiler carcasses during processing

Campylobacter is the most commonly reported gastrointestinal bacterial pathogen in humans in many developed countries. During slaughter of broiler flocks, it is difficult to avoid contamination of broiler carcasses. This study aimed to quantify Campylobacter contamination on broiler carcasses at 5 points in the slaughter processing during the slaughter of a Campylobacter-colonized flock by real-time PCR and conventional enumeration. In addition, the decontamination effect of neutral electrolyzed oxidizing (EO) water and 1.5% lactic acid (pH 2.0) were evaluated. During processing, the Campylobacter counts on the carcasses declined toward the end of the processing line. The log counts on the carcasses as determined by quantitative real-time PCR (qPCR), decreased from 9.37 after scalding to 8.08 after the last cooling step. Enumeration of the campylobacters on plates revealed the same trend, although the counts per carcass were generally 3 logs lower. After scalding, a mean of 6.86 log cfu/carcass were counted, which decreased to 4.83 log cfu/carcass after the last cooling step. Submerging carcasses after scalding in EO water gave a significant reduction of 1.31 log cfu/carcass by enumeration on plates and a not significant reduction of 0.53 log cfu/carcass by qPCR. Treatment of the carcasses after the inside-outside bird washer led to reductions from 0.09 to 0.91 log cfu/carcass, although not significant. After submerging the carcasses in a 1.5% lactic acid solution, significant reductions of 1.62 and 1.24 log cfu/carcass by qPCR and enumeration, respectively, were observed. Spraying the carcasses with lactic acid led to nonsignificant reductions of 0.68 log cfu/carcass determined by qPCR and 0.26 log cfu/carcass by enumeration. Both EO water and lactic acid seem promising for implementation in poultry processing plants

Characterization of Arthrospira / Spirulina strains: Molecular Aspects

We present the results of a phylogenetic study, based on ARDRA of the rDNA operon, of 38 Arthrospira (‘Spirulina’) cultivated clonal strains from four continents. In addition, duplicates from different culture collections or markedly different morphotypes of particular strains established as clonal cultures were treated as separate entries, resulting in a total of 54 tested cultures. Three living samples from Earthrise Farms ponds (September 1997), four freeze-dried samples from EF ponds (August 1996, February and March 1997) and a powder of ‘Spirulina pacifica’ were also included in the study. The strain Spirulina laxissima SAG 256.80 was used as outgroup. The 16S rRNA genes appeared too conserved for discrimination of the strains by ARDRA, and thus the Internal Transcribed Spacer (ITS) was selected as a molecular taxonomic marker. The ITS sequences situated between the 16S and the 23S rRNA genes were amplified by PCR and yielded amplicons of about 540 bp. The amplicons were digested with four restriction enzymes (EcoR V, Hha I, Hinf I, Mse I) and the banding patterns obtained were analyzed. Cluster analysis showed the separation of all the strains into two main clusters (Clusters I and II), of which Cluster I was divided into Subclusters I.A and I.B. Four freeze-dried samples from EF cultivation ponds (Summer 1996 and Winter 1997), as well as a sample of powder sent as ‘Spirulina pacifica’ appeared to contain a mixture of genotypes from Clusters I and II. No clear relationships could be observed between this division into two clusters and the geographic origin of the strains, or their designation in the culture collections, or their morphology. Direct use of cells for PCR seemed to inhibit the amplification reaction. This was overcome by the design of a crude lysis protocol and addition of BSA (Bovine Serum Albumin) in the PCR mix. In order to study in more depth the genotypic relationships of Arthrospira, we have obtained the ITS sequence of 19 cultures and 7 samples (living or freeze-dried samples from EF ponds, dried natural samples and one commercial pill). The data confirmed the existence of Clusters I and II, but also subdivided each of them into two Subclusters (A and B). In three cultures, simultaneous presence of types II.A and II.B was detected. It is likely that sequences of both types are contained in different copies of the ITS and that the three cultures represent cryptic duplicates of one unique genotype. The strains cultivated in the EF ponds belong to types I.A, II.A and II.B, while the winter ponds samples were a mixture of types I and II. Though there was surprisingly little sequence variability in the ITS sequences, we designed PCR primers which are specific for the two clusters (44 different positions) and for the four subclusters (2 to 4 different positions)

Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms

The presence of highly heat-resistant spores of Bacillus sporothermodurans in ultrahigh-temperature or sterilized consumer milk has emerged as an important item in the dairy industry. Their presence is considered undesirable since they hamper the achievement of commercial sterility requirements. By using a selective 30-min heat treatment at 100°C, 17 Belgian dairy farms were screened to evaluate the presence, sources, and nature of potentially highly heat-resistant spores in raw milk. High numbers of these spores were detected in the filter cloth of the milking equipment and in green crop and fodder samples. About 700 strains were isolated after the selective heating, of which 635 could be screened by fatty acid methyl ester analysis. Representative strains were subjected to amplified ribosomal DNA restriction analysis, 16S rRNA gene sequencing, percent G+C content, and DNA-DNA reassociations for further identification. The strain collection showed a remarkable diversity, with representatives of seven aerobic spore-forming genera. Bacillus licheniformis and Bacillus pallidus were the most predominant species overall. Twenty-three percent of the 603 spore-forming isolates proved to belong to 18 separate novel species. These findings suggest that the selective heating revealed a pool of unknown organisms with a higher heat-resistant character. This study showed that high spore counts can occur at the dairy farm and that feed and milking equipment can act as reservoirs or entry points for potentially highly heat-resistant spores into raw milk. Lowering this spore load by good hygienic measures could probably further reduce the contamination level of raw milk, in this way minimizing the aerobic spore-forming bacteria that could lead to spoilage of milk and dairy products. Assessment and characterization of this particular flora are of great importance to allow the dairy or food industry to adequately deal with newly arising microbiological problems