The certification of a reference material for the evaluation of methods for the enumeration of Bacillus cereus

A reference material containing Bacillus cereus was certified by the Community Bureau of Reference (BCR) for its number of colony-forming particles (cfp) in 0.1 ml reconstituted capsule solution. To this end, a batch of approximately 15,000 capsules was produced and tested for its homogeneity and stability. The variation in the number of cfp between capsules (homogeneity) was not found to be significantly different from a Poisson distribution. Stability was tested for extended periods at storage temperature (-20 degrees C) and at various higher temperatures up to 37 degrees C for 4 weeks to simulate transport conditions. Only at 37 degrees C did a small but significant decrease in the number of cfp occur. At -20 degrees C, no decrease in the number of cfp was observed over a period of about 4 years. For certification, 12 laboratories determined the number of cfp on two agars: Mannitol Egg-Yolk Polymyxin agar (MEYP, incubated at 30 degrees C) and Polymyxin pyruvate Egg-yolk Bromothymol blue Agar (PEMBA, incubated at 37 degrees C). The certified geometric mean value on MEYP after 24 h of incubation was 53.4 cfp 0.1 ml-1 of the reconstituted capsule solution (95% confidence interval 51.7-55.2) and on PEMBA, 55.0 (95% confidence interval 52.8-57.4). Based on these certified values, user tables were constructed specifying the 95% confidence limits when testing a smaller number of capsules, as would be done in individual laboratories. Based on the information on homogeneity, stability and the certification study, the BCR decided to certify the material as CRM 528.     

A computer-controlled system to simulate conditions of the large intestine with peristaltic mixing, water absorption and absorption of fermentation products

This paper introduces a new type of system to simulate conditions in the large intestine. This system combines removal of metabolites and water with peristaltic mixing to obtain and handle physiological concentrations of microorganisms, dry matter and microbial metabolites. The system has been designed to be complementary to the dynamic multi-compartmental system that simulates conditions in the stomach and small intestine described by Minekus et al. [Minekus M, Marteau P, Havenaar R, Huis in't Veld JHJ (1995) ATLA 23:197–209]. High densities of microorganisms, comparable to those found in the colon in vivo, were achieved by absorption of water and dialysis of metabolites through hollow-fibre membranes inside the reactor compartments. The dense chyme was mixed and transported by peristaltic movements. The potential of the system as a tool to study fermentation was demonstrated in experiments with pectin, fructo-oligosaccharide, lactulose and lactitol as substrates. Parameters such as total acid production and short-chain fatty acid (SCFA) patterns were determined with time to characterize the fermentation. The stability of the microflora in the system was tested after inoculation with fresh fecal samples and after inoculation with a microflora that was main-tained in a fermenter. Both approaches resulted in total anaerobic bacterial counts higher than 10¹⁰ colony-forming units/ml with physiological levels of Bifidobacterium, Lactobacillus, Enterobacteriaceae and Clostridium. The dry matter content was approximately 10%, while the total SCFA concentration was maintained at physiological concentrations with similar molar ratios for acetic acid, propionic acid and butyric acid as measured in vivo. Received: 4 February 1999 / Received revision: 4 June 1999 / Accepted: 4 June 1999     

Organization and concerted evolution of the ampliconic Y-chromosomal TSPY genes from cattle

The Y-chromosomal gene TSPY (testis-specific protein Y-encoded) is probably involved in early spermatogenesis and has a variable copy number in different mammalian species. Analysis of bovine BAC clones leads to an estimate of 90 TSPY loci on the bovine Y chromosome. Half of these loci (TSPY-M1 and TSPY-M2) contain a single copy, while the other loci (TSPY-C) contain a cluster of three, possibly four, truncated pseudogenes. Fluorescence in situ hybridization indicated that the TSPY loci are located mainly on the short arm (Yp). The TSPY genes appear to account for about 2.5% of the Y chromosome and contain several published bovine Y-chromosomal microsatellites. The homology of TSPY and the major Y-chromosomal repetitive elements BRY.2 from cattle and OY.1 from sheep (80-85% similarity) further illustrates how the Y chromosome is shaped by rearrangements and horizontal spreading of the most abundant sequences. A comparison of TSPY-M1 sequences from different BAC clones and from related bovine species suggests concerted evolution as one of the mechanisms of the rapid evolution of the mammalian Y chromosome.     

Comparison of media for enumeration of Clostridium perfringens from foods

Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin polymyxin sulfadiazine perfringens (OPSP) agar] were compared in a prestudy, of which four (IS, TSC, SCA, and DCA) were selected for an international collaborative trial. Recovery of 15 pure strains was tested in the prestudy and recovery of one strain from foodstuffs was tested in the collaborative trial. Results from the prestudy did reveal statistical difference of the media but recoveries on all media were within the microbiological limits (+/-30%) of IS, which was set as a reference medium. Recoveries on the media tested in the collaborative trial were statistically different as well, but these differences were of no microbiological-analytical relevance. Food matrices did not affect the recovery of C. perfringens in general. DCA and SCA, in particular, are labor-intensive to prepare and DCA frequently failed to produce black colonies; gray colonies were quite common. Since IS medium is nonselective, it was concluded that TSC was the most favorable medium for the enumeration of C. perfringens from foods.     

Phytophthora gemini sp. nov., a new species isolated from the halophilic plant Zostera marina in the Netherlands

Eight strains belonging to the Oomycete genus Phytophthora were isolated from Zostera marina (seagrass) in The Netherlands over the past 25 y. Based on morphology, isozymes, temperature-growth relationships and ITS sequences, these strains were found to belong to two different Phytophthora species. Five strains, four of them isolated from rotting seeds and one isolated from decaying plants, could not be assigned to a known species and hence belong to a new species for which we propose the name Phytophthora gemini sp. nov. Three strains were isolated from decaying plants and were identified as Phytophthora inundata, thereby expanding the known habitat range of this species from fresh to brackish-saline areas. The possible role of both Phytophthora species in the decline of Z. marina in The Netherlands and the evolutionary significance of the presence of Phytophthora species in marine environments are discussed.     

Beta Cell Count Instead of Beta Cell Mass to Assess and Localize Growth in Beta Cell Population following Pancreatic Duct Ligation in Mice

Background

Pancreatic-tail duct ligation (PDL) in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates.

Methodology

Total beta cell number and its distribution over small (<50 µm), medium, large (>100 µm) clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham.

Principal findings

PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number) which contained a higher percent proliferating beta cells (1.9% Ki67+cells) than sham tails (<0.2%); their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters.

Conclusions/significance

The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.     

Genome sequence of Neisseria meningitidis serogroup B strain H44/76

Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.     

Gap junctions between pancreatic B-cells are modulated by cyclic AMP

Gap junction morphology was studied on freeze fracture replicas of pancreatic islet tissue, using morphometric techniques. In rat islets in situ, 60 percent of the connexions were polygonally packed in gap junctions, whereas the remaining part occurred in linear strands. After collagenase isolation, the islets presented similar numbers of gap junctions but contained virtually no linear strands. The distribution of connexions over polygonal or linear arrays also varied with the culture conditions: at 11.2 mM glucose, a higher percentage of particles occurred in gap junctions than at 5.6 mM glucose; this was also the case in other conditions with elevated cellular cyclic AMP levels. The total number of connexions increased when islets were cultured with dibutyryl cyclic AMP or with a phosphodiesterase inhibitor; conditions with an augmented number of gap junctions also displayed an elevated islet cyclic AMP content. A similar association was noted in newly formed aggregates of pancreatic B-cells purified by autofluorescence-activated. cell sorting. These results indicate that the number of classically defined gap junctions is not only dependent on the total number of connexions but also on their organization within the membrane. It is suggested that the distribution of connexions over polygonal and linear arrays follows a dynamic equilibrium varying with the extracellular conditions. Cyclic AMP appears to modulate the number of gap junctions between pancreatic B-cells both through an induction of new connexions and through an assembly of linearly organized particles into polygonal arrays.