Survival and gene expression of enterotoxigenic Escherichia coli during long‐term incubation in sea water and freshwater

Aims: In this study, the main objective was to verify the hypothesis of induction of ‘viable but non‐culturable’ (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. Methods and Results: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat‐labile (LT) or heat‐stable (ST) enterotoxins was observed using GM1‐ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real‐time RT‐PCR on cDNA derived from the bacteria. Conclusions: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. Significance and Impact of the Study: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long‐term incubation in water.     

Sperm dimorphism and spermatozeugmata in the commensal bivalve Pseudopythina macrophthalmensis (Galeommatoidea, Kelliidae)

The bivalve Pseudopythina macrophthalmensis (Galeommatoidea) is a commensal with the crab Macrophthalmus convexus (Ocypodidae) in Okinawa Prefecture, Japan. It is a protandric hermaphrodite which incubates the 65-μm large ova in the suprabranchial cavity. The species produces two types of sperm, which were studied with the electron microscope. The euspermatozoon has an elongate 2.8-μm-long, pointed acrosome, a slender 12- to 13-μm-long nucleus and a middlepiece containing several closely packed mitochondria arranged as a 5.5- to 6.0-μm-long sheath around the basis of the flagellum. The paraspermato- zoon is vermiform, 220-μm-long and up to 5-μm-broad. Anteriorly there is a ca 7-μm-long bullet-shaped acrosome followed by a subcylindrical 3.0- to 4.7-μm-long nucleus. Adjacent to the nucleus occurs a bundle of 26–42 40-μm-long flagella. The cytoplasm is packed with spherical lipid droplets and ovoid granules of unknown composition. Sperm of both types aggregate to form spermatozeugmata, which were found in the posterior mantle cavity or in paired seminal receptacles. Within the receptacles the euspermatozoa dissociate themselves from the spermatozeugma and become attached to the epithelial lining of the receptacle whereas the paraspermatozoa presumably disintegrate. The possible significance of the two types of sperm is discussed in the light of their presumed functions in gastropods. Accepted: 9 November 2000     

Optimization of sulphide production in an anaerobic continuous biofilm process with sulphate reducing bacteria

Different biofilm reactors for sulphide production by sulphate reducing bacteria were compared in a packed bed reactor and in two suspended carrier biofilm reactors. Lactate was used as carbon source in the experiments. The process was reversibly inhibited by free sulphide at 14 mM. The packed bed reactor was more efficient and less sensitive to changes. The maximum load in the system was 5.3 g sulphate/l.d.     

The evolution of highly variable immunity genes across a passerine bird radiation

To survive, individuals must be able to recognize and eliminate pathogens. The genes of the major histocompatibility complex (MHC) play an essential role in this process in vertebrates as their diversity affects the repertoire of pathogens that can be recognized by the immune system. Emerging evidence suggests that birds within the parvorder Passerida possess an exceptionally high number of MHC genes. However, this has yet to be directly investigated using a consistent framework, and the question of how this MHC diversity has evolved has not been addressed. We used next‐generation sequencing to investigate how MHC class I gene copy number and sequence diversity varies across the Passerida radiation using twelve species chosen to represent the phylogenetic range of this group. Additionally, we performed phylogenetic analyses on this data to identify, for the first time, the evolutionary model that best describes how MHC class I gene diversity has evolved within Passerida. We found evidence of multiple MHC class I genes in every family tested, with an extremely broad range in gene copy number across Passerida. There was a strong phylogenetic signal in MHC gene copy number and diversity, and these traits appear to have evolved through a process of Brownian motion in the species studied, that is following the pattern of genetic drift or fluctuating selection, as opposed to towards a single optimal value or through evolutionary ‘bursts’. By characterizing MHC class I gene diversity across Passerida in a systematic framework, this study provides a first step towards understanding this huge variation.     

Gastroprotective and blood pressure lowering effects of dietary nitrate are abolished by an antiseptic mouthwash

Recently, it has been suggested that the supposedly inert nitrite anion is reduced in vivo to form bioactive nitric oxide with physiological and therapeutic implications in the gastrointestinal and cardiovascular systems. Intake of nitrate-rich food such as vegetables results in increased levels of circulating nitrite in a process suggested to involve nitrate-reducing bacteria in the oral cavity. Here we investigated the importance of the oral microflora and dietary nitrate in regulation of gastric mucosal defense and blood pressure. Rats were treated twice daily with a commercial antiseptic mouthwash while they were given nitrate-supplemented drinking water. The mouthwash greatly reduced the number of nitrate-reducing oral bacteria and as a consequence, nitrate-induced increases in gastric NO and circulating nitrite levels were markedly reduced. With the mouthwash the observed nitrate-induced increase in gastric mucus thickness was attenuated and the gastroprotective effect against an ulcerogenic compound was lost. Furthermore, the decrease in systemic blood pressure seen during nitrate supplementation was now absent. These results suggest that oral symbiotic bacteria modulate gastrointestinal and cardiovascular function via bioactivation of salivary nitrate. Excessive use of antiseptic mouthwashes may attenuate the bioactivity of dietary nitrate.     

Nitrated oleic acid up-regulates PPARγ and attenuates experimental inflammatory bowel disease

Nitric oxide and its metabolites undergo nitration reactions with unsaturated fatty acids during oxidative inflammatory conditions, forming electrophilic nitro-fatty acid derivatives. These endogenous electrophilic mediators activate anti-inflammatory signaling reactions, serving as high-affinity ligands for peroxisome proliferator-activated receptor γ (PPARγ). Here we examined the therapeutic effects of 9- or 10-nitro-octadecenoic oleic acid (OA-NO₂) and native oleic acid (OA) in a mouse model of colitis. OA-NO₂ reduced the disease activity index and completely prevented dextran sulfate sodium-induced colon shortening and the increase in colonic p65 expression. Increased PPARγ expression was observed in colon samples as well as in cells after OA-NO₂ administration, whereas no effect was seen with OA. This induction of PPARγ expression was completely abolished by the PPARγ antagonist GW9662. 5-Aminosalicylic acid, an anti-inflammatory drug routinely used in the management of inflammatory bowel disease, also increased PPARγ expression but to a lesser extent. Altogether, these findings demonstrate that administration of OA-NO₂ attenuates colonic inflammation and improves clinical symptoms in experimental inflammatory bowel disease. This protection involves activation of colonic PPARγ.     

Differential effects of polymers PVP90 and Ficoll400 on storage stability and viability of Lactobacillus coryniformis Si3 freeze‐dried in sucrose

Aims: To investigate the effect of freeze‐dried Lactobacillus coryniformis Si3 on storage stability by adding polymers to sucrose‐based formulations and to examine the relationship between amorphous matrix stability and cell viability. Methods and Results: The resistance to moisture‐induced sucrose crystallization and effects on the glass transition temperature (T_g) by the addition of polymers to the formulation were determined by different calorimetric techniques. Both polymers increased the amorphous matrix stability compared to the control, and poly(vinyl)pyrrolidone K90 was more effective in increasing amorphous stability than Ficoll 400. The viability of Lact. coryniformis Si3 after storage was investigated by plate counts following exposure to different moisture levels and temperatures for up to 3 months. The polymers enhanced the cellular viability to different degrees, dependent upon polymer and storage condition. Conclusions: Polymers can be used to enhance the stability of freeze‐dried Lact. coryniformis Si3 products, but cell viability and matrix stability do not always correlate. The general rule of thumb to keep a highly amorphous product 50° below its T_g for overall stability seemed to apply for this type of bacterial products. We showed that by combining thermal analysis with plate counts, it was possible to determine storage conditions where cell viability and matrix stability were kept high. Significance and Impact of the Study: The results will aid in the rational formulation design and proper determination of storage conditions for freeze‐dried and highly amorphous lactic acid bacteria formulations. We propose a hypothesis of reason for different stabilizing effects on the cells by the different polymers based on our findings and previous findings.