Validation of alternative methods for the analysis of drinking water and their application to Escherichia coli

In Europe, the Drinking Water Directive of the European Commission indicates which methods (most of which are CEN/ISO-standardized methods) should be used for the analysis of microbiological parameters (European Commission, Environment, Council Directive 98/83/EC of 3 November 1998). According to the Directive, alternative methods “may be used, providing it can be demonstrated that the results obtained are at least as reliable as those produced by the methods specified.” The prerequisite for the routine use of any alternative method is to provide evidence that this method performs equivalently to the corresponding reference method. In this respect, the ISO 16140 standard (ISO, ISO 16140. Microbiology of Food and Animal Feeding Stuffs—Protocol for the Validation of Alternative Methods, 2003) represents a key issue in generating such a procedure based on an interlaboratory study. A new statistical tool, called the accuracy profile, has been developed to better interpret the data. The study presented here is based upon the enumeration of Escherichia coli bacteria in water. The reference method may require up to 72 h to provide a confirmed result. The aim of this publication is to present data for an alternative method by which results can be obtained in 18 h (Colilert-18/Quanti-Tray) based upon defined substrate technology (DST). The accuracy profile is a statistical and graphical decision-making tool and consists of simultaneously combining, in a single graphic, β expectation tolerance intervals (β-ETIs) and acceptability limits (λ). The study presents the validation criteria calculated at the three levels of contamination used in the trial for a β equal to 80% and a λ equal to ±0.3 and combines the accuracy profiles of Escherichia coli for a λ of ±0.3 log₁₀ unit/100 ml, a λ of ±0.4 log₁₀ unit/100 ml, and a β of 80% or 90%. Several interesting conclusions can be drawn from these data. The accuracy profile method has been applied to the validation of the Colilert-18/Quanti-Tray method against reference method ISO 9308-1 (ISO, ISO 9308-1. Water Quality—Detection and Enumeration of Escherichia coli and Coliform Bacteria. Part 1. Membrane Filtration Method, 2000), using a β of 80% and a λ of 0.4; the alternative method can be validated between 1.00 and 2.05 log₁₀ units/100 ml, equivalent to 10 to 112 CFU/100 ml.     

Case study of a biological control: <Emphasis Type="Italic">Geobacillus caldoxylosilyticus</Emphasis> (IRD) contributes to alleviate salt stress in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants

The inevitable exposure of crop plants to salt stress is a major environmental problem emerged from the presence of excess NaCl radicals in the soil. Handling the problem in maize plants using a biological agent was the main interest of the present study. The non-pathogenic, halophytic, facultative aerobic bacterium Geobacillus caldoxylosilyticus IRD that was isolated from Marakopara pond in the Atoll Tikehau (French Polynesian, 2005) and found tolerant to salt stress until 3.5% NaCl (w/v). An artificial symbiosis was achieved by inoculating Geobacillus sp. into 5-day-old maize cultivars of triple hybrids (321 and 310) and singlet hybrids (10 and 162). Thereafter, maize seedlings were exposed to 350 mmol NaCl for 10 days. The data revealed that Geobacillus sp. had interacted with salinized maize and improved maize overall growth, dry weight and relative water content. Na⁺ accumulation was six times less and Cl⁻ accumulation was 13 times less in the tips of salinized maize seedlings upon Geobacillus sp. inoculation. Salinized maize without Geobacillus viewed decayed cortical cells of seedlings. In addition, proline content was two times higher in salinized seedlings lacking Geobacillus. Photosynthetic pigments and antioxidant enzymes were significantly regulated upon inoculation. Beyond this study, we presented a novel insight into a possible role of Geobacillus caldoxylosilyticus bacteria in controlling/protecting maize plants against high salt stress.     

Growth Differentiation Factor-15 (GDF-15) Levels Are Associated with Cardiac and Renal Injury in Patients Undergoing Coronary Artery Bypass Grafting with Cardiopulmonary Bypass

Objective

Growth differentiation factor-15 (GDF-15) has been identified as a strong marker of cardiovascular disease; however, no data are available concerning the role of GDF-15 in the occurrence of organ dysfunction during coronary artery bypass grafting (CABG) associated with cardiopulmonary bypass (CPB).

Methods

Five arterial blood samples were taken sequentially in 34 patients from anesthesia induction (IND) until 24 h after arrival at the intensive care unit (ICU). Plasma levels of GDF-15, follistatin-like 1 (FLST1), myeloperoxidases (MPO), hydroperoxides and plasma antioxidant status (PAS) were measured at each time-point. Markers of cardiac (cardiac-troponin I, cTnI) and renal dysfunction (neutrophil gelatinase-associated lipocalin, NGAL) and other classical biological factors and clinical data were measured.

Results

Plasma GDF-15 levels increased gradually during and after surgery, reaching nearly three times the IND levels in the ICU (3,075±284 ng/L vs. 1,061±90 ng/L, p<0.001). Plasma MPO levels increased dramatically during surgery, attaining their highest level after unclamping (UNCLAMP) (49±11 ng/mL vs. 1,679±153 ng/mL, p<0.001) while PAS significantly decreased between IND and UNCLAMP (p<0.05), confirming the high oxidative status induced by this surgical procedure. ICU levels of GDF-15 correlated positively with cTnI and NGAL (p = 0.006 and p = 0.036, respectively), and also with hemoglobin and estimated glomerular filtration rate (eGFR). Among all the post-operative biomarkers available, only eGFR, NGAL and GDF-15 measured at ICU arrival were significantly associated with the onset of acute kidney injury (AKI). Patients with a EuroSCORE >3 were shown to have higher GDF-15 levels.

Conclusions

During cardiac surgery associated with CPB, GDF-15 levels increased substantially and were associated with markers of cardiac injury and renal dysfunction.     

Isolation, phylogeny and evolution of the SymRK gene in the legume genus Lupinus L

SymRK is one of the key genes involved in initial steps of legume symbiotic association with fungi (mycorrhization) and nitrogen-fixing bacteria (nodulation). A large portion of the sequence encoding the extracellular domain of SYMRK was obtained for 38 lupine accessions and 2 outgroups in order to characterize this region, to evaluate its phylogenetic utility, and to examine whether its molecular evolutionary pattern is correlated with rhizobial diversity and specificity in Lupinus. The data suggested that, in Lupinus, SymRK is a single copy gene that shows good phylogenetic potential. Accordingly, SymRK provided additional support to previous molecular phylogenies, and shed additional light on relationships within the Old World group of Lupinus, especially among the African species. Similar to results of other studies, analyses of SymRK sequences were unable to resolve placement of the Florida unifoliolate lineage, whose relationship was weakly supported to either the Old or the New World lupines. Our data are consistent with strong purifying selection operating on SymRK in Lupinus, preserving rather than diversifying its function. Thus, although SymRK was demonstrated to be a vital gene in the early stages of the root-bacterial symbiotic associations, no evidence from present analyses indicate that this gene is involved in changes in rhizobial specificity in Lupinus.     

Diffusive oxygen shunting between vessels in the preglomerular renal vasculature: anatomic observations and computational modeling

To understand how geometric factors affect arterial-to-venous (AV) oxygen shunting, a mathematical model of diffusive oxygen transport in the renal cortex was developed. Preglomerular vascular geometry was investigated using light microscopy (providing vein shape, AV separation, and capillary density near arteries) and published micro-computed tomography (CT) data (providing vessel size and AV separation; Nordsletten DA, Blackett S, Bentley MD, Ritman EL, Smith NP. IUPS Physiome Project. http://www.physiome.org.nz/publications/nordsletten_blackett_ritman_bentley_smith_2005/folder_contents). A "U-shaped" relationship was observed between the arterial radius and the distance between the arterial and venous lumens. Veins were found to partially wrap around the artery more consistently for larger rather than smaller arteries. Intrarenal arteries were surrounded by an area of fibrous tissue, lacking capillaries, the thickness of which increased from ～5 μm for the smallest arteries (<16-μm diameter) to ～20 μm for the largest arteries (>200-μm diameter). Capillary density was greater near smaller arteries than larger arteries. No capillaries were observed between wrapped AV vessel pairs. The computational model comprised a single AV pair in cross section. Geometric parameters critical in renal oxygen transport were altered according to variations observed by CT and light microscopy. Lumen separation and wrapping of the vein around the artery were found to be the critical geometric factors determining the amount of oxygen shunted between AV pairs. AV oxygen shunting increases both as lumen separation decreases and as the degree of wrapping increases. The model also predicts that capillaries not only deliver oxygen, but can also remove oxygen from the cortical parenchyma close to an AV pair. Thus the presence of oxygen sinks (capillaries or tubules) near arteries would reduce the effectiveness of AV oxygen shunting. Collectively, these data suggest that AV oxygen shunting would be favored in larger vessels common to the cortical and medullary circulations (i.e., arcuate and proximal interlobular arteries) rather than the smaller vessels specific to the cortical circulation (distal interlobular arteries and afferent arterioles).     

Rhizobial diversity associated with the spontaneous legume <Emphasis Type="Italic">Genista saharae</Emphasis> in the northeastern Algerian Sahara

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N₂-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4 % (w/v) NaCl and grew at 45 °C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.     

Otx1 and Otx2 activities are required for the normal development of the mouse inner ear

The Otx1 and Otx2 genes are two murine orthologues of the Orthodenticle (Otd) gene in Drosophila. In the developing mouse embryo, both Otx genes are expressed in the rostral head region and in certain sense organs such as the inner ear. Previous studies have shown that mice lacking Otx1 display abnormal patterning of the brain, whereas embryos lacking Otx2 develop without heads. In this study, we examined, at different developmental stages, the inner ears of mice lacking both Otx1 and Otx2 genes. In wild-type inner ears, Otx1, but not Otx2, was expressed in the lateral canal and ampulla, as well as part of the utricle. Ventral to the mid-level of the presumptive utricle, Otx1 and Otx2 were co-expressed, in regions such as the saccule and cochlea. Paint-filled membranous labyrinths of Otx1-/- mutants showed an absence of the lateral semicircular canal, lateral ampulla, utriculosaccular duct and cochleosaccular duct, and a poorly defined hook (the proximal part) of the cochlea. Defects in the shape of the saccule and cochlea were variable in Otx1-/- mice and were much more severe in an Otx1-/-;Otx2(+/)- background. Histological and in situ hybridization experiments of both Otx1-/- and Otx1-/-;Otx2(+/)- mutants revealed that the lateral crista was absent. In addition, the maculae of the utricle and saccule were partially fused. In mutant mice in which both copies of the Otx1 gene were replaced with a human Otx2 cDNA (hOtx2(1)/ hOtx2(1)), most of the defects associated with Otx1-/- mutants were rescued. However, within the inner ear, hOtx2 expression failed to rescue the lateral canal and ampulla phenotypes, and only variable rescues were observed in regions where both Otx1 and Otx2 are normally expressed. These results suggest that both Otx genes play important and differing roles in the morphogenesis of the mouse inner ear and the development of its sensory organs.     

Correlation between alterations of the penicillin-binding protein 2 and modifications of the peptidoglycan structure in Neisseria meningitidis with reduced susceptibility to penicillin G

Reduced susceptibility to penicillin G in Neisseria meningitidis is directly correlated with alterations in the penA gene, which encodes the penicillin-binding protein 2 (PBP2). Using purified PBP2s from different backgrounds, we confirmed that the reduced susceptibility to penicillin G is associated with a decreased affinity of altered PBP2s for penicillin G. Infrared spectroscopy analysis using isogenic penicillin-susceptible strains and strains with reduced susceptibility to penicillin G suggested that the meningococcal cell wall is also modified in a penA-dependent manner. Moreover, reverse-phase high pressure liquid chromatography and mass spectrometry analysis of these meningococcal strains confirmed the modifications of peptidoglycan components and showed an increase in the peaks corresponding to pentapeptide-containing muropeptides. These results suggest that the D,D-transpeptidase and/or D,D-carboxypeptidase activities of PBP2 are modified by the changes in penA gene.