Methods for microbiological quality assessment in drinking water: a comparative study.

The present study aimed to compare several methods for quantifying and discriminating between the different physiological states of a bacterial population present in drinking water. Flow cytometry (FCM), solid-phase cytometry (SPC), epifluorescence microscopy (MSP) and culture method performances were assessed by comparing the results obtained for different water samples. These samples, including chlorinated and non-chlorinated water, were collected in a drinking water treatment plant. Total bacteria were quantified by using SYBR Green II (for FCM) and 4',6'-diamino-2-phenylindole (DAPI) (for MSP), viable and non-viable bacteria were distinguished by using SYBR Green II and propidium iodide dual staining (for FCM), and active cells were distinguished by using CTC (for MSP) and Chemchrome V6 (for FCM and SPC). In our conditions, counts using microscopy and FCM were significantly correlated regarding total bacteria and active cells. Conversely, counts were not significantly similar using solid-phase and FCM for active bacteria. Moreover, the R2A medium showed that bacterial culturability could be recovered after chlorination. This study highlights that FCM appears to be a useful and powerful technique for drinking water production monitoring.

Comparison of two filtration-elution procedures to improve the standard methods ISO 10705-1 & 2 for bacteriophage detection in groundwater, surface water and finished water samples.

AIM: To select a reliable method for bacteriophage concentration prior detection by culture from surface water, groundwater and drinking water to enhance the sensitivity of the standard methods ISO 10705-1 & 2. METHODS AND RESULTS: Artificially contaminated (groundwater and drinking water) and naturally contaminated (surface water) 1-litre samples were processed for bacteriophages detection. The spiked samples were inoculated with about 150 PFU of F-specific RNA bacteriophages and somatic coliphages using wastewater. Bacteriophage detection in the water samples was achieved using the standard method without and with a concentration step (electropositive Anodisc membrane or a pretreated electronegative Micro Filtration membrane, MF). For artificially contaminated matrices (drinking and ground waters), recovery rates using the concentration step were superior to 70% whilst analyses without concentration step mainly led to false negative results. Besides, the MF membrane presented higher performances compared with the Anodisc membrane. CONCLUSION: The concentration of a large volume of water (up to one litre) on a filter membrane avoids false negative results obtained by direct analysis as it allows detecting low number of bacteriophages in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of concentration step before applying the standard method could be useful to enhance the reliability of bacteriophages monitoring in water samples as bio-indicators to highlight faecal pollution.

Nitrated type III collagen as a biological marker of nitric oxide-mediated synovial tissue metabolism in osteoarthritis.

OBJECTIVES: Nitric oxide (NO) is a major mediator of joint tissue inflammation and damage in osteoarthritis (OA) and mediates the nitration of tyrosine (Y*) residues in proteins. We investigated the nitration of type III collagen, a major constituent of synovial membrane, in knee OA. METHODS: A polyclonal antibody directed against the nitrated QY*DSY*DVKSG sequence from type III collagen N-telopeptide was generated. Synovial tissues from patients with knee OA (n=4) and rheumatoid arthritis (RA, n=4) were analyzed by immunohistochemistry for IIINys. Serum IIINys levels were measured by enzyme-linked immunosorbent assay in 87 patients with painful knee OA (mean age: 63.0+/-8.0 years, Kellgren-Lawrence score II-III) and in 40 sex and age-matched healthy controls. RESULTS: Competition experiments using various nitrated and un-nitrated type III collagen and derived sequences, showed that the antibody was highly specific for the nitrated IIINys sequence. High IIINys immunoreactivity was detected in the synovial tissues from all patients with OA and RA with a preferential localization in the intimal layer. Serum IIINys levels were on average 1.5-fold higher (P<0.0001) in patients with knee OA than in healthy controls and significantly correlated with C-reactive protein values (r=0.40, P<0.005). CONCLUSIONS: Nitration of tyrosine residues of type III collagen N-telopeptide is increased in the synovial tissue of patients with knee OA. Measurements of serum IIINys level may be useful for the clinical investigation of oxidative-related alterations of synovial tissue metabolism in OA.

The type II collagen fragments Helix-II and CTX-II reveal different enzymatic pathways of human cartilage collagen degradation.

OBJECTIVE: Cartilage degradation in osteoarthritis (OA) generates the type II collagen fragments, Helix-II and CTX-II that can be used as clinical biological markers. Helix-II and C-telopeptide of type II collagen (CTX-II) levels are associated independently with progression of OA suggesting that they may be generated through different collagenolytic pathways. In this study we analyzed the release of Helix-II and CTX-II from human cartilage collagen by the proteinases reported to play a role in cartilage degradation. METHODS: In vitro, human articular cartilage extract was incubated with activated human recombinant cathepsins (Cats) and matrix-metalloproteases (MMPs). Next, we analyzed the spontaneous release of Helix-II and CTX-II from cartilage sections of patients with knee OA who were immediately deep frozen after joint replacement to preserve endogenous enzyme activity until assay. Cartilage sections were then incubated for up to 84 h in the presence or absence of E-64 and GM6001, inhibitors of cysteine proteases and MMPs, respectively. RESULTS: In vitro, Cats K, L and S generated large amount of Helix-II, but not CTX-II. Cat B generated CTX-II fragment, but destroyed Helix-II immunoreactivity. Cat D was unable to digest intact cartilage. MMPs-1, -3, -7, -9, and -13 efficiently released CTX-II, but only small amount of Helix-II. Neither CTX-II nor Helix-II alone was able to reflect accurately the collagenolytic activity of Cats and MMPs as reflected by the release of hydroxyproline. In OA cartilage explants, E-64 blunted the release of Helix-II whereas the release of CTX-II could be completely abrogated by GM6001 and only partly by E-64. CONCLUSION: These in vitro and ex vivo experiments of human cartilage suggest that Helix-II and CTX-II could be released in part by different enzymatic pathways. Helix-II and CTX-II alone reflect only partially overall cartilage collagen degradation. These findings may explain why these two biological markers could provide complementary information on disease progression in OA.

PIIANP and HELIXII diurnal variation.

OBJECTIVE: Serum levels of procollagen type IIA N-terminal propeptide (sPIIANP) and type-II collagen helical peptide (sHELIXII) biomarkers were evaluated for variation diurnally and with physical activity and food in participants with osteoarthritis (OA) of the knee. METHODS: Forty participants with OA of at least one knee were admitted overnight to the General Clinical Research Center for serial serum sampling. Samples were obtained on the evening (6-8 pm) of Day 1 (T3, n=40); prior to rising (8 am) from bed (T0, n=40); 1 h after rising (9 am) without food consumption (T1a, n=20); 1-2 h after rising (9-10 am) with food consumption (T1, n=40); and additionally at noon, 4 h after rising (T2, n=20). sPIIANP and sHELIXII were measured by enzyme-linked immunosorbent assay. Results were analyzed using non-parametric Freidman's test with Dunn's post-hoc multiple comparison test. RESULTS: Normalized mean concentrations for sPIIANP and sHELIXII increased significantly from T0 to T1 (P<0.05). CONCLUSIONS: This is the first study to demonstrate diurnal variation of these collagen-II biomarkers in individuals with knee OA. These results suggest that serum sampling for these markers should be standardized for purposes of clinical trials.

Biochemical markers of type II collagen breakdown and synthesis are positioned at specific sites in human osteoarthritic knee cartilage.

OBJECTIVE: To investigate whether type II collagen turnover markers used for osteoarthritis (OA) activity evaluation in body fluids can be detected at the level of specific histological features of OA cartilage tissue, as well as how they relate with each other at this level. METHODS: Adjacent sections were obtained from full-depth cartilage biopsies from 32 OA knees. Immunohistochemistry was performed for Helix-II and CTX-II, which are type II collagen fragments originating from the triple helix and the telopeptide region, respectively, and believed to reflect distinct breakdown events, as well as for type IIA N propeptide (PIIANP), a biochemical marker reflecting synthesis of type IIA collagen. RESULTS: Helix-II and CTX-II were detected in areas where collagen damage was reported previously, most frequently around chondrocytes, but also frequently in regions not previously investigated such as the margin area and close to subchondral bone, including vascularization sites and bone-cartilage interface. The latter is CTX-II's prevailing position and shows rarely Helix-II. PIIANP co-localized with Helix-II and CTX-II on a limited number of features, mainly in deep zone cartilage. Overall, our analysis highlights clear patterns of association of the markers with specific histological features, and shows that they spread to these features in an ordered way. CONCLUSION: Helix-II and CTX-II show to some degree differential selectivity for specific features in cartilage tissue. CTX-II detection close to bone may be relevant to the possible role of subchondral bone in OA. The restricted co-localization of breakdown markers and PIIANP suggests that collagen fragments can result only partially from newly synthesized collagen. Our study strengthens the interest for the question whether combining several markers reflecting different regional cartilage contributions or metabolic processes should allow a broader detection of OA activity.