Importance of Local and Regional Scales in Shaping Mycobacterial Abundance in Freshwater Lakes.

Biogeographical studies considering the entire bacterial community may underestimate mechanisms of bacterial assemblages at lower taxonomic levels. In this context, the study aimed to identify factors affecting the spatial and temporal dynamic of the Mycobacterium, a genus widespread in aquatic ecosystems. Nontuberculous mycobacteria (NTM) density variations were quantified in the water column of freshwater lakes at the regional scale (annual monitoring of 49 lakes in the Paris area) and at the local scale (2-year monthly monitoring in Créteil Lake) by real-time quantitative PCR targeting the atpE gene. At the regional scale, mycobacteria densities in water samples ranged from 6.7 × 103 to 1.9 × 108 genome units per liter. Density variations were primarily explained by water pH, labile iron, and dispersal processes through the connection of the lakes to a river. In Créteil Lake, no spatial variation of mycobacterial densities was noticed over the 2-year monthly survey, except after large rainfall events. Indeed, storm sewer effluents locally and temporarily increased NTM densities in the water column. The temporal dynamic of the NTM densities in Créteil Lake was associated with suspended solid concentrations. No clear seasonal variation was noticed despite a shift in NTM densities observed over the 2012-2013 winter. Temporal NTM densities fluctuations were well predicted by the neutral community model, suggesting a random balance between loss and gain of mycobacterial taxa within Créteil Lake. This study highlights the importance of considering multiple spatial scales for understanding the spatio-temporal dynamic of bacterial populations in natural environments.

atpE gene as a new useful specific molecular target to quantify Mycobacterium in environmental samples.

BACKGROUND: The environment is the likely source of many pathogenic mycobacterial species but detection of mycobacteria by bacteriological tools is generally difficult and time-consuming. Consequently, several molecular targets based on the sequences of housekeeping genes, non-functional RNA and structural ribosomal RNAs have been proposed for the detection and identification of mycobacteria in clinical or environmental samples. While certain of these targets were proposed as specific for this genus, most are prone to false positive results in complex environmental samples that include related, but distinct, bacterial genera. Nowadays the increased number of sequenced genomes and the availability of software for genomic comparison provide tools to develop novel, mycobacteria-specific targets, and the associated molecular probes and primers. Consequently, we conducted an in silico search for proteins exclusive to Mycobacterium spp. genomes in order to design sensitive and specific molecular targets. RESULTS: Among the 3989 predicted proteins from M. tuberculosis H37Rv, only 11 proteins showed 80% to 100% of similarity with Mycobacterium spp. genomes, and less than 50% of similarity with genomes of closely related Corynebacterium, Nocardia and Rhodococcus genera. Based on DNA sequence alignments, we designed primer pairs and a probe that specifically detect the atpE gene of mycobacteria, as verified by quantitative real-time PCR on a collection of mycobacteria and non-mycobacterial species. The real-time PCR method we developed was successfully used to detect mycobacteria in tap water and lake samples. CONCLUSIONS: The results indicate that this real-time PCR method targeting the atpE gene can serve for highly specific detection and precise quantification of Mycobacterium spp. in environmental samples.

Mycobacterium behavior in wastewater treatment plant, a bacterial model distinct from Escherichia coli and Enterococci.

Mycobacteria are waterborne emerging pathogens causing infections in human. Mycobacteria have been previously isolated from wastewater and sludge, but their densities were not estimated due to cultural biases. In order to evaluate the impact of wastewater treatment processes on mycobacteria removal, we used a real time PCR method. First we compared six DNA extraction methods and second we used the more efficient DNA extraction procedure (i.e., enzymatic lysis combined with hexadecyltrimethylammonium bromide-NaCl procedure) in order to quantify Mycobacterium. With the aim to identify parameters that could serve as indicator of mycobacterial behavior, mycobacterial densities were measured in parallel to those of Escherichia coli and enterococci, and to concentrations of chemical parameters usually monitored in wastewater. Mycobacterium reached 5.5 × 105 ± 3.9 × 105 copies/L in the influent, but was not detected in the effluent after decantation and biofiltration. Most mycobacteria (98.6 ± 2.7%, i.e. 2.4 ± 0.7 log10) were removed by the physical-chemical decantation, and the remaining mycobacteria were removed by biofiltration. In contrast, enterococci and E. coli were lightly removed by decantation step and mainly removed by biofiltration. Our results showed that Mycobacterium corresponds to a hydrophobic behavior linked to insoluble compound removal, whereas enterococci and E. coli refer to hydrophilic behaviors linked to soluble compound removals.

Development of a real-time qPCR method for detection and enumeration of Mycobacterium spp. in surface water.

A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).

Comparison of culture methods for isolation of nontuberculous mycobacteria from surface waters.

The environment is the likely source of most nontuberculous mycobacteria (NTM) involved in human infections, especially pulmonary, skin, and soft tissue infections. In order to measure the prevalence of NTM in different aquatic ecosystems, we tried to standardize the culture methods used for surface water testing since many procedures have been described previously. Cultivation of mycobacteria requires long-term incubation in rich media and inactivation of rapidly growing microorganisms whose growth impedes observation of mycobacterial colonies. Consequently, the two criteria used for evaluation of the methods examined were (i) the rate of inhibition of nontarget microorganisms and (ii) the efficiency of recovery of mycobacteria. We compared the competitive growth of Mycobacterium chelonae and M. avium with nontarget microorganisms on rich Middlebrook 7H11-mycobactin medium after treatment by several chemical decontamination methods that included acids, bases, detergent, or cetylpyridinium chloride (CPC) with and without an antibiotic cocktail, either PANTA (40 U/ml polymyxin, 4 microg/ml amphotericin B, 16 microg/ml nalidixic acid, 4 microg/ml trimethoprim, and 4 microg/ml azlocillin) or PANTAV (PANTA plus 10 microg/ml vancomycin). Our results showed that treatment for 30 min with CPC (final concentration, 0.05%) of water concentrated by centrifugation, followed by culture on a rich medium supplemented with PANTA, significantly decreased the growth of nontarget microorganisms (the concentrations were 6.2 +/- 0.4 log(10) CFU/liter on Middlebrook 7H11j medium and 4.2 +/- 0.2 log(10) CFU/liter on Middlebrook 7H11j medium containing PANTA [P < 0.001]), while the effect of this procedure on NTM was not as great (the concentrations of M. chelonae on the two media were 7.0 +/- 0.0 log(10) CFU/liter and 6.9 +/- 0.0 log(10) CFU/liter, respectively, and the concentrations of M. avium were 9.1 +/- 0.0 log(10) CFU/liter and 8.9 +/- 0.0 log(10) CFU/liter, respectively). We propose that this standardized culture procedure could be used for detection of NTM in aquatic samples.

Neutral community model explains the bacterial community assembly in freshwater lakes.

Over the past decade, neutral theory has gained attention and recognition for its capacity to explain bacterial community structure (BCS) in addition to deterministic processes. However, no clear consensus has been drawn so far on their relative importance. In a metacommunity analysis, we explored at the regional and local scale the effects of these processes on the bacterial community assembly within the water column of 49 freshwater lakes. The BCS was assessed using terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA genes. At the regional scales, results indicated that the neutral community model well predicted the spatial community structure (R(2) mean = 76%) compared with the deterministic factors - which explained only a small fraction of the BCS total variance (less than 14%). This suggests that the bacterial compartment was notably driven by stochastic processes, through loss and gain of taxa. At the local scale, the bacterial community appeared to be spatially structured by stochastic processes (R(2) mean = 65%) and temporally governed by the water temperature, a deterministic factor, even if some bacterial taxa were driven by neutral dynamics. Therefore, at both regional and local scales the neutral community model appeared to be relevant in explaining the bacterial assemblage structure.

Detection of enterovirus in environmental waters: a new optimized method compared to commercial real-time RT-qPCR kits.

Despite the progress in water and wastewater treatment technologies, waterborne diseases are still a major concern of public health. In the reported water-related outbreaks, viruses constitute one of the main causal agents. Enteroviruses are one of the most viruses monitored in water and are often used as an indicator of viral pollution. Isolation and identification of this virus are now regularly based on molecular tools. However published or commercial protocols for detection of these viruses in water are frequently lacking of validation processes and performance evaluation in such complex samples. A method for enterovirus detection in environmental water has been developed, its performance has been evaluated and compared with several commercial kits. The sensitivity of commercial methods in clinical samples, ranged between 89% and 100%, while the sensitivity in seeded environmental matrices fell between 16% and 91%. This method showed the best performance in environmental samples and was subsequently applied on surface and treated wastewater. The results showed the large dissemination of enteroviruses in an urbanized river. The results also emphasized the importance of good knowledge of the method's limits for its utilization in environmental samples in order to minimize false negatives and to avoid underestimating viral concentration.

Variation of raw wastewater microbiological quality in dry and wet weather conditions.

The microbiological quality of urban wastewaters presents important environmental, sanitary, and political challenges. However, the variability of untreated wastewater quality is seldom known when it comes to microbial parameters. This study aims to evaluate the variability of microbiological quality in wastewater influents from different wastewater treatment plants connected to combined and partially separate sewer networks in the Parisian area and to evaluate the impact of this variability on the treatment efficiency and on the accuracy of wastewater effluent monitoring. The densities of fecal indicator bacteria (FIB), Escherichia coli and intestinal enterococci, and their partitioning on settleable particles were analyzed at the inlet of two wastewater treatment plants during dry weather (130 composite samples and 7 days sampled every 2 hours) and storm events (39 composite samples, and 7 rain courses) from 2008 to 2012. The results showed that fecal indicator densities vary according to the network characteristics and according to the meteorological conditions. During storm events, a significant dilution of E. coli and enterococci was observed, as well as a decrease in the settleable fraction of E. coli during the maximal impact of the storm. However, storm events did not significantly impact the regular FIB monitoring. FIB removals by primary and secondary treatment were significantly correlated with FIB densities in influent wastewater; however, meteorological conditions also influenced the removal of FIB.

Brood size modifications affect plumage bacterial assemblages of European starlings.

During reproduction, birds face trade-offs between time and energy devoted to parental effort and traits associated with self-maintenance. We manipulated brood sizes to investigate the effects of such trade-offs on feather bacterial densities and the structure of bacterial assemblages on feathers in adult European starlings, Sturnus vulgaris, and in vitro feather degradation. As predicted by a trade-off between parental effort and self-maintenance, we found that birds with enlarged broods had more free-living bacteria on their feathers than birds with reduced broods. Furthermore, we found a significant interaction between brood manipulation and original brood size on free-living bacterial densities suggesting that the trade-off is mediated by the adults' initial reproductive investment. In contrast, brood size manipulations had no significant effect on densities of attached bacteria. Using ribosomal intergenic spacer analysis (RISA), we demonstrated that brood manipulations significantly modified the structure (band pattern) of feather-degrading bacterial assemblages, but had no significant effect on their richness (number of bands) or the in vitro feather degradation. In vitro feather degradation varied in relation to the premanipulation brood size and positively with the richness of the feather degrading bacterial community. Besides brood manipulation effect, we found that ecological factors and individual traits, such as the age, the nest location or the capture date, shaped bacterial assemblages and feather degradation capacities.