Effect of land application of manure from enrofloxacin-treated chickens on ciprofloxacin resistance of Enterobacteriaceae in soil.

A field plot experiment was carried out to evaluate the impact of spreading chicken manure containing enrofloxacin (ENR) and its metabolite ciprofloxacin (CIP), on the levels of CIP-resistant Enterobacteriaceae in soil. The manures from chickens treated with ENR and from untreated control chickens were applied on six plots. Total and CIP-resistant Enterobacteriaceae were counted on Violet Red Bile Glucose medium containing 0 to 16mg L(-1) of CIP. A total of 145 isolates were genotyped by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). The minimum inhibitory concentration (MIC) of CIP for the isolates of each ERIC-PCR profile was determined. The most frequently isolated Enterobacteriaceae included Escherichia coli, and to a lesser extent, Enterobacter and 5 other genera from environmental origin. The composition of the E. coli community differed between manure and manured soil suggesting that the E. coli genotypes determined by ERIC-PCR varied significantly in their ability to survive in soil. One of these genotypes, including both susceptible and resistant isolates, was detected up to 89 days after the manure was applied. Most of the E. coli isolated in soil amended with manure from treated chickens was resistant to CIP (with a MIC ranging between 2 and 32mg L(-1)). In contrast, despite the presence of ENR in soil at concentrations ranging from 13-518µg kg(-1), the environmental Enterobacteriaceae isolates had a CIP MIC≤0.064mg L(-1), except one isolate which had a MIC of 0.25mg L(-1), These results showed that spreading manure from ENR-treated chickens enabled CIP-resistant E. coli to persist for at least three months in the soil. However, neither the presence of fluoroquinolones, nor the persistence of CIP-resistant E. coli, increased the CIP-susceptibility of environmental Enterobacteriaceae.

Origin of fecal contamination in waters from contrasted areas: stanols as Microbial Source Tracking markers.

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 5ß-cholestan-3ß-ol (coprostanol), 5ß-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3ß-ol (campestanol), 24-ethyl-5α-cholestan-3ß-ol (sitostanol), 24-ethyl-5ß-cholestan-3ß-ol (24-ethylcoprostanol) and 24-ethyl-5ß-cholestan-3α-ol (24-ethylepicoprostanol)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in "Microbial Source Tracking (MST)" methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of "pure" human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate.

Relative decay of fecal indicator bacteria and human-associated markers: a microcosm study simulating wastewater input into seawater and freshwater.

Fecal contaminations of inland and coastal waters induce risks to human health and economic losses. To improve water management, specific markers have been developed to differentiate between sources of contamination. This study investigates the relative decay of fecal indicator bacteria (FIB, Escherichia coli and enterococci) and six human-associated markers (two bacterial markers: Bacteroidales HF183 (HF183) and Bifidobacterium adolescentis (BifAd); one viral marker: genogroup II F-specific RNA bacteriophages (FRNAPH II); three chemical markers: caffeine and two fecal stanol ratios) in freshwater and seawater microcosms seeded with human wastewater. These experiments were performed in darkness, at 20 °C and under aerobic conditions. The modeling of the decay curves allows us (i) to compare FIB and markers and (ii) to classify markers according to their persistence in seawater (FRNAPH II < HF183, stanol ratios < BifAd, caffeine) and in freshwater (HF183, stanol ratios < FRNAPH II < BifAd < caffeine). Although those results depend on the experimental conditions, this study represents a necessary step to develop and validate an interdisciplinary toolbox for the investigation of the sources of fecal contaminations.

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France.

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.