Thierry Berthe

Evidence for a complex relationship between antibiotics and antibiotic-resistant Escherichia coli: from medical center patients to a receiving environment.

The aim of this study was to investigate the relationship between antibiotics and antibiotic-resistant fecal bacteria (E. coli) in water along a medical center-wastewater treatment plant-river continuum (4 km). A multiresidue chemical analysis methodology, using solid phase extraction coupled with liquid chromatography tandem mass spectrometry, was performed to detect whether low levels of contamination by 34 antibiotics were related to antibiotic resistance of E. coli and antibiotic use. The contamination of water by antibiotics and antibiotic-resistant E. coli decreased along the continuum. Although amoxicillin was predominantly prescribed, only ofloxacin (1 ng·L(-1)) and sulfamethoxazole (4 ng·L(-1)) persisted in the river. At the retirement home, in the medical center, even though no tetracycline and sulfamethoxazole were consumed, the highest occurrences of antibiotic resistance were in classes of quinolones (42.0%), sulfonamides (24.0%), tetracyclines (38.0%), and penicillins (38.0%), mainly due to the presence of multiple antibiotic-resistance genes on class 1 integrons. Along the continuum, the occurrence of E. coli resistant to antibiotics and those carrying class 1 integrons decreased in water samples (p-value <0.001). Interestingly, in the river, only persistent antibiotic compounds (ofloxacin, sulfamethoxazole) were found, but they did not correspond to the major resistances (tetracycline, amoxicillin) of E. coli.

Evidence for coexistence of distinct Escherichia coli populations in various aquatic environments and their survival in estuary water.

ABSTRACT Escherichia coli, a commensal bacterium from the intestinal tracts of humans and vertebrate animals, has been used as one of two bacterial indicators of fecal contamination, along with intestinal enterococci, to monitor the microbiological quality of water. However, water environments are now recognized as a secondary habitat where some strains can survive. We investigated the survival of E. coli isolates collected from bodies of water in France exhibiting distinct profiles of contamination, defined according to the following criteria: vicinity of the point sources of contamination, land use, hydrology, and physicochemical characteristics of the receiving water. We selected 88 E. coli strains among a collection of 352 strains to carry out a microcosm experiment in filtered estuarine water for 14 days at 10°C. The relationship between the survival of E. coli strains and genotypic and phenotypic characteristics was analyzed. This work showed that distinct E. coli survival types, able to survive from between 7 and 14 days to less than 2 days, coexisted in the water. E. coli isolates that rapidly lost their culturability were more frequently isolated in water recently contaminated by fecal bacteria of human origin, and most were multiresistant to antibiotics and harbored several virulence factors. In contrast, persistent strains able to survive from 4 to 14 days were more often found in water with low levels of fecal bacteria, belonged mainly to the B1 phylogroup, often harbored only one virulence factor, kspE or ompT, and were able to grow at 7°C.

Influence of hydrological conditions on the Escherichia coli population structure in the water of a creek on a rural watershed

BackgroundEscherichia coli is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, although the aquatic environment could be a secondary habitat. The aim of this study was to investigate the effect of hydrological conditions on the structure of the E. coli population in the water of a creek on a small rural watershed in France composed of pasture and with human occupation.ResultsIt became apparent, after studying the distribution in the four main E. coli phylo-groups (A, B1, B2, D), the presence of the hly (hemolysin) gene and the antibiotic resistance pattern, that the E. coli population structure was modified not only by the hydrological conditions (dry versus wet periods, rainfall events), but also by how the watershed was used (presence or absence of cattle). Isolates of the B1 phylo-group devoid of hly and sensitive to antibiotics were particularly abundant during the dry period. During the wet period and the rainfall events, contamination from human sources was predominantly characterized by strains of the A phylo-group, whereas contamination by cattle mainly involved B1 phylo-group strains resistant to antibiotics and exhibiting hly. As E. coli B1 was the main phylo-group isolated in water, the diversity of 112 E. coli B1 isolates was further investigated by studying uidA alleles (beta-D-glucuronidase), the presence of hly, the O-type, and antibiotic resistance. Among the forty epidemiolgical types (ETs) identified, five E. coli B1 ETs were more abundant in slightly contaminated water.ConclusionsThe structure of an E. coli population in water is not stable, but depends on the hydrological conditions and on current use of the land on the watershed. In our study it was the ratio of A to B1 phylo-groups that changed. However, a set of B1 phylo-group isolates seems to be persistent in water, strengthening the hypothesis that they may correspond to specifically adapted strains.

Intensive exploitation of a karst aquifer leads to Cryptosporidium water supply contamination

Groundwater from karst aquifers is an important source of drinking water worldwide. Outbreaks of cryptosporidiosis linked to surface water and treated public water are regularly reported. Cryptosporidium oocysts are resistant to conventional drinking water disinfectants and are a major concern for the water industry. Here, we examined conditions associated with oocyst transport along a karstic hydrosystem, and the impact of intensive exploitation on Cryptosporidium oocyst contamination of the water supply. We studied a well-characterized karstic hydrosystem composed of a sinkhole, a spring and a wellbore. Thirty-six surface water and groundwater samples were analyzed for suspended particulate matter, turbidity, electrical conductivity, and Cryptosporidium and Giardia (oo)cyst concentrations. (Oo)cysts were identified and counted by means of solid-phase cytometry (ChemScan RDI(®)), a highly sensitive method. Cryptosporidium oocysts were detected in 78% of both surface water and groundwater samples, while Giardia cysts were found in respectively 22% and 8% of surface water and groundwater samples. Mean Cryptosporidium oocyst concentrations were 29, 13 and 4/100 L at the sinkhole, spring and wellbore, respectively. Cryptosporidium oocysts were transported from the sinkhole to the spring and the wellbore, with respective release rates of 45% and 14%, suggesting that oocysts are subject to storage and remobilization in karst conduits. Principal components analysis showed that Cryptosporidium oocyst concentrations depended on variations in hydrological forcing factors. All water samples collected during intensive exploitation contained oocysts. Control of Cryptosporidium oocyst contamination during intensive exploitation is therefore necessary to ensure drinking water quality.

Comparative effects of mercury contamination and wastewater effluent input on Gram-negative merA gene abundance in mudflats of an anthropized estuary (Seine, France): a microcosm approach

The macrotidal Seine estuary (France) is one of the most man-altered and mercury-contaminated European estuaries. Molecular quantification by competitive PCR has shown that the highest quantities of Gram-negative merA genes in intertidal freshwater mudflat sediments are located in recent sediment deposits independently of mercury concentrations, suggesting that particle-attached allochtonous mercury-resistant merA bacteria are deposited on mudflat surfaces. To investigate this hypothesis, a microcosm experiment was carried out to evaluate the respective contributions of (i) the input of allochtonous merA bacteria supplied by WWTP-treated effluents and (ii) merA gene abundance corresponding to a response of the sediments autochthonous bacterial community to mercury contamination. Gram-negative merA gene quantification and T-RFLP analysis of both 16S rDNA and merA genes demonstrated that deposited allochtonous bacteria did not develop in estuarine sediments, whereas mercury contamination (10microg g(-1) wet sediment) selected an autochthonous mercury-resistant merA bacterial community. Thus, in mudflats of highly anthropized macrotidal estuaries, i.e. those subjected to intense hydrosedimentary processes and continuously contaminated by mercury and fecal bacteria, inputs of allochtonous merA bacteria are largely responsible for the high quantities of merA genes on the surface of mudflat sediments.

Relationships between hydrosedimentary processes and occurrence of mercury-resistant bacteria (merA) in estuary mudflats (Seine, France)

The Seine estuary (France) is one of the worlds macrotidal systems that is most contaminated with heavy metals. To study the mercury-resistant bacterial community in such an environment, we have developed a molecular tool, based on competitive PCR, enabling the quantification of Gram-negative merA gene abundance. The occurrence of the Gram-negative merA gene in relation with the topology (erosion/deposit periods) and the mercury contamination of three contrasted mudflats was investigated through a multidisciplinary approach and compared with a non-anthropized site (Authie, France). The higher abundance of the Gram-negative merA gene in the Seine estuary mudflats indicates a relationship between the degree of anthropization and the abundance of the merA gene in the mudflat sediments. In the Seine mudflats, the maxima of abundance are always located in fresh sediment deposits. Therefore, the abundance is closely related with the hydrosedimentary processes, which thus seem to be determining factors in the occurrence of the Gram-negative merA gene in the surface sediments of the Seines mudflat.

Evidence of methylmercury production and modification of the microbial community structure in estuary sediments contaminated with wastewater treatment plant effluents.

The Seines estuary (France) waters are the receptacle of effluents originating from wastewater treatment plants (WWTP). In this estuary, mudflats are deposition zones for sediments and their associated contaminants, and play an essential role in the mercury (Hg) biogeochemical cycle mainly due to indigenous microorganisms. Microcosms were used to assess the impact of WWTP-effluents on mercury methylation by monitoring Hg species (total dissolved Hg in porewater, methylmercury and total mercury) and on microbial communities in sediments. After effluent amendment, methylmercury (MeHg) concentrations increased in relation with the total Hg and organic matter content of the WWTP-effluents. A correlation was observed between MeHg and acid-volatile-sulfides concentrations. Quantification of sulfate-reducing microorganisms involved in Hg methylation showed no increase of their abundance but their activity was probably enhanced by the organic matter supplied with the effluents. WWTP-effluent spiking modified the bacterial community fingerprint, mainly influenced by Hg contamination and the organic matter amendment.

Distinct diversity of the czcA gene in two sedimentary horizons from a contaminated estuarine core

In estuarine ecosystems, trace metals are mainly associated with fine grain sediments which settle on mudflats. Over time, the layers of sediments accumulate and are then transformed by diagenetic processes, recording the history of the estuary’s chemical contamination. In such a specific environment, we investigated to what extent a chronic exposure to contaminants could affect metal-resistant sedimentary bacteria in subsurface sediments. The occurrence and diversity of cadmium resistance genes (cadA, czcA) was investigated in 5- and 33-year-old sediments from a highly contaminated estuary (Seine France). Primers were designed to detect a 252-bp fragment of the czcA gene, specifically targeting a transmembrane helice domain (TMH IV) involved in the proton substrate antiport of this efflux pump. Although the cadA gene was not detected, the highest diversity of the sequence of the czcA gene was observed in the 5-year-old sediment. According to the percentage of identity at the amino acid level, the closest CzcA relatives were identified among Proteobacteria (α, β, γ, and δ), Verrucomicrobia, Nitrospirae, and Bacteroidetes. The most abundant sequences were affiliated with Stenotrophomonas. In contrast, in the 33-year-old sediment, CzcA sequences were mainly related to Rhodanobacter thiooxydans and Stenotrophomonas, suggesting a shaping of the metal-resistant microbial communities over time by both diagenetic processes and trace metal contamination.

Change in the Structure of Escherichia coli Population and the Pattern of Virulence Genes along a Rural Aquatic Continuum

The aim of this study was to investigate the diversity of the Escherichia coli population, focusing on the occurrence of pathogenic E. coli, in surface water draining a rural catchment. Two sampling campaigns were carried out in similar hydrological conditions (wet period, low flow) along a river continuum, characterized by two opposite density gradients of animals (cattle and wild animals) and human populations. While the abundance of E. coli slightly increased along the river continuum, the abundance of both human and ruminant-associated Bacteroidales markers, as well as the number of E. coli multi-resistant to antibiotics, evidenced a fecal contamination originating from animals at upstream rural sites, and from humans at downstream urban sites. A strong spatial modification of the structure of the E. coli population was observed. At the upstream site close to a forest, a higher abundance of the B2 phylogroup and Escherichia clade strains were observed. At the pasture upstream site, a greater proportion of both E and B1 phylogroups was detected, therefore suggesting a fecal contamination of mainly bovine origin. Conversely, in downstream urban sites, A, D, and F phylogroups were more abundant. To assess the occurrence of intestinal pathogenic strains, virulence factors [afaD, stx1, stx2, eltB (LT), estA (ST), ipaH, bfpA, eae, aaiC and aatA] were screened among 651 E. coli isolates. Intestinal pathogenic strains STEC O174:H21 (stx2) and EHEC O26:H11 (eae, stx1) were isolated in water and sediments close to the pasture site. In contrast, in the downstream urban site aEPEC/EAEC and DAEC of human origin, as well as extra-intestinal pathogenic E. coli belonging to clonal group A of D phylogroup, were sampled. Even if the estimated input of STEC (Shiga toxin-producing E. coli) – released in water at the upstream pasture site – at the downstream site was low, we show that STEC could persist in sediment. These results show that, the run-off of small cattle farms contributed, as much as the wastewater effluent, in the dissemination of pathogenic E. coli in both water and sediments, even if the microbiological quality of the water was good or to average quality according to the French water index.

Fate of Antibiotics and Antibiotic-Resistant Fecal Bacteria in Water and Sediments from the Contamination Source to the Estuary: Impact and/or Resilience? Resilience to Contamination by Antibiotics

The aim of this study based on a multidisciplinary research program (FLASH) (FLASH (FLuxes of Antibiotic and Antibiotic-resistant bacteria and the corresponding genes in Surface Hydrosystems) a research project founded by CNRS EC2CO/GIP Seine-Aval), associating chemists, hydrologists, and clinical and environmental microbiologists, was to monitor the impact of antibiotic prescription in human and veterinary practices on water and sediment contamination by antibiotic and Escherichia coli (antibiotic resistance, integrons) and Enterococci (diversity, antibiotic resistance, and the corresponding genes) along a medical center–WWTP–river continuum. For this purpose, a multi-residue chemical methodology was developed in order to detect low levels of 34 antibiotics. In the medical center, the main prescribed antibiotic (amoxicillin) was weakly found in effluents. Along the continuum, contamination of water by antibiotics decreased from 160 μg.L−1 (cefotaxime) in hospital effluents to 1 ng.L−1 (ofloxacin) in the river. These concentrations were too low to exert a selective pressure (mg.L−1) on antibiotic-resistant bacteria. In the same samples, occurrence of antibiotic-resistant E. coli and those harboring a class 1 integrons was significantly (p-value < 0.001) decreased along the continuum. Among Enterococcus populations, E. faecium was mainly isolated (from 89 to 98 %). All E. faecium isolates from medical center effluents were multiple antibiotic resistant, containing erm(B) and mef(A) genes, and belonged to the hospital-adapted clonal complex 17 (CC17). The relative proportion of CC17 decreased in favor of other subpopulations, less resistant to antibiotics. In water, only persistent compounds were found (quinolones, macrolides, sulfonamides), but they did not correspond to the major resistances in E. coli and Enterococcus (penicillins, tetracyclines).