Fabienne Petit

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.

Estimation of the abundance of the cadmium resistance gene cadA in microbial communities in polluted estuary water

We describe herein a molecular method for estimating the abundance of the cadA gene, which encodes a Cd2+/ATPase protein transporter, in bacterial DNA extracted from samples of environmental water. Competitive polymerase chain reaction (cPCR) may be the most appropriate technique for assessing the prevalence of the cadA gene in microbial communities in highly heterogeneous and polluted environments, such as the Seine estuary (France). We describe the development of this method: (i) the choice of two specific primers, based on the sequences encoding the cadmium binding site and the ion channel domains; (ii) the construction of a competitor sequence and assessment of its amplification efficiency; and (iii) the estimation of the copy number of the cadA gene. The cadA content in the bacterial community is expressed as the number of gene copies per ng of total DNA extracted, which is independent of the DNA extraction yield. This molecular procedure was improved to analyze cadA levels in bacterial DNA extracted from estuary water accidentally contaminated with cadmium. Results revealed a subsequent increase in the copy number of the cadA gene in the microbial community.

Distribution and diversity of a cadmium resistance (cadA) determinant and occurrence of IS257 insertion sequences in staphylococcal bacteria isolated from a contaminated estuary (Seine, France)

Water and sediments of the Seine estuary are contaminated by chemicals, especially cadmium, which favors survival and growth of cadmium-resistant bacteria. We investigated the diversity of the cadA gene, which encodes a Cd(2+)/ATPase protein transporter, in the microbial community of the Seine estuary. The cadA gene first isolated from S. aureus (pI258) was prevalent, with a conservation better than 98% identity, despite its presence in host bacteria of diverse species and genera. We report for the first time, eleven distinct Staphylococcus species, and also bacteria belonging to the Micrococcus and Halobacillus genera carrying the cadA gene. This cadA determinant was mostly plasmid-borne in the Staphylococcus genus, and IS257 sequences, which are known to participate in antibiotic resistance gene dissemination in S. aureus, were found to be located near to the cadA gene in 16/31 cadmium-resistant Staphylococcus strains and one Micrococcus strain. This suggests that IS257 has also contributed to the dissemination of the cadA resistance gene among staphylococci. These findings also emphasize on the existence of Staphylococcal bacteria in contaminated natural niches outside hospital environments.

Changes in enterococcal populations and related antibiotic resistance along a medical center-wastewater treatment plant-river continuum.

ABSTRACT To determine if hospital effluent input has an ecological impact on downstream aquatic environment, antibiotic resistance in Enterococcus spp. along a medical center-retirement home-wastewater treatment plant-river continuum in France was determined using a culture-based method. Data on antibiotic consumption among hospitalized and general populations and levels of water contamination by antibiotics were collected. All isolated enterococci were genotypically identified to the species level, tested for in vitro antibiotic susceptibility, and typed by multilocus sequence typing. The erm(B) and mef(A) (macrolide resistance) and tet(M) (tetracycline resistance) genes were detected by PCR. Along the continuum, from 89 to 98% of enterococci, according to the sampled site, were identified as Enterococcus faecium. All E. faecium isolates from hospital and retirement home effluents were multiply resistant to antibiotics, contained erm(B) and mef(A) genes, and belonged to hospital-adapted clonal complex 17 (CC17). Even though this species remained dominant in the downstream continuum, the relative proportion of CC17 isolates progressively decreased in favor of other subpopulations of E. faecium that were more diverse, less resistant to antibiotics, and devoid of the classical macrolide resistance genes and that belonged to various sequence types. Antibiotic concentrations in waters were far below the MICs for susceptible isolates. CC17 E. faecium was probably selected in the gastrointestinal tract of patients under the pressure of administered antibiotics and then excreted together with the resistance genes in waters to progressively decrease along the continuum.

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.

The gene encoding the β-1,4-endoglucanase (CelA) from Myxococcus xanthus: evidence for independent acquisition by horizontal transfer of binding and catalytic domains from actinomycetes

The celA gene encoding a beta-1,4 endoglucanase (CelA) from Myxococcus xanthus has been cloned in Escherichia coli and sequenced. The C-terminal region of CelA displayed a high level of similarity with the catalytic domain of several Egl belonging to the glycosyl hydrolases family 6 (CenA from Cellulomonas fimi, CelA from Microbispora bispora, E2 from Thermonospora fusca, CasA from Streptomyces KSM9 and CelA1 from Streptomyces halstedii) and less similarity to the cellobiohydrolases of the fungi Trichoderma reesei and Agaricus bisporus. Using PCR amplification we found in another myxobacterium, Stigmatella aurantiaca, a part of a glycosyl hydrolase belonging to the same family. The N-terminal part of CelA displayed significant similarities with the cellulose-binding domain of other cellulases belonging to a rare subset of family II, such as the avicelase I from Streptomyces reticuli, both tandem repeats N1 and N2 of the cellulase CenC from Cellulomonas fimi, and the N-terminal part of the Egl E1 from Thermonospora fusca. Analyses of the multiple alignments and reconstruction of phylogenetic trees strongly suggest that both domains of CelA were acquired by independent horizontal transfers between Gram+ soil bacteria and scavenging myxobacteria followed by domain shuffling.

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.