In order to evaluate the impact of an urban effluent on antibiotic resistance of freshwater bacterial populations, water samples were collected from the Arga river (Spain), upstream and downstream from the wastewater discharge of the city of Pamplona. Strains of Enterobacteriaceae (representative of the human and animal commensal flora) (110 isolates) and Aeromonas (typically waterborne bacteria) (118 isolates) were selected for antibiotic susceptibility testing. Most of the Aeromonas strains (72%) and many of the Enterobacteriaceae (20%) were resistant to nalidixic acid. Singly nalidixic acid-resistant strains were frequent regardless of the sampling site for Aeromonas, whereas they were more common upstream from the discharge for enterobacteria. The most common resistances to antibiotics other than quinolones were to tetracycline (24.3%) and beta-lactams (20.5%) for Enterobacteriaceae and to tetracycline (27.5%) and co-trimoxazole (26.6%) for Aeromonas. The rates of these antibiotic resistances increased downstream from the discharge at similar degrees for the two bacterial groups; it remained at high levels for enterobacteria but decreased along the 30-km study zone for Aeromonas. Genetic analysis of representative strains demonstrated that these resistances were mostly (enterobacteria) or exclusively (Aeromonas) chromosomally mediated. Moreover, a reference strain of Aeromonas caviae (CIP 7616) could not be transformed with conjugative R plasmids of enterobacteria. Thus, the urban effluent resulted in an increase of the rates of resistance to antibiotics other than quinolones in the riverine bacterial populations, despite limited genetic exchanges between enterobacteria and Aeromonas. Quinolone resistance probably was selected by heavy antibiotic discharges of unknown origin upstream from the urban effluent.

Tributyltin (TBT) is a toxic agent used in marine antifouling paints. Among the bacterial flora of a polluted harbor, TBT-resistant strains of Pseudomonas stutzeri have been isolated. In the strain 5MP1 (TBT minimum inhibitory concentration (MIC) v 1000 mg l 31 ), TBT resistance was found to be associated with the presence of the operon tbtABM, homologous to the resistance^nodulation^cell division (RND) efflux pump family, as demonstrated by cloning in Escherichia coli. TbtABM exhibited the greatest homology (60.98 4.9%) with the TtgDEF and SrpABC systems, both involved in aromatic compound tolerance in P. putida. TbtABM conferred multidrug resistance (MDR) including to n-hexane, nalidixic acid, chloramphenicol, and sulfamethoxazole (antibiotic MICsU4 for the E. coli host strain carrying the operon). By polymerase chain reaction amplification and hybridization experiments, the presence of tbtABM was detected in the TBT-sensitive P. stutzeri 3MP1 (TBT MIC 25 mg l 31 ). However, the latter strain did not seem to express TbtABM. This is the first description of a MDR efflux pump in P. stutzeri, and of a new kind of substrate, TBT, for the RND family of transporters.

Most Aeromonas strains isolated from two European rivers were previously found to be resistant to nalidixic acid. In order to elucidate the mechanism of this resistance, 20 strains of Aeromonas caviae (n ‫؍‬ 10), A. hydrophila (n ‫؍‬ 5), and A. sobria (n ‫؍‬ 5) complexes, including 3 reference strains and 17 environmental isolates, were investigated. Fragments of the gyrA, gyrB, parC, and parE genes encompassing the quinolone resistance-determining regions (QRDRs) were amplified by PCR and sequenced. Results obtained for the six sensitive strains showed that the GyrA, GyrB, ParC, and ParE QRDR fragments of Aeromonas spp. were highly conserved (>96.1% identity), despite some genetic polymorphism; they were most closely related to those of Vibrio spp., Pseudomonas spp., and members of the family Enterobacteriaceae (72.4 to 97.1% homology). All 14 environmental resistant strains carried a point mutation in the GyrA QRDR at codon 83, leading to the substitution Ser-833Ile (10 strains) or Ser-833Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-803Ile (three strains) or Ser-803Arg change, or at position 84, yielding a Glu-843Lys modification. No amino acid alterations were discovered in the GyrB and ParE QRDRs. Double gyrA-parC missense mutations were associated with higher levels of quinolone resistance compared with the levels associated with single gyrA mutations. The most resistant strains probably had an additional mechanism(s) of resistance, such as decreased accumulation of the drugs. Our data suggest that, in mesophilic Aeromonas spp., as in other gram-negative bacteria, gyrase and topoisomerase IV are the primary and secondary targets for quinolones, respectively.

Tributyltin (TBT) resistance of aerobic and anaerobic bacteria isolated from TBT-contaminated coastal sediments in Arcachon Bay, France, was determined and compared with taxonomically similar bacteria originating from diverse non-TBT-contaminated environments. The minimum inhibitory concentrations (MICs) showed that TBT was toxic to most of the bacteria studied at concentrations above 2.1 μM (0.7 mg/L) for clostridia, 76 μM (25 mg/L) for pseudomonads, and 3 mM (1000 mg/L) for enterobacteria. The aerobic or facultative anaerobic Gram-negative bacteria (pseudomonads and enterobacteria) presented the highest TBT tolerance. No difference in TBT resistance was observed between strains originating from the polluted site and strains originating from non-TBT-polluted environments (thermal muds, Hospital of Bordeaux, etc.), suggesting that a TBT-polluted environment did not select for more specific resistant bacteria. It should be noted that the bacteria tested were found to be tolerant to TBT concentrations 1000 times higher than those found in the polluted site. Further, all strains presented the same pattern of metal and pharmaceutical antibiotic sensitivity despite the origin of the environment (TBT-polluted or non-TBT-polluted sediments). Six strains of Pseudomonas stutzeri isolated from Arcachon Harbor and one strain of Pseudomonas aeruginosa originating from the bacterial collection of Bordeaux Hospital have been selected for their TBT resistance. As antibiotic resistance, metal or organometal resistance can be plasmid mediated. However, among these TBT-resistant strains no plasmid was detected.Key words: TBT resistance, coastal environment, clostridia, pseudomonads, enterobacteria.

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The Arga River is an interesting system in which to study the impact of urban effluent pollution because it receives a single effluent in the form of wastewater discharge from the city of Pamplona. To analyze the extent of this discharge, total bacteria, culturable heterotrophic bacteria, and Gram-negative heterotrophic bacteria were enumerated and 409 isolates of the latter were identified. One sampling station was located upstream from the inflow, while five were located up to 30 km downstream. Bacterial counts increased drastically downstream from the wastewater inflow. Total bacterial numbers decreased along the 30 km downstream, the last station attaining similar values to those recorded upstream from the discharge. However, culturable heterotrophic and Gram-negative heterotrophic bacteria levels generally remained significantly higher within the 30 km zone investigated. Among the 409 isolates identified, Aeromonas spp. were the most frequent both upstream and downstream from the discharge. In contrast, although strains belonging to different genera of Enterobacteriaceae were found in all stations, their occurrence was significantly higher just downstream from the polluted discharge. Acinetobacter spp., which were never found upstream, were detected in all stations below the discharge. Our results clearly show that the bacterial community structure changes definitively downstream from the discharge and that Aeromonas were common throughout the sampling zone. Thus they cannot be considered good indicators of pollution in this setting compared to some genera of Enterobacteriaceae or some species of Acinetobacter, the distribution of which correlated better with the distance from the wastewater discharge.

The Arga River is an interesting system in which to study the impact of urban effluent pollution because it receives a single effluent in the form of wastewater discharge from the city of Pamplona. To analyze the extent of this discharge, total bacteria, culturable heterotrophic bacteria, and Gram-negative heterotrophic bacteria were enumerated and 409 isolates of the latter were identified. One sampling station was located upstream from the inflow, while five were located up to 30 km downstream. Bacterial counts increased drastically downstream from the wastewater inflow. Total bacterial numbers decreased along the 30 km downstream, the last station attaining similar values to those recorded upstream from the discharge. However, culturable heterotrophic and Gram-negative heterotrophic bacteria levels generally remained significantly higher within the 30 km zone investigated. Among the 409 isolates identified, Aeromonas spp. were the most frequent both upstream and downstream from the discharge. In contrast, although strains belonging to different genera of Enterobacteriaceae were found in all stations, their occurrence was significantly higher just downstream from the polluted discharge. Acinetobacter spp., which were never found upstream, were detected in all stations below the discharge. Our results clearly show that the bacterial community structure changes definitively downstream from the discharge and that Aeromonas were common throughout the sampling zone. Thus they cannot be considered good indicators of pollution in this setting compared to some genera of Enterobacteriaceae or some species of Acinetobacter, the distribution of which correlated better with the distance from the wastewater discharge.

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