Pierre Caumette

Impact of an Urban Effluent on Antibiotic Resistance of Riverine Enterobacteriaceae and Aeromonas spp.

ABSTRACT 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 ofEnterobacteriaceae (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 forAeromonas, 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%) forAeromonas. 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.

Effects of Heavy Fuel Oil on the Bacterial Community Structure of a Pristine Microbial Mat

ABSTRACT The effects of petroleum contamination on the bacterial community of a pristine microbial mat from Salins-de-Giraud (Camargue, France) have been investigated. Mats were maintained as microcosms and contaminated with no. 2 fuel oil from the wreck of the Erika. The evolution of the complex bacterial community was monitored by combining analyses based on 16S rRNA genes and their transcripts. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) analyses clearly showed the effects of the heavy fuel oil after 60 days of incubation. At the end of the experiment, the initial community structure was recovered, illustrating the resilience of this microbial ecosystem. In addition, the responses of the metabolically active bacterial community were evaluated by T-RFLP and clone library analyses based on 16S rRNA. Immediately after the heavy fuel oil was added to the microcosms, the structure of the active bacterial community was modified, indicating a rapid microbial mat response. Members of the Gammaproteobacteria were initially dominant in the contaminated microcosms. Pseudomonas and Acinetobacter were the main genera representative of this class. After 90 days of incubation, the Gammaproteobacteria were superseded by “Bacilli” and Alphaproteobacteria. This study shows the major changes that occur in the microbial mat community at different time periods following contamination. At the conclusion of the experiment, the RNA approach also demonstrated the resilience of the microbial mat community in resisting environmental stress resulting from oil pollution.

Isolation and Characterization of Desulfovibrio halophilus sp. nov., a Halophilic Sulfate-reducing Bacterium Isolated from Solar Lake (Sinai)

Summary From samples collected in the anoxic part of the photosynthetic microbial mat of Solar Lake, sulfate-reducing bacteria were enriched with lactate and sulfate in basal medium supplemented with 10% NaCl and 1% MgCl2.6H2O. A few strains were isolated and maintained in pure culture. Strain SL 8903 which showed the highest NaCl tolerance (up to 18%) consisted of motile non-sporulating vibrioid cells and resembled the genus Desulfovibrio. However, on the basis of physiological differences to all described species of this genus, strain SL 8903 was described as a new species and named Desulfovibrio halophilus, sp. nov.

Microbial Mats on the Orkney Islands Revisited: Microenvironment and Microbial Community Composition

The microenvironment and community composition of microbial mats developing on beaches in Scapa Flow (Orkney Islands) were investigated. Analysis of characteristic biomarkers (major fatty acids, hydrocarbons, alcohols, and alkenones) revealed the presence of different groups of bacteria and microalgae in mats from Waulkmill and Swanbister beach, including diatoms, Haptophyceae, cyanobacteria, and sulfate-reducing bacteria. These analyses also indicated the presence of methanogens, especially in Swanbister beach mats, and therefore a possible role of methanogenesis for the carbon cycle of these sediments. High amounts of algal lipids and slightly higher numbers (genera, abundances) of cyanobacteria were found in Waulkmill Bay mats. However, overall only a few genera and low numbers of unicellular and filamentous cyanobacteria were present in mats from Waulkmill and Swanbister beach, as deduced from CLSM (confocal laser scanning microscopy) analysis. Spectral scalar irradiance measurements with fiber-optic microprobes indicated a pronounced heterogeneity concerning zonation and density of mainly anoxygenic phototrophs in Swanbister Bay mats. By microsensor and T-RFLP (terminal restriction fragment length polymorphism) analysis in Swanbister beach mats, the depth distribution of different populations of purple and sulfate-reducing bacteria could be related to the microenvironmental conditions. Oxygen, but also sulfide and other (inorganic and organic) sulfur compounds, seems to play an important role in the stratification and diversity of these two major bacterial groups involved in sulfur cycling in Swanbister beach mats.

Desulfotomaculum halophilum sp. nov., a halophilic sulfate-reducing bacterium isolated from oil production facilities

A halophilic endospore-forming, sulfate-reducing bacterium was isolated from an oilfield brine in France. The strain, designated SEBR 3139, was composed of long, straight to curved rods. It grew in 1-14% NaCl with an optimum at 6%. On the basis of morphological, physiological and phylogenetical characteristics, strain SEBR 3139 should be classified in the genus Desulfotomaculum. However, it is sufficiently different from the hitherto described Desulfotomaculum species to be considered as a new species. Strain SEBR 3139T (= DSM 11559T) represents the first moderate halophilic species of the genus Desulfotomaculum. The name Desulfotomaculum halophilum sp. nov. is proposed.

Type II Topoisomerase Quinolone Resistance-Determining Regions of Aeromonas caviae, A. hydrophila, and A. sobria Complexes and Mutations Associated with Quinolone Resistance

ABSTRACT 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-83→Ile (10 strains) or Ser-83→Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-80→Ile (three strains) or Ser-80→Arg change, or at position 84, yielding a Glu-84→Lys 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.

Thiocapsa halophila sp. nov., a new halophilic phototrophic purple sulfur bacterium

A new phototrophic sulfur bacterium has been isolated from a red layer in a laminated mat occurring underneath a gypsum crust in the mediterranean salterns of Salin-de-Giraud (Camargue, France). Single cells were coccus-shaped, non motile, without gas vacuoles and contained sulfur globules. Bacteriochlorophyll a and okenone were present as major photosynthetic pigments. These properties and the G+C content of DNA (65.9–66.6 mol% G+C) are typical characteristics of the genus Thiocapsa. However, the new isolate differs from known species in the genus, particularly in NaCl requirement (optimum, 7% NaCl; range, 3–20% NaCl) and some physiological characteristics. Therefore, a new species is proposed, Thiocapsa halophila, sp. nov.

Characterization of Chromatium salexigens sp. nov., a halophilic Chromatiaceae isolated from mediterranean salinas

Summary In the course of ecological studies in salinas and salt-works of Salin de Giraud (Camargue, France), a halophilic strain of the Chromatiaceae was isolated from a red layer below the gypsum crust. Because of its morphology, this strain (SG 3201) was assigned to the genus Chromatium , resembling Chromatium buderi and Chromatium vinosum . However it differed from these species in some physiological characteristics, in particular pigment composition, salinity range with 10% NaCl requirement for optimum growth, and utilization of some substrates. It is the first described Chromatium strain with high NaCl requirement. Therefore, a new species, Chromatium salexigens , is proposed.

TbtABM, a multidrug efflux pump associated with tributyltin resistance in Pseudomonas stutzeri

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) > or =1000 mg l(-1)), 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 (60.9-84.9%) 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 MICsx4 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(-1)). 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.

Rapid identification of Listeria species by using restriction fragment length polymorphism of PCR-amplified 23S rRNA gene fragments.

ABSTRACT A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.