Long-Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Rhizobacteria Occupying Nodules of Soybeans (Glycine max).

Antibiotics are entrained in agricultural soil through the application of manures from medicated animals. In the present study, a series of small field plots was established in 1999 that receive annual spring applications of a mixture of tylosin, sulfamethazine, and chlortetracycline at concentrations ranging from 0.1 to 10 mg · kg-1 soil. These antibiotics are commonly used in commercial swine production. The field plots were cropped continuously for soybeans, and in 2012, after 14 annual antibiotic applications, the nodules from soybean roots were sampled and the occupying bradyrhizobia were characterized. Nodules and isolates were serotyped, and isolates were distinguished using 16S rRNA gene and 16S to 23S rRNA gene intergenic spacer region sequencing, multilocus sequence typing, and RSα fingerprinting. Treatment with the antibiotic mixture skewed the population of bradyrhizobia dominating the nodule occupancy, with a significantly larger proportion of Bradyrhizobium liaoningense organisms even at the lowest dose of 0.1 mg · kg-1 soil. Likewise, all doses of antibiotics altered the distribution of RSα fingerprint types. Bradyrhizobia were phenotypically evaluated for their sensitivity to the antibiotics, and there was no association between in situ treatment and a decreased sensitivity to the drugs. Overall, long-term exposure to the antibiotic mixture altered the composition of bradyrhizobial populations occupying nitrogen-fixing nodules, apparently through an indirect effect not associated with the sensitivity to the drugs. Further work evaluating agronomic impacts is warranted.IMPORTANCE Antibiotics are entrained in agricultural soil through the application of animal or human waste or by irrigation with reused wastewater. Soybeans obtain nitrogen through symbiotic nitrogen fixation. Here, we evaluated the impact of 14 annual exposures to antibiotics commonly used in swine production on the distribution of bradyrhizobia occupying nitrogen-fixing nodules on soybean roots in a long-term field experiment. By means of various sequencing and genomic fingerprinting techniques, the repeated exposure to a mixture of tylosin, sulfamethazine, and chlortetracycline each at a nominal soil concentration of 0.1 mg · kg-1 soil was found to modify the diversity and identity of bradyrhizobia occupying the nodules. Nodule occupancy was not associated with the level of sensitivity to the antibiotics, indicating that the observed effects were not due to the direct toxicity of the antibiotics on bradyrhizobia. Altogether, these results indicate the potential for long-term impacts of antibiotics on this agronomically important symbiosis.

Survival of Listeria monocytogenes in Soil Requires AgrA-Mediated Regulation.

In a recent paper, we demonstrated that inactivation of the Agr system affects the patterns of survival of Listeria monocytogenes (A.-L. Vivant, D. Garmyn, L. Gal, and P. Piveteau, Front Cell Infect Microbiol 4:160, http://dx.doi.org/10.3389/fcimb.2014.00160). In this study, we investigated whether the Agr-mediated response is triggered during adaptation in soil, and we compared survival patterns in a set of 10 soils. The fate of the parental strain L. monocytogenes L9 (a rifampin-resistant mutant of L. monocytogenes EGD-e) and that of a ΔagrA deletion mutant were compared in a collection of 10 soil microcosms. The ΔagrA mutant displayed significantly reduced survival in these biotic soil microcosms, and differential transcriptome analyses showed large alterations of the transcriptome when AgrA was not functional, while the variations in the transcriptomes between the wild type and the ΔagrA deletion mutant were modest under abiotic conditions. Indeed, in biotic soil environments, 578 protein-coding genes and an extensive repertoire of noncoding RNAs (ncRNAs) were differentially transcribed. The transcription of genes coding for proteins involved in cell envelope and cellular processes, including the phosphotransferase system and ABC transporters, and proteins involved in resistance to antimicrobial peptides was affected. Under sterilized soil conditions, the differences were limited to 86 genes and 29 ncRNAs. These results suggest that the response regulator AgrA of the Agr communication system plays important roles during the saprophytic life of L. monocytogenes in soil.

Amperometric detection of extended-spectrum ß-lactamase activity: application to the characterization of resistant E. coli strains.

The amperometric detection of extended-spectrum ß-lactamase (ESBL) with carbon screen-printed sensors was investigated in the presence of the Nitrocefin, a commercially-available ß-lactamase chromogenic cephalosporin substrate. Using an ESBL isolated from a clinical sample, it was shown for the first time that the intensity of a specific anodic pic current (EP = ∼+0.3 V vs. Ag/AgCl) resulting from the catalytic hydrolysis of the ß-lactam ring was proportional to the amount of ESBL. The proof-of-principle of a novel susceptibility assay for the rapid and accurate identification of ESBL- producing bacteria was then demonstrated. The detection scheme relied on (i) the culture of the sample in a medium containing the cefotaxime supplemented or not with the clavulanic acid inhibitor to allow the specific determination of ESBL producers (ii) followed by the incubation of the bacteria with the Nitrocefin and (iii) the measurement of the enzyme product by cyclic voltammetry. The amperometric assay was further applied to the characterization of E. coli strains and to the quantification of the ESBL producers. A detection limit of 5 × 10(4) cfu mL(-1) ESBL-producing E. coli was achieved after a 10 min incubation time. In contrast to the approved routine assays, the electrochemical approach, which did not require isolated colonies to be performed, provided quantified results regarding ESBL activity within a few hours. Finally, owing to its cost-effectiveness, portability and simplicity, this test holds great promise for clinical and environmental applications.

Complete genome sequence of Bradyrhizobium ottawaense strain MIAE 01942 isolated from soybean nodules grown in antibiotic-amended soil.

Bradyrhizobium ottawaense MIAE 01942 is a symbiotic nitrogen-fixing bacterium isolated from the root nodules of soybeans grown in agricultural soils amended with veterinary antibiotics. The genome consists of a single 8.45 Mb circular chromosome that harbors genes involved in nitrogen fixation, denitrification, and antibiotic and metal resistance.

An antiplasmid system drives antibiotic resistance gene integration in carbapenemase-producing Escherichia coli lineages.

Plasmids carrying antibiotic resistance genes (ARG) are the main mechanism of resistance dissemination in Enterobacterales. However, the fitness-resistance trade-off may result in their elimination. Chromosomal integration of ARGs preserves resistance advantage while relieving the selective pressure for keeping costly plasmids. In some bacterial lineages, such as carbapenemase producing sequence type ST38 Escherichia coli, most ARGs are chromosomally integrated. Here we reproduce by experimental evolution the mobilisation of the carbapenemase blaOXA-48 gene from the pOXA-48 plasmid into the chromosome. We demonstrate that this integration depends on a plasmid-induced fitness cost, a mobile genetic structure embedding the ARG and a novel antiplasmid system ApsAB actively involved in pOXA-48 destabilization. We show that ApsAB targets high and low-copy number plasmids. ApsAB combines a nuclease/helicase protein and a novel type of Argonaute-like protein. It belongs to a family of defense systems broadly distributed among bacteria, which might have a strong ecological impact on plasmid diffusion.

Accelerated Biodegradation of Veterinary Antibiotics in Agricultural Soil following Long-Term Exposure, and Isolation of a Sulfamethazine-degrading sp.

The World Health Organization has identified antibiotic resistance as one of the top three threats to global health. There is concern that the use of antibiotics as growth promoting agents in livestock production contributes to the increasingly problematic development of antibiotic resistance. Many antibiotics are excreted at high rates, and the land application of animal manures represents a significant source of environmental exposure to these agents. To evaluate the long-term effects of antibiotic exposure on soil microbial populations, a series of field plots were established in 1999 that have since received annual applications of a mixture of sulfamethazine (SMZ), tylosin (TYL), and chlortetracycline (CTC). During the first 6 yr (1999-2004) soils were treated at concentrations of 0, 0.01 0.1, and 1.0 mg kg soil, in subsequent years at concentrations of 0, 0.1, 1.0, and 10 mg kg soil. The lower end of this concentration range is within that which would result from an annual application of manure from medicated swine. Following ten annual applications, the fate of the drugs in the soil was evaluated. Residues of SMZ and TYL, but not CTC were removed much more rapidly in soil with a history of exposure to 10 mg/kg drugs than in untreated control soil. Residues of C-SMZ were rapidly and thoroughly mineralized to CO in the historically treated soils, but not in the untreated soil. A SMZ-degrading sp. was isolated from the treated soil. Overall, these results indicate that soil bacteria adapt to long-term exposure to some veterinary antibiotics resulting in sharply reduced persistence. Accelerated biodegradation of antibiotics in matrices exposed to agricultural, wastewater, or pharmaceutical manufacturing effluents would attenuate environmental exposure to antibiotics, and merits investigation in the context of assessing potential risks of antibiotic resistance development in environmental matrices.

Rapid electrochemical detection of Mycobacterium tuberculosis in sputum by measuring Ag85 activity with disposable carbon sensors.

An electrochemical assay for the detection of the enzymatic activity of the antigen 85 (Ag85) tuberculosis (TB) biomarker was developed and evaluated for the qualitative detection of Mycobacterium tuberculosis in decontaminated sputum. For this purpose, the electroactive properties of both synthetic p-aminophenyl-6-O-octanoyl-3-d-glucopyranoside (p-APOG) substrate and p-aminophenyl-6-3-d-glucopyranoside (p-APG) product released after the removal of the octanoyl fatty acid by the Ag85 were investigated with disposable carbon screen-printed electrodes by cyclic voltammetry. Since specific anodic responses were obtained for the p-APOG substrate and the p-APG product, the intensity of the oxidation peak of the p-APG (E = + 0.35 V vs. Ag/AgCl) was selected as the analytical response for the detection of the Ag85 acyltransferase activity. Once the proof of concept of the Ag85 electrochemical assay was validated with a commercially-available Ag85B protein, its specificity was further assessed by analyzing pure cultures of various bacteria including tuberculous and non-tuberculous mycobacteria as well as different species found in patients' sputum. Finally, with a specificity of 78% and a sensitivity of 89%, the method was successfully compared to microscopy and culture routine tests for TB testing in 36 frozen fluidized and decontaminated sputum. This suggests that owing to its convenience, rapidity, low-cost and portability, the reported Ag85 electrochemical assay is a promising tool to screen patients for TB.

A comparison of enrichment and direct-plating methods for isolation of Listeria monocytogenes from surface water.

Listeria monocytogenes is a facultative intracellular pathogen distributed in the environment. Reference isolation methods include an enrichment step. We compared 2 protocols: one based on enrichment and the other on direct plating, with respect to the efficiency of isolation of L. monocytogenes from surface water samples. From March to May 2006, 126 surface water samples were collected biweekly from 24 discrete sampling sites located in the South Nation River basin (Ontario). Presumptive Listeria spp. were isolated using the 2 methods, and L. monocytogenes isolates were confirmed based on colony morphology and amplification of 3 pathogenicity genes: iap, inlA, and hlyA. Listeria monocytogenes was detected in 31 water samples using the enrichment approach and in 27 using the direct-plating approach, with only 13 samples positive using both approaches. No correlation was observed between the ability of the enrichment procedure to fail in the detection of the pathogenic bacterium and the abundance of several indicator bacteria. The proportion of samples carrying isolates from serovar groups 1/2a, 3a; 1/2b, 3b, 7; 4b, 4d, 4e; and Listeria spp. was significantly different between the 2 approaches. This data indicate that a direct-plating approach would be suitable for the detection of L. monocytogenes in water samples, but that enrichment and direct-plating protocols both have specific biases.

Evidence of autoinduction heterogeneity via expression of the Agr system of Listeria monocytogenes at the single-cell level.

To investigate if the primary function of the Agr system of Listeria monocytogenes is to monitor cell density, we followed Agr expression in batch cultures, in which the autoinducer concentration was uniform, and in biofilms. Expression was heterogeneous, suggesting that the primary function of Agr is not to monitor population density.

A voltammetric test for the rapid discrimination of ß-lactamase-producing Enterobacteriaceae in blood cultures.

The accurate identification of ß-lactamases produced by Enterobacteriaceae is a major challenge in clinical laboratories in order to optimize antimicrobial treatment and patient care. We describe here a rapid voltammetric-based method to detect and to discriminate ß-lactamase activity in Enterobacteriaceae i.e., penicillinase, cephalosporinase (inducible or overproduced), extended-spectrum beta-lactamase and carbapenemase producers. After a 2-h growth step of the sample under three separate conditions: 1) LB (Luria-Bertani) medium, 2) LB supplemented with 4 µg/mL cefotaxime and 3) LB supplemented with 4 µg/mL cefotaxime and 100 µg/mL potassium clavulanate, the ß-lactamase activity was measured by incubating a 0.5 mM nitrocefin solution for 15 min followed by the voltammetric detection of the hydrolyzed nitrocefin with disposable carbon screen-printed sensors. The development and the calibration of the method were carried out by analyzing pure cultures of fifty-seven strains with well characterized ß-lactam-resistance phenotypes. Thanks to the combination of the three currents (i1, i2, i3) recorded for each tested bacteria, the proposed procedure allowed to distinguish the different classes of ß-lactamase producers. In the second part of the study, the method was applied to the analysis of one hundred and fifteen samples Enterobacteriaceae-positive blood culture samples of bacteraemic patients. Overall data showed that the voltammetric method offered a sensitivity of 100% and a specificity of 80%. Interestingly, all of sixteen samples infected by a third-generation cephalosporins-resistant bacteria (i.e. ESBL and overproduced cephalosporinase producers) were detected. This study clearly demonstrated that the voltammetric assay is an efficient alternative technique for the rapid discrimination of ß-lactamases-producing Enterobacteriaceae in blood culture. In contrast to the approved routine assays, the electrochemical test did not require isolated colonies to be performed and was thus carried out in less than 3 h which could allow early administration of an appropriate antibiotic therapy.

Dissemination of CTX-M-Producing Escherichia coli in Freshwater Fishes From a French Watershed (Burgundy).

The burden of extended-spectrum ß-lactamases producing Escherichia coli (ESBL-Ec), has increased over several decades. Freshwater ecosystems are suspected to play an important ecological and evolutionary role in driving the dissemination of antimicrobial resistance. The aim of our study was to decipher the occurrence of ESBL-Ec in a small watershed (Ouche river, Burgundy, France), targeting environmental matrices and fishes. Among cefotaxime resistant E. coli (ctxR Ec) isolates, we detected and characterized 36 ESBL-Ec from water, biofilm and fish guts. ctxR Ec and ESBL-Ec were found in samples from sites near the first small town, located downstream from the watershed which was studied. Treatment of urban wastewater by waste water treatment plants (WWTP), might therefore be a major potential source of ctxR Ec and thus of ESBL-Ec. Prevalence of total E. coli and ctxR Ec in fish guts ranged between 0 to 92% and 0 to 85%; respectively, depending on the sampling site and the fish species. The diet of fish (predator or omnivore) seems to strongly influence the prevalence of total E. coli and ESBL-Ec. Extended spectrum beta-lactamases produced by the isolates from this study belonged to the CTX-M family (CTX-M group 1 and 9). Moreover, some environmental ESBL-Ec proved to share genotypic features (MLST types) with isolates which originated from 8 WWTP effluents discharged in the Ouche river and with the sequence type ST131, which is widely described in clinical isolates. Ninety-seven % (97%) of ESBL-Ec from the study harbored additional antibiotic resistances and can thus be considered as multi drug resistant (MDR) bacteria. Finally, 53% of the ESBL-Ec strains harbored class 1 integron-integrase (intl1). These results are discussed with the perspective of defining indicators of antibiotic resistance contamination in freshwater ecosystems.

Impact of temperature and soil type on Mycobacterium bovis survival in the environment.

Mycobacterium bovis, the causative agent of the bovine tuberculosis (bTB), mainly affects cattle, its natural reservoir, but also a wide range of domestic and wild mammals. Besides direct transmission via contaminated aerosols, indirect transmission of the M. bovis between wildlife and livestock might occur by inhalation or ingestion of environmental substrates contaminated through infected animal shedding. We monitored the survival of M. bovis in two soil samples chosen for their contrasted physical and-chemical properties (i.e. pH, clay content). The population of M. bovis spiked in sterile soils was enumerated by a culture-based method after 14, 30, 60, 90, 120 and 150 days of incubation at 4°C and 22°C. A qPCR based assay targeting the IS1561' locus was also performed to monitor M. bovis in both sterile and biotic spiked soils. The analysis of survival profiles using culture-based method showed that M. bovis survived longer at lower temperature (4°C versus 22°C) whereas the impact of soil characteristics on M. bovis persistence was not obvious. Furthermore, qPCR-based assay detected M. bovis for a longer period of time than the culture based method with higher gene copy numbers observed in sterile soils than in biotic ones. Impact of soil type on M. bovis persistence need to be deepened in order to fill the gap of knowledge concerning indirect transmission of the disease.

A nitrocefin-based amperometric assay for the rapid quantification of extended-spectrum ß-lactamase-producing Escherichia coli in wastewaters.

A sensitive and inexpensive amperometric assay based on the electrochemical detection of the ß-lactamase activity using the nitrocefin as substrate was developed for the rapid and quantitative detection of extended spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) in urban wastewaters. The specific detection of ESBL-EC was achieved by culturing the filtered sample in a medium containing the cefotaxime supplemented or not with the potassium clavulanate inhibitor. This step was followed by the incubation of each subculture filtrate with the nitrocefin substrate which hydrolysis was monitored by amperometry using disposable carbon screen-printed sensors. Current intensities iCef and iClav correspond to the intensity of the anodic current measured (∼+ 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The intensity value i = iCef - iClav was chosen as the analytical response. ESBL-EC calibration plots were established with artificially contaminated wastewater samples. This assay allowed the detection of ESBL-EC amounts as low as 10 cfu in treated effluents and 100 cfu in raw wastewaters with short time analysis of 5.5 h and 4.5 h, respectively. The amperometric method was applied to the analysis of 38 wastewater samples and the results were in good agreement with CFU counts on a selective chromogenic medium for 24 h. Owing to its rapidity, convenience, low-cost and portability, this assay is a promising tool to obtain quantitative data on antimicrobial-resistant E. coli in wastewater effluents. Furthermore, this assay might be used to improve wastewater treatment plant processes in order to minimize the release of antibiotic resistant bacteria into the aquatic environment.

Conservation of type III secretion system genes in Bradyrhizobium isolated from soybean.

The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.

Rapid dissemination of Mycobacterium bovis from cattle dung to soil by the earthworm Lumbricus terrestris.

Indirect transmission of Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), between wildlife and livestock is thought to occur by inhalation or ingestion of environmental substrates contaminated through animal shedding. The role of the soil fauna, such as earthworms, in the circulation of M. bovis from contaminated animal feces is of interest in the epidemiology of bTB. The objective of this study was to assess the impact of earthworm activity on M. bovis transfer from animal dung to castings and the surrounding soil. For this purpose, microcosms of soil containing the anecic earthworms Lumbricus terrestris were prepared and covered with cattle feces spiked with the M. bovis BCG strain Pasteur to carry out two separate experiments. The dissemination, the gut carriage and the excretion of M. bovis were all monitored using a specific qPCR-based assay. Our results showed that the earthworm L. terrestris was able to rapidly disseminate M. bovis from the contaminated cattle feces to the surrounding soil through casting egestion. Moreover, contaminated earthworms were shown to shed the bacteria for 4 days when transferred to a M. bovis-free soil. This study highlights for the first time the possible role of earthworms in the dissemination and the persistence of M. bovis in soils within bTB endemic areas.

Occurence of ArmA and RmtB Aminoglycoside Resistance 16S rRNA Methylases in Extended-Spectrum ß-Lactamases Producing Escherichia coli in Algerian Hospitals.

The aim of this study, was to characterize the extended-spectrum-ß-lactamases (ESBLs) producing clinical strains of Escherichia coli isolated between January 2009 and June 2012 from Algerian hospitals and to determine the prevalence of 16S rRNA methylase among them. Sixty-seven ESBL-producers were detected among the 239 isolates included: 52 CTX-M-15-producers, 5 CTX-M-3-producers, 5 CTX-M-1-producers, 2 CTX-M-14-producers, 2 SHV-12-producers and one TEM-167-producer. Among the ESBL-producing strains twelve harbored 16S rRNA methylase genes: 8 rmtB and 4 armA. rmtB was located on a IncFIA plasmid and armA was located either on a IncL/M or a IncFIA plasmid. RmtB-producing isolates were genotypically related and belonged to the sequence type ST 405 whereas ArmA-producing isolates belonged to ST10, ST 167, and ST 117. This first description of 16S rRNA methylases among E. coli in Algerian hospitals pointed out the necessity to establish control measures to avoid their dissemination.

Free water surface constructed wetlands limit the dissemination of extended-spectrum beta-lactamase producing Escherichia coli in the natural environment.

The fates of Escherichia coli and extended-spectrum beta-lactamase-producing E. coli (ESBL E. coli) were studied over a period of one year in a free water surface constructed wetland (FWS CW) with a succession of open water zones and vegetation ponds (Typha or Phragmites), that received the effluent from a wastewater treatment plant. ESBL E. coli were detected and isolated from all sampling areas of the FWS CW throughout the study period. They represented 1‰ of the total E. coli population regardless of the origin of samples. Two main factors affected the log removal of E. coli and of ESBL E. coli: the season and the presence of vegetation. Between the inlet and the outlet of the FWS CW, the log removal of E. coli ranged from 1.5 in the warmer season (summer and fall) to 3.0 in the colder season (winter and spring). The concentrations of E. coli decreased significantly in the vegetated areas during the colder season, but increased in the warmer season, suggesting an effect of the plant growth stage on the survival of E. coli. Among the 369 ESBL E. coli isolates collected during our study, 84% harbored the CTX-M-ESBL type and 55.3% carried bla genes on plasmid DNA. Furthermore, 93% of the ESBL E. coli isolates were multidrug resistant but the proportion of resistant strains did not change significantly along the FWS CW. ESBL E. coli were characterized by MLST analysis using the 7 genes based Achtman Scheme. ESBL E. coli isolated from water, sediments, roots and feces of myocastors collected in the FWS CW and in the recipient river were genotypically related, suggesting persistence and circulation of the ESBL producing E. coli throughout the FWS CW and in the receiving river. Overall, these observations show that FWS CW could be an efficient treatment for ESBL E. coli disinfection of wastewater and could limit their dissemination in the aquatic environment.

Effect of natamycin on the enumeration, genetic structure and composition of bacterial community isolated from soils and soybean rhizosphere.

Natamycin is commonly used to control fungal growth on agar media used for bacterial enumeration or strain isolation. However, there is no conclusive report on the possible effect of this antibiotic on bacterial growth or on the diversity of the recovered soil bacteria. Therefore, the possible effects of natamycin on the numbers of bacteria isolated at 12 degrees C from three different soils and soybean rhizosphere soil were investigated using natamycin concentrations ranging from 0 to 200 mg l(-1). Our results demonstrate that natamycin concentrations, which inhibit the growth of fungi on the media, have a small but significant inhibitory effect on the number of bacterial colony forming units. A natamycin concentration of 50-200 mg l(-1) is required for an efficient control of fungal growth on media in our experimental conditions depending on the soil type. Bacterial community structure was assessed on culturable cells (cells washed from enumeration plates: plate-wash approach) obtained at 12 degrees C from soybean rhizosphere soil by performing Ribosomal Intergenic Spacer Analysis (RISA) fingerprinting. We demonstrate that all natamycin concentrations used alter the structure of the recovered, culturable bacterial community, compared to control without natamycin. Using ARDRA (amplification of the 16S rDNA gene and restriction analysis) genotyping of individual isolates, some differences were observed between the bacterial isolates obtained in the presence or absence of natamycin. Bacterial isolates recovered in the presence of natamycin are more tolerant (maximal growth rate and lag phase) to this compound than those isolated without natamycin, indicating a possible selection of resistant strains. Therefore, high concentration of natamycin cannot be used for isolation of bacterial strains with the aim of studying biodiversity and could bias a selection of strains for practical applications.

Horizontal gene transfer of atrazine-degrading genes (atz) from Agrobacterium tumefaciens St96-4 pADP1::Tn5 to bacteria of maize-cultivated soil.

The plasmid pADP1::Tn5 derived from pADP1[Atr+] carrying a Tn5 transposon conferring kanamycin and streptomycin resistances was constructed and introduced in Agrobacterium tumefaciens St96-4. This genetically modified strain was inoculated (approximately 10(8) cfu g(-1)) in potted soils planted with maize and treated or not with atrazine (1.5 mg kg(-1)). Bulk and maize rhizosphere soils were sampled 39 days after planting to look for soil indigenous bacteria that had acquired pADP1::Tn5. Four transconjugants were isolated from four different soil samples. The estimated transfer frequency of pADP1::Tn5 was 10(-4) per donor. Maize rhizosphere and atrazine treatment had no obvious effect on pADP1::Tn5 transfer frequency. The sequencing of the 16S rDNA sequences of the transconjugants revealed that they were almost identical and highly similar to that of Variovorax spp (97%). In addition, their characterization suggested that the atzA and atzB genes had been transferred from pADP1::Tn5 to the bacterial chromosome in two of the four transconjugants. These data suggest that the atz degrading genes are horizontally transferred in soil and possibly subjected to gene rearrangement.