vanI: a novel D-Ala-D-Lac vancomycin resistance gene cluster found in Desulfitobacterium hafniense.

The glycopeptide vancomycin was until recently considered a drug of last resort against Gram-positive bacteria. Increasing numbers of bacteria, however, are found to carry genes that confer resistance to this antibiotic. So far, 10 different vancomycin resistance clusters have been described. A chromosomal vancomycin resistance gene cluster was previously described for the anaerobic Desulfitobacterium hafniense Y51. We demonstrate that this gene cluster, characterized by its d-Ala-d-Lac ligase-encoding vanI gene, is present in all strains of D. hafniense, D. chlororespirans and some strains of Desulfosporosinus spp. This gene cluster was not found in vancomycin-sensitive Desulfitobacterium or Desulfosporosinus spp., and we show that this antibiotic resistance can be exploited as an intrinsic selection marker for Desulfitobacterium hafniense and D. chlororespirans. The gene cluster containing vanI is phylogenetically only distantly related with those described from soil and gut bacteria, but clusters instead with vancomycin resistance genes found within the phylum Actinobacteria that include several vancomycin-producing bacteria. It lacks a vanH homologue, encoding a D-lactate dehydrogenase, previously thought to always be present within vancomycin resistance gene clusters. The location of vanH outside the resistance gene cluster likely hinders horizontal gene transfer. Hence, the vancomycin resistance cluster in D. hafniense should be regarded a novel one that we here designated vanI after its unique d-Ala-d-Lac ligase.

Quantitative analysis of the relative transcript levels of four chlorophenol reductive dehalogenase genes in Desulfitobacterium hafniense PCP-1 exposed to chlorophenols.

Relative to those of unexposed cultures, the transcript levels of the four CprA-type reductive dehalogenase genes (cprA2, cprA3, cprA4, and cprA5) in Desulfitobacterium hafniense PCP-1 were measured in cultures exposed to chlorophenols. In 2,4,6-trichlorophenol-amended cultures, cprA2 and cprA3 were upregulated, as was cprA5, but concomitantly with the appearance of 2,4-dichlorophenol (DCP). In 3,5-DCP-amended cultures, only cprA5 was upregulated. In pentachlorophenol-amended cultures grown for 12 h, cprA2 and cprA3 were upregulated but not cprA5. cprA4 was not upregulated significantly in cultures containing any tested chlorophenols.

Effect of 2,4,6-trichlorophenol on the microbial activity of adapted anaerobic granular sludge bioaugmented with Desulfitobacterium strains.

The anaerobic degradation of 2,4,6-trichlorophenol (246TCP) has been studied in batch experiments. Granular sludges previously acclimated to 2,4-dichlorophenol (24DCP) and then adapted to at a load of 330 µM 246TCPd(-1) in two expanded granular sludge bed (EGSB) reactors were used. One of the reactors had been bioaugmented with Desulfitobacterium strains whereas the other served as control. 246TCP was tested at concentrations between 250 and 760 µM. The study focused on the fate of both fermentation products and chlorophenols derived from dechlorination of 246TCP. This compound mainly affected the biodegradation of acetate and propionate, which were inhibited at 246TCP concentrations above 380 µM. Lactate and ethanol were also accumulated at 760 µM 246TCP. Methanogenesis was strongly inhibited at 246TCP concentrations higher than 380 µM. A diauxic production of methane was observed, which can be described by a kinetic model in which acetoclastic methanogenesis was inhibited, whereas hydrogenotrophic methanogenesis was hardly affected by 246TCP. The similarity of the kinetic parameters obtained for the control and the bioaugmented sludges (K(i)=175-200 µM 246TCP and n=7) suggests that methanogenesis is not affected by the bioaugmentation. Moreover, the 246TCP dechlorination occurred mainly at ortho position, successively generating 24DCP and 4-chlorophenol (4CP), which was identified as final product. The bioaugmentation does not significantly improve the anaerobic biodegradation of 246TCP. It has been shown that the active biomass is capable of bioaccumulating 246TCP and products from dechlorination, which are subsequently excreted to the bulk medium when the biomass becomes active again. A kinetic model is proposed which simultaneously explains 246TCP and 24DCP reductive dechlorinations and includes the 246TCP bioaccumulation. The values of the kinetic parameters for 246TCP dechlorination were not affected by bioaugmentation (V(max)=5.3 and 5.1 µM h(-1) and K(s)=5.8 and 13.1 µM for control and bioaugmented sludges, respectively).

Noncanonical vancomycin resistance cluster from Desulfitobacterium hafniense Y51.

The glycopeptide vancomycin is a drug of last resort for infection with gram-positive organisms, and three genes are vital to resistance: vanH, vanA, and vanX. These genes are found in a vanHAX cluster, which is conserved across pathogenic bacteria, glycopeptide antibiotic producers, and other environmental bacteria. The genome sequence of the anaerobic, gram-positive, dehalogenating bacterium Desulfitobacterium hafniense Y51 revealed a predicted vanA homolog; however, it exists in a vanAWK-murFX cluster, unlike those of other vancomycin-resistant organisms. Using purified recombinant VanA from D. hafniense Y51, we determined its substrate specificity and found it to have a 42-fold preference for D-lactate over D-alanine, confirming its activity as a D-Ala-D-Lac ligase and its annotation as VanA. Furthermore, we showed that D. hafniense Y51 is highly resistant to vancomycin, with a MIC for growth of 64 microg/ml. Finally, vanA(Dh) is expressed during growth in vancomycin, as demonstrated by reverse transcription-PCR. This finding represents a new glycopeptide antibiotic resistance gene cluster and expands the genetic diversity of resistance to this important class of antibiotic.

Effects of chloromethanes on growth of and deletion of the pce gene cluster in dehalorespiring Desulfitobacterium hafniense strain Y51.

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 microM, but not carbon dichloride even at 1 mM.

Exposure to sulfide causes populations shifts in sulfate-reducing consortia.

The shift in the community structure of a mixed culture of sulfate-reducing bacteria (SRB) at 0.5, 0.75, 1, and 1.5 kg m(-3) sulfide loadings was investigated in an anaerobic continuous bioreactor used for treatment of sulfate-containing wastewater by fluorescence in situ hybridization (FISH), using SRB species-specific and group-specific 16S rRNA-targeting probes. Hybridization analysis using these 16S rRNA-targeted oligonucleotide probes revealed that sulfide was toxic for Desulfonema, Desulfobulbus spp. and the Desulfobacteriaceae group, although it was not toxic for Desulfobacter, Desulfotomaculum, Desulfobacterium spp. or the Desulfovibrionaceae group. On the other hand, only a high concentration of sulfide of 1.5 kg m(-3) was found to be toxic for the Desulfococcus group in the bioreactor. When the sulfide in the feed was 1.00 kg m(-3) the sulfate-reducing capacity of the system decreased, and this decrease was more pronounced when the inlet sulfide was further increased to 1.5 kg m(-3).

Response of 1,2-dichloroethane-adapted microbial communities to ex-situ biostimulation of polluted groundwater.

The microbial community of a groundwater system contaminated by 1,2-dichloroethane (1,2-DCA), a toxic and persistent chlorinated hydrocarbon, has been investigated for its response to biostimulation finalized to 1,2-DCA removal by reductive dehalogenation. The microbial population profile of samples from different wells in the aquifer and from microcosms enriched in the laboratory with different organic electron donors was analyzed by ARISA (Amplified Ribosomal Intergenic Spacer Analysis) and DGGE (Denaturing Gradient Gel Electrophoresis) of 16S rRNA genes. 1,2-DCA was completely removed with release of ethene from most of the microcosms supplemented with lactate, acetate plus formate, while cheese whey supported 1,2-DCA dehalogenation only after a lag period. Microbial species richness deduced from ARISA profiles of the microbial community before and after electron donor amendments indicated that the response of the community to biostimulation was heterogeneous and depended on the well from which groundwater was sampled. Sequencing of 16S rRNA genes separated by DGGE indicated the presence of bacteria previously associated with soils and groundwater polluted by halogenated hydrocarbons or present in consortia active in the removal of these compounds. A PCR assay specific for Desulfitobacterium sp. showed the enrichment of this genus in some of the microcosms. The dehalogenation potential of the microbial community was confirmed by the amplification of dehalogenase-related sequences from the most active microcosms. Cloning and sequencing of PCR products indicated the presence in the metagenome of the bacterial community of a new dehalogenase potentially involved in 1,2-DCA reductive dechlorination.