Elisabeth Grohmann

Conjugative Plasmid Transfer in Gram-Positive Bacteria

SUMMARY Conjugative transfer of bacterial plasmids is the most efficient way of horizontal gene spread, and it is therefore considered one of the major reasons for the increase in the number of bacteria exhibiting multiple-antibiotic resistance. Thus, conjugation and spread of antibiotic resistance represents a severe problem in antibiotic treatment, especially of immunosuppressed patients and in intensive care units. While conjugation in gram-negative bacteria has been studied in great detail over the last decades, the transfer mechanisms of antibiotic resistance plasmids in gram-positive bacteria remained obscure. In the last few years, the entire nucleotide sequences of several large conjugative plasmids from gram-positive bacteria have been determined. Sequence analyses and data bank comparisons of their putative transfer (tra) regions have revealed significant similarities to tra regions of plasmids from gram-negative bacteria with regard to the respective DNA relaxases and their targets, the origins of transfer (oriT), and putative nucleoside triphosphatases NTP-ases with homologies to type IV secretion systems. In contrast, a single gene encoding a septal DNA translocator protein is involved in plasmid transfer between micelle-forming streptomycetes. Based on these clues, we propose the existence of two fundamentally different plasmid-mediated conjugative mechanisms in gram-positive microorganisms, namely, the mechanism taking place in unicellular gram-positive bacteria, which is functionally similar to that in gram-negative bacteria, and a second type that occurs in multicellular gram-positive bacteria, which seems to be characterized by double-stranded DNA transfer.

A Type IV-Secretion-Like System Is Required for Conjugative DNA Transport of Broad-Host-Range Plasmid pIP501 in Gram-Positive Bacteria

Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopeć, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.

Accumulation of Pharmaceuticals, Enterococcus, and Resistance Genes in Soils Irrigated with Wastewater for Zero to 100 Years in Central Mexico

Irrigation with wastewater releases pharmaceuticals, pathogenic bacteria, and resistance genes, but little is known about the accumulation of these contaminants in the environment when wastewater is applied for decades. We sampled a chronosequence of soils that were variously irrigated with wastewater from zero up to 100 years in the Mezquital Valley, Mexico, and investigated the accumulation of ciprofloxacin, enrofloxacin, sulfamethoxazole, trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen, diclofenac, as well as the occurrence of Enterococcus spp., and sul and qnr resistance genes. Total concentrations of ciprofloxacin, sulfamethoxazole, and carbamazepine increased with irrigation duration reaching 95% of their upper limit of 1.4 µg/kg (ciprofloxacin), 4.3 µg/kg (sulfamethoxazole), and 5.4 µg/kg (carbamazepine) in soils irrigated for 19–28 years. Accumulation was soil-type-specific, with largest accumulation rates in Leptosols and no time-trend in Vertisols. Acidic pharmaceuticals (diclofenac, naproxen, bezafibrate) were not retained and thus did not accumulate in soils. We did not detect qnrA genes, but qnrS and qnrB genes were found in two of the irrigated soils. Relative concentrations of sul1 genes in irrigated soils were two orders of magnitude larger (3.15×10−3±0.22×10−3 copies/16S rDNA) than in non-irrigated soils (4.35×10−5±1.00×10−5 copies/16S rDNA), while those of sul2 exceeded the ones in non-irrigated soils still by a factor of 22 (6.61×10–4±0.59×10−4 versus 2.99×10−5±0.26×10−5 copies/16S rDNA). Absolute numbers of sul genes continued to increase with prolonging irrigation together with Enterococcus spp. 23S rDNA and total 16S rDNA contents. Increasing total concentrations of antibiotics in soil are not accompanied by increasing relative abundances of resistance genes. Nevertheless, wastewater irrigation enlarges the absolute concentration of resistance genes in soils due to a long-term increase in total microbial biomass.

Quantitative PCR Monitoring of Antibiotic Resistance Genes and Bacterial Pathogens in Three European Artificial Groundwater Recharge Systems

ABSTRACT Aquifer recharge presents advantages for integrated water management in the anthropic cycle, namely, advanced treatment of reclaimed water and additional dilution of pollutants due to mixing with natural groundwater. Nevertheless, this practice represents a health and environmental hazard because of the presence of pathogenic microorganisms and chemical contaminants. To assess the quality of water extracted from recharged aquifers, the groundwater recharge systems in Torreele, Belgium, Sabadell, Spain, and Nardò, Italy, were investigated for fecal-contamination indicators, bacterial pathogens, and antibiotic resistance genes over the period of 1 year. Real-time quantitative PCR assays for Helicobacter pylori, Yersinia enterocolitica, and Mycobacterium avium subsp. paratuberculosis, human pathogens with long-time survival capacity in water, and for the resistance genes ermB, mecA, blaSHV-5, ampC, tetO, and vanA were adapted or developed for water samples differing in pollutant content. The resistance genes and pathogen concentrations were determined at five or six sampling points for each recharge system. In drinking and irrigation water, none of the pathogens were detected. tetO and ermB were found frequently in reclaimed water from Sabadell and Nardò. mecA was detected only once in reclaimed water from Sabadell. The three aquifer recharge systems demonstrated different capacities for removal of fecal contaminators and antibiotic resistance genes. Ultrafiltration and reverse osmosis in the Torreele plant proved to be very efficient barriers for the elimination of both contaminant types, whereas aquifer passage followed by UV treatment and chlorination at Sabadell and the fractured and permeable aquifer at Nardò posed only partial barriers for bacterial contaminants.

A new enzymatic method for the detachment of particle associated soil bacteria

A new enzymatic technique for the detachment of bacteria from soil particles was developed and applied to different soil samples taken at various sampling sites and depths. Many soil microorganisms are closely associated with the organic matrix of soil particles. They produce extracellular polymeric substances (EPS), which promote the irreversible adhesion of cells to soil particulates. To characterize the EPS, a prestaining of the soil samples with different lectins was performed. Samples from a sewage field, an urban park, a farmland, a mixed forest and garden mold were stained with a set of FITC-labelled lectins from Triticum vulgaris, Ulex europaeus, Concanavalin A and Pseudomonas aeruginosa. Based on the results, a combination of alpha-glucosidase, beta-galactosidase and a lipase was chosen for degradation of the EPS structures, followed by gentle mechanical and chemical dispersion in a modified sodium pyrophosphate buffer. The samples were fixed with formaldehyde and total cell counts were determined by DAPI staining. With the exception of the wheat field sample, this technique revealed up to 22-fold higher total cell counts for all investigated soil samples compared to the conventional detachment method, a simple dispersion with sodium pyrophosphate buffer. Efficiency of the technique was assessed by scanning electron microscopy. These images showed convincingly that the enzymatic treatment followed by sonication efficiently detached the bacteria and left the soil particles almost blank.

Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe

Managed Aquifer Recharge (MAR) is becoming an attractive option for water storage in water reuse processes as it provides an additional treatment barrier to improve recharged water quality and buffers seasonal variations of water supply and demand. To achieve a better understanding about the level of pathogenic microorganisms and their relation with microbial indicators in these systems, five waterborne pathogens and four microbial indicators were monitored over one year in three European MAR sites operated with reclaimed wastewater. Giardia and Cryptosporidium (oo)cysts were found in 63.2 and 36.7% of the samples respectively. Salmonella spp. and helminth eggs were more rarely detected (16.3% and 12.5% of the samples respectively) and Campylobacter cells were only found in 2% of samples. At the Belgian site advanced tertiary treatment technology prior to soil aquifer treatment (SAT) produced effluent of drinking water quality, with no presence of the analysed pathogens. At the Spanish and Italian sites amelioration of microbiological water quality was observed between the MAR injectant and the recovered water. In particular Giardia levels decreased from 0.24-6.14 cysts/L to 0-0.01 cysts/L and from 0.4-6.2 cysts/L to 0-0.07 cysts/L in the Spanish and Italian sites respectively. Salmonella gene copies and Giardia cysts were however found in the water for final use and/or the recovered groundwater water at the two sites. Significant positive Spearman correlations (p<0.05, r(s) range: 0.45-0.95) were obtained, in all the three sites, between Giardia cysts and the most resistant microbial markers, Clostridium spores and bacteriophages.

Intergeneric transfer of the Enterococcus faecalis plasmid pIP501 to Escherichia coli and Streptomyces lividans and sequence analysis of its tra region

The nucleotide sequence of the transfer (tra) region of the multiresistance broad-host-range Inc18 plasmid pIP501 was completed. The 8629-bp DNA sequence encodes 10 open reading frames (orf), 9 of them are possibly involved in pIP501 conjugative transfer. The putative pIP501 tra gene products show highest similarity to the respective ORFs of the conjugative Enterococcus faecalis plasmids pRE25 and pAMbeta1, and the Streptococcus pyogenes plasmid pSM19035, respectively. ORF7 and ORF10 encode putative homologues of type IV secretion systems involved in transport of effector molecules from pathogens to host cells and in conjugative plasmid transfer in Gram-negative (G-) bacteria. pIP501 mobilized non-selftransmissible plasmids such as pMV158 between different E. faecalis strains and from E. faecalis to Bacillus subtilis. Evidence for the very broad-host-range of pIP501 was obtained by intergeneric conjugative transfer of pIP501 to a multicellular Gram-positive (G+) bacterium, Streptomyces lividans, and to G- Escherichia coli. We proved for the first time pIP501 replication, expression of its antibiotic resistance genes as well as functionality of the pIP501 tra genes in S. lividans and E. coli.

Conjugative type IV secretion systems in Gram-positive bacteria.

Highlights • The conjugative transfer mechanism of broad-host-range, Enterococcus sex pheromone and Clostridium plasmids is reviewed.• Comparisons with Gram-negative type IV secretion systems are presented.• The current understanding of the unique Streptomyces double stranded DNA transfer mechanism is reviewed.

Environmental protection strategies for sustainable development

1. Environmental Protection Strategies: An overview 2. The potential of rhizospheric microorganisms to promote the plant growth in disturbed soils 3. Sustainable solutions for agro processing waste management: An overview 4. Dyes - Environmental Impact and Remediation 5. Molecular detection of resistance and transfer genes in environmental samples Elisabeth Grohmann and Karsten Arends 6. Key biochemical attributes to assess soil ecosystem sustainability 7. Methods for Genotoxicity testing of environmental pollutants 8. Trends in biological degradation of cyanobacteria and toxins 9. Bioremediation of pesticides from soil and wastewater 10. Isolation and characterization of rhizobacteria antagonistic to Macrophomina phaseolina (Tassi) Goid., causal agent of alfalfa damping-off 11. Biofilm formation by environmental bacteria 12. Biochemical processes of rhizobacteria and their application in biotechnology 13. Pulp and paper industry-manufacturing process, wastewater generation and treatment 14. A review of environmental contamination and remediation strategies for heavy metals at shooting range soils 15. Peroxidases as a potential tool for the decolorization and removal of synthetic dyes from polluted water 16. Solid waste management options and their impacts on climate change and human health 17. Potential of biopesticides in sustainable agriculture

The 2.5 Å Structure of the Enterococcus Conjugation Protein TraM resembles VirB8 Type IV Secretion Proteins

Background: Conjugative plasmid transfer is the prevalent means for spreading antibiotic resistance genes among bacteria. Results: Surface exposure of transfer protein TraM from the Gram-positive (G+) plasmid pIP501 was confirmed, and its crystal structure was solved. Conclusion: Structural relations to type IV secretion (T4S) proteins provide a novel classification scheme. Significance: The novel classification will help elucidate structure-function relationships in G+ T4S systems. Conjugative plasmid transfer is the most important means of spreading antibiotic resistance and virulence genes among bacteria and therefore presents a serious threat to human health. The process requires direct cell-cell contact made possible by a multiprotein complex that spans cellular membranes and serves as a channel for macromolecular secretion. Thus far, well studied conjugative type IV secretion systems (T4SS) are of Gram-negative (G−) origin. Although many medically relevant pathogens (e.g., enterococci, staphylococci, and streptococci) are Gram-positive (G+), their conjugation systems have received little attention. This study provides structural information for the transfer protein TraM of the G+ broad host range Enterococcus conjugative plasmid pIP501. Immunolocalization demonstrated that the protein localizes to the cell wall. We then used opsonophagocytosis as a novel tool to verify that TraM was exposed on the cell surface. In these assays, antibodies generated to TraM recruited macrophages and enabled killing of pIP501 harboring Enteroccocus faecalis cells. The crystal structure of the C-terminal, surface-exposed domain of TraM was determined to 2.5 Å resolution. The structure, molecular dynamics, and cross-linking studies indicated that a TraM trimer acts as the biological unit. Despite the absence of sequence-based similarity, TraM unexpectedly displayed a fold similar to the T4SS VirB8 proteins from Agrobacterium tumefaciens and Brucella suis (G−) and to the transfer protein TcpC from Clostridium perfringens plasmid pCW3 (G+). Based on the alignments of secondary structure elements of VirB8-like proteins from mobile genetic elements and chromosomally encoded T4SS from G+ and G− bacteria, we propose a new classification scheme of VirB8-like proteins.