Melanie Broszat

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.

TraG Encoded by the pIP501 Type IV Secretion System Is a Two-Domain Peptidoglycan-Degrading Enzyme Essential for Conjugative Transfer

pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.

Wastewater Irrigation Increases the Abundance of Potentially Harmful Gammaproteobacteria in Soils in Mezquital Valley, Mexico

ABSTRACT Wastewater contains large amounts of pharmaceuticals, pathogens, and antimicrobial resistance determinants. Only a little is known about the dissemination of resistance determinants and changes in soil microbial communities affected by wastewater irrigation. Community DNAs from Mezquital Valley soils under irrigation with untreated wastewater for 0 to 100 years were analyzed by quantitative real-time PCR for the presence of sul genes, encoding resistance to sulfonamides. Amplicon sequencing of bacterial 16S rRNA genes from community DNAs from soils irrigated for 0, 8, 10, 85, and 100 years was performed and revealed a 14% increase of the relative abundance of Proteobacteria in rainy season soils and a 26.7% increase in dry season soils for soils irrigated for 100 years with wastewater. In particular, Gammaproteobacteria, including potential pathogens, such as Pseudomonas, Stenotrophomonas, and Acinetobacter spp., were found in wastewater-irrigated fields. 16S rRNA gene sequencing of 96 isolates from soils irrigated with wastewater for 100 years (48 from dry and 48 from rainy season soils) revealed that 46% were affiliated with the Gammaproteobacteria (mainly potentially pathogenic Stenotrophomonas strains) and 50% with the Bacilli, whereas all 96 isolates from rain-fed soils (48 from dry and 48 from rainy season soils) were affiliated with the Bacilli. Up to six types of antibiotic resistance were found in isolates from wastewater-irrigated soils; sulfamethoxazole resistance was the most abundant (33.3% of the isolates), followed by oxacillin resistance (21.9% of the isolates). In summary, we detected an increase of potentially harmful bacteria and a larger incidence of resistance determinants in wastewater-irrigated soils, which might result in health risks for farm workers and consumers of wastewater-irrigated crops.

Effects of 100 years wastewater irrigation on resistance genes, class 1 integrons and IncP-1 plasmids in Mexican soil

Long-term irrigation with untreated wastewater can lead to an accumulation of antibiotic substances and antibiotic resistance genes in soil. However, little is known so far about effects of wastewater, applied for decades, on the abundance of IncP-1 plasmids and class 1 integrons which may contribute to the accumulation and spread of resistance genes in the environment, and their correlation with heavy metal concentrations. Therefore, a chronosequence of soils that were irrigated with wastewater from 0 to 100 years was sampled in the Mezquital Valley in Mexico in the dry season. The total community DNA was extracted and the absolute and relative abundance (relative to 16S rRNA genes) of antibiotic resistance genes (tet(W), tet(Q), aadA), class 1 integrons (intI1), quaternary ammonium compound resistance genes (qacE+qacEΔ1) and IncP-1 plasmids (korB) were quantified by real-time PCR. Except for intI1 and qacE+qacEΔ1 the abundances of selected genes were below the detection limit in non-irrigated soil. Confirming the results of a previous study, the absolute abundance of 16S rRNA genes in the samples increased significantly over time (linear regression model, p < 0.05) suggesting an increase in bacterial biomass due to repeated irrigation with wastewater. Correspondingly, all tested antibiotic resistance genes as well as intI1 and korB significantly increased in abundance over the period of 100 years of irrigation. In parallel, concentrations of the heavy metals Zn, Cu, Pb, Ni, and Cr significantly increased. However, no significant positive correlations were observed between the relative abundance of selected genes and years of irrigation, indicating no enrichment in the soil bacterial community due to repeated wastewater irrigation or due to a potential co-selection by increasing concentrations of heavy metals.

New antimicrobial contact catalyst killing antibiotic resistant clinical and waterborne pathogens

Microbial growth on medical and technical devices is a big health issue, particularly when microorganisms aggregate to form biofilms. Moreover, the occurrence of antibiotic-resistant bacteria in the clinical environment is dramatically growing, making treatment of bacterial infections very challenging. In search of an alternative, we studied a novel antimicrobial surface coating based on micro galvanic elements formed by silver and ruthenium with surface catalytic properties. The antimicrobial coating efficiently inhibited the growth of the nosocomial pathogens Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium as demonstrated by the growth inhibition on agar surface and in biofilms of antibiotic resistant clinical E. faecalis, E. faecium, and S. aureus isolates. It also strongly reduced the growth of Legionella in a drinking water pipeline and of Escherichia coli in urine. We postulate a mode of action of the antimicrobial material, which is independent of the release of silver ions. Thus, the novel antimicrobial coating could represent an alternative to combat microbial growth avoiding the toxic side effects of high levels of silver ions on eukaryotic cells.

Spread of Antibiotic Resistance in the Environment: Impact on Human Health

Antibiotic-resistant pathogenic bacteria pose a high threat to human health, but the environmental reservoirs of resistance genes are poorly understood. The origins of antibiotic resistance in the environment are relevant to human health because of the increasing importance of zoonotic diseases as well as the requirement for predicting emerging resistant pathogens. Only little is known about the antibiotic resistomes of the great majority of environmental bacteria, although there have been calls for a greater understanding of the environmental reservoirs of antibiotic resistance. The data on antibiotic resistance before the antibiotic era and in soil show how far away we are from a complete picture about the ecology of antibiotic resistance genes (ARGs). Most of the natural antibiotic producers reside in soil, but soil is a particularly challenging habitat due to its chemical and physical heterogeneity. The prevalence and diversity of ARGs in the environment led to hypotheses about the native roles of resistance genes in natural microbial communities.

Water flow paths are hotspots for the dissemination of antibiotic resistance in soil

Antibiotic resistance genes in soil pose a potential risk for human health. They can enter the soil by irrigation with untreated or insufficiently treated waste water. We hypothesized that water flow paths trigger the formation of antibiotic resistance, since they transport antibiotics, multi-resistant bacteria and free resistance genes through the soil. To test this, we irrigated soil cores once or twice with waste water only, or with waste water added with sulfamethoxazole (SMX) and ciprofloxacin (CIP). The treatments also contained a dye to stain the water flow paths and allowed to sample these separately from unstained bulk soil. The fate of SMX and CIP was assessed by sorption experiments, leachate analyses and the quantification of total and extractable SMX and CIP in soil. The abundance of resistance genes to SMX (sul1 and sul2) and to CIP (qnrB and qnrS) was quantified by qPCR. The sorption of CIP was larger than the dye and SMX. Ciprofloxacin accumulated exclusively in the water flow paths but the resistance genes qnrB and qnrS were not detectable. The SMX concentration in the water flow paths doubled the concentration of the bulk soil, as did the abundance of sul genes, particularly sul1 gene. These results suggest that flow paths do function as hotspots for the accumulation of antibiotics and trigger the formation of resistance genes in soil. Their dissemination also depends on the mobility of the antibiotic, which was much larger for SMX than for CIP.

Enterococcus faecalis Glycolipids Modulate Lipoprotein-Content of the Bacterial Cell Membrane and Host Immune Response

Reference 1. Theilacker C, Diederich A-K, Otto A, Sava IG, Wobser D, Bao Y, et al. (2015) Enterococcus faecalis Glycolipids Modulate Lipoprotein-Content of the Bacterial Cell Membrane and Host Immune Response. PLoS ONE 10(7): e0132949. doi: 10.1371/journal.pone.0132949 PMID: 26172831 Fig 3. Stimulation of TNF-α production in RAW 264.7 mousemacrophages by lipoprotein-enriched cell membrane fractions of E. faecaliswild type and ΔbgsA.RAW264.7 cells were incubated with lipoproteinenriched Triton X-114 extracts from total membrane protein fractions derived from the indicated E. faecalis strains. The concentration of lipoprotein extracts was measured photometrically and normalized to a bacterial cfu:RAW 264.7 cell ratio of 10,000:1. At 16 h, supernatants were collected and TNF-α concentrations were quantified by ELISA. LPS at a concentration of 100 ng/ml was used as positive control. Data represent mean ± SEM of triplicates. * p < 0.001 12030 ΔbgsA versus 12030WT.

Horizontal Gene Transfer in Planktonic and Biofilm Modes

Horizontal gene transfer (HGT) is an important means to obtain and maintain plasticity of microbial genomes. Basically, bacteria apply three different modes to horizontally exchange genetic material: (1) conjugative transfer mediated by mobile genetic elements (MGE), (2) DNA uptake via transformation, and (3) transduction. The three modes rely on different prerequisites of the participating cells: conjugative transfer depends on close cell to cell contact between a donor and a recipient cell and is mediated through multi-protein complexes, denominated type IV secretion systems (T4SS), and DNA transformation does not rely on cell–cell contact but is the uptake of free DNA from the environment by a competent bacterial cell. In some bacteria it is also mediated by a T4SS. The third mechanism depends on the presence of a bacteriophage, which can transfer genomic DNA from one host cell to another. Experimental evidence exists that all three modes occur in planktonic cultures and recent data have also been provided for the occurrence of all three ways in biofilms. Regulation of these HGT events and their consequences for the acting microbes and the biofilms they live in are discussed in this chapter. Additionally, we focus on modern techniques to visualize and to quantify HGT in planktonic and biofilm modes.