High antibiotic releases from manufacturing facilities have been identified as a risk factor for antibiotic resistance development in bacterial pathogens. However, the role of antibiotic pollution in selection and transferability of antibiotic resistance genes (ARGs) is still limited. In this study, we analyzed effluents from azithromycin-synthesis and veterinary-drug formulation facilities as well as sediments from receiving river and creek taken at the effluent discharge sites, upstream and downstream of discharge. Culturing showed that the effluent discharge significantly increased the proportion of antibiotic resistant bacteria in exposed sediments compared to the upstream ones. Quantitative real-time PCR revealed that effluents from both industries contained high and similar relative abundances of resistance genes [sul1, sul2, qacE/qacEΔ1, tet(A)], class 1 integrons (intI1) and IncP-1 plasmids (korB). Consequently, these genes significantly increased in relative abundances in receiving sediments, with more pronounced effects being observed for river than for creek sediments due to lower background levels of the investigated genes in the river. In addition, effluent discharge considerably increased transfer frequencies of captured ARGs from exposed sediments into Escherichia coli CV601 recipient as shown by biparental mating experiments. Most plasmids exogenously captured from effluent and polluted sediments belonged to the broad host range IncP-1ε plasmid group, conferred multiple antibiotic resistance and harbored class 1 integrons. Discharge of pharmaceutical waste from antibiotic manufacturing sites thus poses a risk for development and dissemination of multi-resistant bacteria, including pathogens.

Manure application to agricultural soil introduces antibiotic residues and increases the abundance of antibiotic-resistant bacteria (ARB) carrying antibiotic resistance genes (ARGs), often located on mobile genetic elements (MGEs). The rhizosphere is regarded as a hotspot of microbial activity and gene transfer, which can alter and prolong the effects of organic fertilizers containing antibiotics. However, not much is known about the influence of plants on the effects of doxycycline applied to soil via manure. In this study, the effects of manure spiked with or without doxycycline on the prokaryotic community composition as well as on the relative abundance of ARGs and MGEs in lettuce rhizosphere and bulk soil were investigated by means of a polyphasic cultivation-independent approach. Samples were taken 42 days after manure application, and total community DNA was extracted. Besides a pronounced manure effect, doxycycline spiking caused an additional enrichment of ARGs and MGEs. High-throughput quantitative PCR revealed an increase in tetracycline, aminoglycoside, and macrolide–lincosamide–streptogramin B (MLS_B) resistance genes associated with the application of manure spiked with doxycycline. This effect was unexpectedly lower in the rhizosphere than in bulk soil, suggesting a faster dissipation of the antibiotic and a more resilient prokaryotic community in the rhizosphere. Interestingly, the tetracycline resistance gene tetA(P) was highly enriched in manure-treated bulk soil and rhizosphere, with highest values observed in doxycycline-treated bulk soil, concurring with an enrichment of Clostridia. Thus, the gene tetA(P) might be a suitable marker of soil contamination by ARB, ARGs, and antibiotics of manure origin. These findings illustrate that the effects of manure and doxycycline on ARGs and MGEs differ between rhizosphere and bulk soil, which needs to be considered when assessing risks for human health connected to the spread of ARGs in the environment.

Produce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets in Germany were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (TET)-resistant Escherichia coli were isolated and plasmids conferring TET resistance were captured by exogenous plasmid isolation. TET-resistant E. coli isolates, transconjugants, and total community DNA (TC-DNA) from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons, and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for IncI and IncF plasmids. TET-resistant E. coli isolated from arugula and cilantro carried IncF, IncI1, IncN, IncHI1, IncU, and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids and bla_CTX-M-1 From mixed salad and cilantro, IncF, IncI1, and IncP-1β plasmids were captured exogenously. Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing into E. coli suggests that they could transfer to gut bacteria as well.

Plants are colonized by diverse microorganisms, which may positively or negatively influence the plant fitness. The positive impact includes nutrient acquisition, enhancement of resistance to biotic and abiotic stresses, both important factors for plant growth and survival, while plant pathogenic bacteria can cause diseases. Plant pathogens are adapted to negate or evade plant defense mechanisms, e.g. by the injection of effector proteins into the host cells or by avoiding the recognition by the host. Plasmids play an important role in the rapid bacterial adaptation to stresses and changing environmental conditions. In the plant environment, plasmids can further provide a selective advantage for the host bacteria, e.g. by carrying genes encoding metabolic pathways, metal and antibiotic resistances, or pathogenicity-related genes. However, we are only beginning to understand the role of mobile genetic elements and horizontal gene transfer for plant-associated bacteria. In this review, we aim to provide a short update on what is known about plasmids and horizontal gene transfer of plant associated bacteria and their role in plant-bacteria interactions. Furthermore, we discuss tools available to study the plant-associated mobilome, its transferability, and its bacterial hosts.

Veterinary antibiotics, bacteria carrying antibiotic resistance determinants located on mobile genetic elements and nutrients are spread on agricultural soil using manure as fertilizer. However, systematic quantitative studies linking antibiotic concentrations and antimicrobial resistance genes (ARGs) in manure and the environment are scarce but needed to assess environmental risks. In this microcosm study, a sandy and a loamy soil were mixed with manure spiked with streptomycin or doxycycline at five concentrations. Total-community DNA was extracted on days 28 and 92, and the abundances of ARGs (aadA, strA, tet(A), tet(M), tet(W), tet(Q), sul1, qacE/qacEΔ1) and class 1 and 2 integron integrase genes (intI1 and intI2) were determined by qPCR relative to 16S rRNA genes. Effects on the bacterial community composition were evaluated by denaturing gradient gel electrophoresis of 16S rRNA gene amplicons. Manure application to the soils strongly increased the relative abundance of most tested genes. Antibiotics caused further enrichments which decreased over time and were mostly seen at high concentrations. Strikingly, the effects on relative gene abundances and soil bacterial community composition were more pronounced in sandy soil. The concept of defining antibiotic threshold concentrations for environmental risk assessments remains challenging due to the various influencing factors. 

Abstract-A series of polyelectrolyte hydrogels ranging from 92-98wt% were synthesized by copolymerization of acrylamide,

AAM with 2-acrylamido-2-methyl propane sulphonic acid, AMPS using 0.001g APS as initiator in the presence of 30wt% H2O

and 1.0wt% ethylene glycol dimethacrylate,EDMA as cross-linking agent. The final copolymers was obtained in the form of

glassy and transparent roads at room temperature, these roads were soaked in water for two days to remove unreacted

monomers. The swelling behavior of the hydrogels was studied in distilled water and salt solutions of 1.5 mol/l each of NaCl

and KCl. The Swelling in water shows decreasing values of q, LE, Ø1, EWC% and increasing polymer volume fraction, Ø2 by

increasing acrylamide, due to increasing hydrophobicity and decreasing the hydrophilicity. The swelling in salt solutions

shows a decreasing in the values of ESSNa%, WCNa%, SCNa%, ESSK%, WCK% and SCK% by increasing acrylamid

monomer in the feed due to increasing the hydrophobicity and decreasing the ionized ionic groups (SO3H). The increasing

values of ESSNa%, WCNa% and SCNa% compared with the values of ESSK%, WCK% and SCK% respectively, is due to

the higher charge density of sodium ion than that of potassium ion.

Six Hakea species varying greatly in seed size were selected for cotyledon damage experiments. The

growth of seedlings with cotyledons partially or completely removed was monitored over 90 days. All

seedlings perished by the fifth week when both cotyledons were removed irrespective of seed size.

Partial removal of cotyledons caused a significant delay in the emergence of the first leaf, and reduction

in root and shoot growth of the large-seeded species. The growth of seedlings of small-seeded species

was less impacted by cotyledon damage. The rate of survival, root and shoot lengths and dry biomass

of the seedlings were determined after 90 days. When seedlings were treated with balanced nutrient

solutions following removal of the cotyledons, survival was 95–98%, but 0% when supplied with

nutrient solutions lacking N or P or with water only. The addition of a balanced nutrient solution failed

to restore complete growth of any species, but the rate of root elongation for the small-seeded species

was maintained. Cotyledons provide nutrients to support early growth of Hakea seedlings, but other

physiological roles for the cotyledons are also implicated. In conclusion, small-seeded Hakea species can

tolerate cotyledons loss better than large-seeded species.

Seed size is a key functional trait that affects plant fitness at the seedling stage and may

vary greatly with species fruit size, growth form and fecundity. Using structural equation

modelling (SEM) and correlated trait evolution analysis, we investigated the interaction network

between seed size and fecundity, postfire regeneration strategy, fruit size, plant height

and serotiny (on-plant seed storage) among 82 species of the woody shrub genus, Hakea,

with a wide spectrum of seed sizes (2–500 mg). Seed size is negatively correlated with fecundity,

while fire-killed species (nonsprouters) produce more seeds than resprouters

though they are of similar size. Seed size is unrelated to plant height and level of serotiny

while it scales allometrically with fruit size. A strong phylogenetic signal in seed size revealed

phylogenetic constraints on seed size variation in Hakea. Our analyses suggest a

causal relationship between seed size, fecundity and postfire regeneration strategy in

Hakea. These results demonstrate that fruit size, fecundity and evolutionary history have

had most control over seed size variation among Hakea species.