High abundances of antibiotic-resistant pathogenic bacteria (ARPB) and antibiotic resistance genes (ARGs) in agricultural soil-plant systems have become serious threats to human health and environmental safety. Therefore, it is crucial to develop targeted technology to control existing antibiotic resistance (AR) contamination and potential dissemination in soil-plant systems. In this work, polyvalent bacteriophage (phage) therapy and biochar amendment were applied separately and in combination to stimulate ARPB/ARG dissipation in a soil-lettuce system. With combined application of biochar and polyvalent phage, the abundance of Escherichia coli K-12 (tetR) and Pseudomonas aeruginosa PAO1 (ampR + fosR) and their corresponding ARGs (tetM, tetQ, tetW, ampC, and fosA) significantly decreased in the soil after 63 days' incubation (p < 0.05). Similar results for endophytic K-12 and PAO1, and ARGs, were also obtained in lettuce tissues following combined treatment. Additionally, high throughput sequencing revealed that biochar and polyvalent phage synergetically improved the structural diversity and functional stability of the indigenous bacterial communities in soil and the endophytic ones in lettuce. Hence, this work proposes a novel biotechnology that combines biochar amendment and polyvalent phage therapy to achieve targeted inactivation of ARPB, which stimulates ARG dissipation in soil-lettuce systems.

The determination of values of abundance of antibiotic resistance genes (ARGs) per mass of soil is extremely useful to assess the potential impacts of relevant sources of antibiotic resistance, such as irrigation with treated wastewater or manure application. Culture-independent methods and, in particular, quantitative PCR (qPCR), have been regarded as suitable approaches for such a purpose. However, it is arguable if these methods are sensitive enough to measure ARGs abundance at levels that may represent a risk for environmental and human health. This study aimed at demonstrating the range of values of ARGs quantification that can be expected based on currently used procedures of DNA extraction and qPCR analyses. The demonstration was based on the use of soil samples spiked with known amounts of wastewater antibiotic resistant bacteria (ARB) (Enterococcus faecalis, Escherichia coli, Acinetobacter johnsonii, or Pseudomonas aeruginosa), harbouring known ARGs, and also on the calculation of expected values determined based on qPCR.The limits of quantification (LOQ) of the ARGs (vanA, qnrS, blaTEM, blaOXA, blaIMP, blaVIM) were observed to be approximately 4 log-units per gram of soil dry weight, irrespective of the type of soil tested. These values were close to the theoretical LOQ values calculated based on currently used DNA extraction methods and qPCR procedures. The observed LOQ values can be considered extremely high to perform an accurate assessment of the impacts of ARGs discharges in soils. A key message is that ARGs accumulation will be noticeable only at very high doses. The assessment of the impacts of ARGs discharges in soils, of associated risks of propagation and potential transmission to humans, must take into consideration this type of evidence, and avoid the simplistic assumption that no detection corresponds to risk absence.

Effects of sulfadiazine and ciprofloxacin on the occurrence of free-living and particle-associated opportunistic pathogens in bulk water of simulated drinking water distribution systems (DWDSs) were investigated. It was found that sulfadiazine and ciprofloxacin greatly promoted the occurrence of opportunistic pathogens including Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium avium and its broader genus Mycobacterium spp., as well as the amoebae Acanthamoeba spp. and Hartmanella vermiformis, in bulk water of DWDSs. Moreover, sulfadiazine and ciprofloxacin exhibited much stronger combined effects on the increase of these opportunistic pathogens. Based on the analysis of the antibiotic resistance genes (ARGs) and extracellular polymeric substances (EPS), it was verified that EPS production was increased by the antibiotic resistant bacteria arising from the effects of sulfadiazine/ciprofloxacin. The combined effects of sulfadiazine and ciprofloxacin induced the greatest increase of EPS production in DWDSs. Furthermore, the increased EPS with higher contents of proteins and secondary structure β-sheet led to greater bacterial aggregation and adsorption. Meanwhile, large numbers of suspended particles were formed, increasing the chlorine-resistance capability, which was responsible for the enhancement of the particle-associated opportunistic pathogens in bulk water of DWDSs with sulfadiazine/ciprofloxacin. Therefore, sulfadiazine and ciprofloxacin promoted the occurrence of particle-associated opportunistic pathogens in bulk water of DWDSs due to the role of EPS produced by the bacteria with ARGs.

In respect to direct and indirect water reuse, the microbiological quality of treated wastewater is highly important. Conventional wastewater treatment plants are normally not equipped with advanced technologies for the elimination of bacteria. Molecular biology analyses were combined with live-dead discrimination analysis of wastewater population using Propidium monoazide (PMA) to study population shifts during ozonation (1 g ozone/g DOC) at a municipal wastewater treatment plant. Escherichia coli, enterococci, and Pseudomonas aeruginosa were quantified by polymerase chain reaction (qPCR) and the whole wastewater population was analyzed by metagenomic sequencing. The PMA-qPCR experiments showed that the abundances of P. aeruginosa didn't change by ozone treatment, whereas a reduction was observed for E. coli and enterococci. Results comparing conventional cultivation experiments with PMA-qPCR underlined the presence of viable but not culturable cells (VBNC) and their regrowth potential after ozone treatment. Illumina HiSeq sequencing results with and without PMA treatment demonstrated high population similarities in water samples originating from ozone inflow sampling sides. Upon using PMA treatment after ozonation, population shifts became visible and also underlined the importance of PMA treatment for the evaluation of elimination and selection processes during ozonation at WWTPs. Amongst a number of 14 most abundant genera identified in the inflow samples, 9 genera were found to be reduced, whereas 4 genera increased in relative abundance and 1 genus almost remained constant. The strongest increase in relative abundance after ozonation was detected for Oscillatoria spp., Microcoleus spp. and Nitrospira spp. Beside this, a continuous release of Pseudomonas spp. (including P. aeruginosa) to the downstream receiving body was confirmed. Regrowth experiments demonstrated a high prevalence of P. aeruginosa as part of the surviving bacterial population. Summing up, molecular biology analyses in combination with live-dead discrimination are comprehensive methods to evaluate the elimination processes targeting specific species and/or whole microbial populations.

International audience | The nature and fate of urban contaminants washed by stormwater events and accumulating in a detention basin (DB) were investigated. Relations between bacterial and chemical contaminants of trapped urban sediments, and field parameters were analyzed. Fecal indicators and some pathogens known to be environmentally transmitted (Nocardia, Pseudomonas aeruginosa, and Aeromonas caviae) were tracked, and their persistence investigated. Six sampling campaigns were carried out over 3 years, using five sites including a settling chamber (SC). Aerosolized bacteria at these sites were also monitored. Deposits in the basin were made of fine particles and their content in chemical pollutants was found highly variable. High polycyclic aromatic hydrocarbon (PAH) contents were measured but only three pesticides, over 22, were detected. Deposits were significantly contaminated by fecal indicator bacteria (FIB), P. aeruginosa, A. caviae, and by Nocardia. Only A. caviae showed significant numbers in aerosolized particles recovered over the detention basin. Nocardia spp. cells heavily contaminated the SC. The efficacy of the detention basin at reducing bacterial counts per rain event and over time were estimated. A slight drop in the counts was monitored for fecal indicators but not for the other bacterial groups. Hydrodynamic parameters had a strong impact on the distribution and features of the deposits. Multiple factors impacted the fate of FIB, P. aeruginosa, A. caviae, and Nocardia cells, but in a group dependent manner. Nocardia counts were found positively correlated with volatile organic matter. FIB appeared highly efficient colonizers of the DB.

In order to examine the health of the coastal waters off Visakhapatnam in terms of prevalence and abundance of heterotrophic (H), indicator and pathogenic (P) bacterial counts (BC) and influence of physical processes on them, time-series observations were conducted during January (winter), March (spring), July (summer) and October (post-monsoon). We noticed the impact of physical forces on substantial variations in abundance and distribution of the HBC, total coliforms, Enterococcus faecalis and Pseudomonas aeruginosa in the study region. Based on our results Escherichia coli and other PBC were not much influenced by the physical conditions. It has been noticed that the perennial existence of the high abundance of IBC and PBC above the standard limits during the entire study period leading to an alarming situation in the coastal waters off Visakhapatnam.

This study provides an alternative solution for the bioremediation of a recalcitrant pharmaceutical micropollutant. Clofibric acid (CLA) was chosen as target molecule, because of its environmental persistence and resistance to wastewater treatment technologies. The aim of this study was to investigate the potential of a phenol-resistant Pseudomonas aeruginosa strain isolated from the activated sludge to degrade CLA. In order to evaluate the effect of acclimation process with glucose as carbon co-substrate, two protocols were performed, in which the transfer of the inoculum is carried out either in the exponential growth phase or in the decline phase. The results showed a removal efficiency of CLA of 35% when cells in the decline phase were used for inoculation. In contrast, a very low removal yield (10%) was achieved when cells harvested in the exponential phase were used as inoculum. This work is the first one reporting on the capability of this bacterium to remove this drug. The obtained data showed that the isolated strain is able to degrade target molecule and might be a promising agent for the elimination of this refractory compound.

Urinary tract infections are the utmost common bacterial infections caused by Proteus mirabilis, Pseudomonas aeruginosa, Escherichia coli, and Serratia marcescens. These uropathogens resist the action of several antibiotics due to their ability to form biofilms. Most of these bacterial pathogens use the quorum sensing (QS) machinery to co-ordinate their cells and regulate several virulence factors and biofilm formation. On the other hand, the anti-quorum sensing (anti-QS) and antibiofilm potential of silver nanoparticles have been well reported against certain bacterial pathogens, but to the best of our knowledge, no report is available against the pathogenicity of uropathogens in particular S. marcescens and P. mirabilis. Therefore, the present study is primarily focused on the anti-QS and antibiofilm potential of Piper betle-based synthesized silver nanoparticles (PbAgNPs) against S. marcescens and P. mirabilis. Initially, the silver nanoparticles were synthesized by the aqueous extract of P. betle and characterized by UV-absorbance spectroscopy, XRD, FT-IR, SEM, TEM, and DLS. The synthesized silver nanoparticles were assessed for their anti-QS activity and the obtained results revealed that the PbAgNPs inhibited the QS-mediated virulence factors such as prodigiosin, protease, biofilm formation, exopolysaccharides and hydrophobicity productions in uropathogens. The gene expression analysis divulged the downregulation of fimA, fimC, flhD, and bsmB genes in S. marcescens and flhB, flhD, and rsbA genes in P. mirabilis, respectively. The in vivo Caenorhabditis elegans assays revealed the non-toxic and anti-adherence efficiency of PbAgNPs. Furthermore, the non-toxic effect of PbAgNPs was also confirmed through peripheral blood mononuclear cells and normal lung epithelial cells. Therefore, the contemporary study demonstrates the use of PbAgNPs as a possible alternative toward conventional antibiotics in controlling QS and biofilm-related uropathogen infections.

Biofouling is a serious issue in membrane-based water and wastewater treatment as it critically compromises the efficacy of the water treatment processes. This investigation demonstrates the antimicrobial and antifouling properties of a nanocomposite membrane system composed of carboxyl-functionalized graphene oxide (COOH-GO) and polyphenylsulfone (PPSU). The PPSU/COOH-GO nanocomposite membrane exhibited excellent antimicrobial properties, achieving maximum bacteriostasis rates of 74.2% and 81.1% against the representative Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa, respectively) and 41.9% against the representative Gram-positive bacterium (Staphylococcus aureus). The PPSU/COOH-GO nanocomposite membrane inhibited the attachment, colonization, and the biofilm formation of three species. Antifouling was assessed through filtration experiments using a model foulant bovine serum albumin (BSA). The fouling mechanisms were investigated by Hermia’s models (complete blocking, intermediate blocking, standard blocking, and cake formation), and the analysis involved fitting the volumetric flux decline experimental data to models. The fouling study revealed a less irreversible fouling and increased flux recovery ratio for the PPSU/COOH-GO nanocomposite membrane. Complete blocking of pores and cake formation were the major fouling mechanisms for the membrane.

Ventilator-associated pneumonia (VAP) is the most frequent infection in intensive care units (ICU). It is associated with high rates of long morbidity and mortality. Management of a case of VAP is often said to add $40,000 to hospital costs USA. All these data directed our interest to study the etiology, risk factors, and antibiotic susceptibility patterns of VAP in ICU of Tanta University Hospital. This study included 36 cases of VAP. Endotracheal aspirates were obtained from all cases and microbiologically analyzed. Samples were collected over 1 year. Forty-two strains were isolated from 28 cases, while eight cases showed no bacterial growth. The most frequent organism was Staphylococcus aureus (30.95%), followed by Acinetobacter baumannii and Pseudomonas aeruginosa (21.43% for each), and the least common was Staphylococcus epidermidis (2.38%). Multi-drug resistance was detected in (50%) of the isolated bacteria in this study. Imipenem, amikacin, linezolid, vancomycin, and levofloxacin are recommended to be the most effective drugs in management of VAP. VAP is a serious problem in ICU carrying many risks for the patient live. Regimens of empirical treatment should take in consideration the update in the bacterial etiology and antibiotic susceptibility patterns of VAP.