Inactivation Of Antibiotic Resistant Bacteria Research Articles

Efficient inactivation of antibiotic resistant bacteria and antibiotic resistance genes by photo-Fenton process under visible LED light and neutral pH

Antibiotic resistance has been recognized as a major threat to public health worldwide. Inactivation of antibiotic resistant bacteria (ARB) and degradation of antibiotic resistance genes (ARGs) are critical to prevent the spread of antibiotic resistance in the environment. Conventional disinfection processes are effective to inactivate water-borne pathogens, yet they are unable to completely eliminate the antibiotic resistance risk. This study explored the potential of the photo-Fenton process to inactivate ARB, and to degrade both extracellular and intracellular ARGs (e-ARGs and i-ARGs, respectively). Using Escherichia coli DH5α with two plasmid-encoded ARGs (tetA and blaTEM-1) as a model ARB, a 6.17 log ARB removal was achieved within 30 min of applying photo-Fenton under visible LED and neutral pH conditions. In addition, no ARB regrowth occurred after 48-h, demonstrating that this process is very effective to induce permanent disinfection on ARB. The photo-Fenton process was validated under various water matrices, including ultrapure water (UPW), simulated wastewater (SWW) and phosphate buffer (PBS). The higher inactivation efficiency was observed in SWW as compared to other matrices. The photo-Fenton process also caused a 6.75 to 8.56-log reduction in eARGs based on quantitative real-time PCR of both short- and long amplicons. Atomic force microscopy (AFM) further confirmed that the extracellular DNA was sheared into short DNA fragments, thus eliminating the risk of the transmission of antibiotic resistance. As compared with e-ARGs, a higher dosage of Fenton reagent was required to damage i-ARGs. In addition, the tetA gene was more easily degraded than the blaTEM-1 gene. Collectively, our results demonstrate the photo-Fenton process is a promising technology for disinfecting water to prevent the spread of antibiotic resistance.

Impacts on antibiotic-resistant bacteria and their horizontal gene transfer by graphene-based TiO2&Ag composite photocatalysts under solar irradiation

In recent years, photocatalysis has been considered as a promising method, which provides measures to environmental pollution. Antibiotic resistant bacteria (ARB) and their antibiotic resistance genes (ARGs), as the emerging environmental pollutants, are released into the environment, resulting in antibiotic resistance spread. TiO2-based nanocomposites, as the most common photocatalytic material, may influence ARB and ARGs under photocatalytic conditions. However, the research on this aspect is rare. A novel nanocomposite synthesized from Ag, TiO2 and graphene oxide (GO), was selected as a representative of nanomaterials for investigation. The experimental results indicated that TiO2/Ag/GO nanocomposites significantly affected ARB vitality. 100 mg/L TiO2/Ag/GO will reduce bacterial survival to 12.2% in 10 min under simulated sunlight irradiation. Chloramphenicol as the most representative antibiotic in the water, reduces the effect of ARB inactivation under photocatalytic conditions. The addition of TiO2/Ag/GO could affect tetracycline antibiotic resistance. The level of bacterial tolerance to tetracycline had a significant reduction. The horizontal gene transfer was promoted from 1 to 2 folds with the addition of TiO2/Ag/GO. Even high TiO2/Ag/GO concentration (100 mg/L) sample had a limited promotion, suggesting that TiO2/Ag/GO will not increase the risk of antibiotic resistance spread compared to other nano materials.

Presence, dissemination and removal of antibiotic resistant bacteria and antibiotic resistance genes in urban drinking water system: A review

Antibiotic resistance in aquatic environment has become an important pollution problem worldwide. In recent years, much attention was paid to antibiotic resistance in urban drinking water systems due to its close relationship with the biosafety of drinking water. This review was focused on the mechanisms of antibiotic resistance, as well as the presence, dissemination and removal of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the urban drinking water system. First, the presence of ARB and ARGs in the drinking water source was discussed. The variation of concentration of ARGs and ARB during coagulation, sedimentation and filtration process were provided subsequently, in which filtration was proved to be a promising technology to remove ARGs. However, biological activated carbon (BAC) process and drinking water distribution systems (DWDSs) could be incubators which promote the antibiotic resistance, due to the enrichment of ARGs and ARB in the biofilms attached to the active carbon and pipe wall. Besides, as for disinfection process, mechanisms of the inactivation of ARB and the promotion of conjugative transfer of ARGs under chlorine, ozone and UV disinfection were described in detail. Here we provide some theoretical support for future researches which aim at antibiotic resistance controlling in drinking water.

Enhanced inactivation of antibiotic-resistant bacteria isolated from secondary effluents by g-C3N4 photocatalysis.

The extensive use of antibiotics has resulted in the development of antibiotic-resistant bacteria (ARB), which may not be completely removed by traditional wastewater treatment processes. More effective approaches to disinfection are needed to prevent the release of ARB into the surface water. The metal-free photocatalyst graphitic carbon nitride (g-C3N4) has aroused great interest as a possible agent for water and wastewater treatment, due to its low cytotoxicity and photoactivity with visible light. In this study, the efficacy of g-C3N4 was assessed as a possible means to enhance ARB inactivation by irradiation. ARB were isolated and purified from secondary effluents in 4 municipal wastewater treatment plants. Of these, 4 typical multi-drug ARB isolates, belonging to Enterobacteriaceae, were selected for irradiation experiments. Inactivation was seen to increase with irradiation time. At 60min, the inactivation of the 4 ARB isolates by light at > 300nm and > 400nm was in the range of 0.25-0.39 log and 0.16-0.19 log, respectively. The use of g-C3N4-mediated photocatalysis at the same wavelengths significantly enhanced that to 0.64-1.26 log and 0.31-0.41 log, respectively. The antibiotic susceptibility of the ARB isolates remained unchanged either prior to or after irradiation and was independent of photon fluence, reaction time, and the presence of g-C3N4. This study establishes a baseline for understanding the effectiveness of g-C3N4 photocatalysis on inactivation of ARB in wastewaters and lays the foundation for further improvement in the use of photocatalysis for wastewater treatment.

Immobilised Cerium-Doped Zinc Oxide as a Photocatalyst for the Degradation of Antibiotics and the Inactivation of Antibiotic-Resistant Bacteria

The threat of antibiotic resistance to the wellbeing of societies is well established. Urban wastewater treatment plants (UWTPs) are recognised sources for antibiotic resistance dissemination in the environment. Herein a novel cerium-doped zinc oxide (Ce-ZnO) photocatalyst is compared to ZnO and the benchmark TiO2-P25 in the immobilised form on a metallic support, to evaluate a photocatalytic process as a possible tertiary treatment in UWTPs. The catalysts were compared for the removal of two antibiotics, trimethoprim (TMP) and sulfamethoxazole (SMX), and for the inactivation of Escherichia coli (E. coli) strain DH5-Alpha in isotonic sodium chloride solution and of autochthonous bacteria in real secondary wastewater. In real wastewater, E. coli and other coliforms were monitored, as well as the respective fractions resistant to ofloxacin and azithromycin. In parallel, Pseudomonas aeruginosa and the respective sub-population resistant to ofloxacin or ciprofloxacin were also monitored. Photocatalysis with both ZnO and Ce-ZnO was faster than using TiO2-P25 at degrading the antibiotics, with Ce-ZnO the fastest against SMX but slower than undoped ZnO in the removal of TMP. Ce-ZnO catalyst reuse in the immobilised form produced somewhat slower kinetics maintained >50% of the initial activity, even after five cycles of use. Approximately 3 log10 inactivation of E. coli in isotonic sodium chloride water was recorded with reproducible results. In the removal of autochthonous bacteria in real wastewater, Ce-ZnO performed better (more than 2 log values higher) than TiO2-P25. In all cases, E. coli and other coliforms, including their resistant subpopulations, were inactivated at a higher rate than P. aeruginosa. With short reaction times no evidence for enrichment of resistance was observed, yet with extended reaction times low levels of bacterial loads were not further inactivated. Overall, Ce-ZnO is an easy and cheap photocatalyst to produce and immobilise and the one that showed higher activity than the industry standard TiO2-P25 against the tested antibiotics and bacteria, including antibiotic-resistant bacteria.

Removal of sulfonamide antibiotic resistant bacterial and intracellular antibiotic resistance genes by UVC-activated peroxymonosulfate

The inactivation of an isolated sulfonamide antibiotic resistant bacteria (ARB) HLS-6 and reduction of intracellular sul1 and intI1 in its genome by UVC irradiation, PMS oxidation and UVC-activated peroxymonosulfate (UVC/PMS) treatments were investigated in this study. The UVC/PMS treatment was superior to the other two methods in the inactivation of ARB and reduction of qPCR-sul1 and qPCR-intI1. The HLS-6 ARB (108 CFU/mL) could be effectively inactivated 5.3 log by UVC (100 μW/cm2)/PMS (1 mg/L), and the reduction rates of qPCR-sul1 and qPCR-intI1 by UVC (100 μW/cm2)/PMS (20 mg/L), reached up to 2.9 log and 3.4 log, respectively within 30 min. qPCR-intI1 reacted faster than qPCR-sul1 in all methods. Sulfate radical was responsible for the reduction of target genes, while hydroxyl radical had negligible effect on that. The dosage of PMS positively affected the reduction of both genes during UVC/PMS, while the initial concentration of ARB could negatively influence the reduction of target genes. The pH 5 of reaction solution was most beneficial to the reduction of ARGs. The reduction rates at pH 5 reached up to 3.1 log (sul1) and 3.3 log (intI1). The reduction of target genes was slightly facilitated in the initial 5 min and suppressed after 5 min with the co-existence of sulfamethoxazole. This study will provide a potential alternative method for controlling the antibiotic resistance in aquatic environment.

Inactivation of antibiotic-resistant bacteria by chlorine dioxide in soil and shifts in community composition.

To study the efficacy of chlorine dioxide in the inactivation of antibiotic-resistant bacteria in soil, bacteria resistant to penicillin, amoxicillin or streptomycin were screened out from the soils around a hennery. The effects of dosage, contact time and pH value on the killing rates were investigated by batch experiments. The community composition before and after inactivation was analyzed by high-throughput genetic sequencing. The results showed that antibiotic-resistant bacteria are common and widespread in soil and the most resistant species is Staphylococcus aureus. More than 99% of antibiotic-resistant bacteria could be killed by chlorine dioxide at 5 mg L−1 within 30 min under neutral conditions. The killing log value declined slightly when the pH was changed from 4 to 9. The dominant genus was Sphingomonas, which was sensitive to chlorine dioxide and could be inactivated easily similar to Arthrobacter and Massilia. However, Micromonosporaceae and Thaumarchaeota were more resistant to chlorine dioxide than other species, and their relative abundance increased after disinfection.

Solar light and the photo-Fenton process against antibiotic resistant bacteria in wastewater: A kinetic study with a Streptomycin-resistant strain

In this work, we assess the potential of solar light and the solar-assisted photo-Fenton process to inactivate antibiotic resistant bacteria (ARB) in wastewater (WW). A systematic investigation on ARB inactivation and regrowth kinetics after solar-based processes is intended, as well as the modification of their antibiotic resistance (AR). As such, a Streptomycin-resistant (SR) E. coli as a model ARB was subjected to solar exposure (with or without UVB light) at various irradiance levels, as well as the solar/H2O2 and the photo-Fenton process at neutral pH. We report the good fit of the SR E.coli to known kinetic models (>96% R2), and analytically present the necessary treatment timed for total inactivation and halting their post-treatment regrowth capability. For all treatment methods, the AR was found to decrease during treatment, prior to loss of cultivability (5–15% lower time for 4-log inactivation). UVB irradiation was determined as the key factor of loss of cultivability, AR and regrowth. Solar/H2O2 and photo-Fenton presented fast inactivation rates, ensured no regrowth of ARB, and indicated moderate effect on modifying the SR of E. coli, with similar inactivation times for both strains. The presence of Streptomycin in WW was found to act synergistically on the faster inactivation by all processes tested. Finally, ∼1h of solar-based AOPs was found to ensure 4-log ARB inactivation and no regrowth, even in absence of the residual H2O2, indicating their suitability as proper WW disinfection processes.

Inactivation of Antibiotic Resistant Bacteria and Resistance Genes by Ozone: From Laboratory Experiments to Full-Scale Wastewater Treatment.

Ozone, a strong oxidant and disinfectant, seems ideal to cope with future challenges of water treatment, such as micropollutants, multiresistant bacteria (MRB) and even intracellular antibiotic resistance genes (ARG), but information on the latter is scarce. In ozonation experiments we simultaneously determined kinetics and dose-dependent inactivation of Escherichia coli and its plasmid-encoded sulfonamide resistance gene sul1 in different water matrixes. Effects in E. coli were compared to an autochthonous wastewater community. Furthermore, resistance elimination by ozonation and post-treatment were studied in full-scale at a wastewater treatment plant (WWTP). Bacterial inactivation (cultivability, membrane damage) and degradation of sul1 were investigated using plate counts, flow cytometry and quantitative real-time PCR. In experiments with E. coli and the more ozone tolerant wastewater community disruption of intracellular genes was observed at specific ozone doses feasible for full-scale application, but flocs seemed to interfere with this effect. At the WWTP, regrowth during postozonation treatment partly compensated inactivation of MRB, and intracellular sul1 seemed unaffected by ozonation. Our findings indicate that ozone doses relevant for micropollutant abatement from wastewater do not eliminate intracellular ARG.

H2O2 and/or TiO2 photocatalysis under UV irradiation for the removal of antibiotic resistant bacteria and their antibiotic resistance genes

Inactivating antibiotic resistant bacteria (ARB) and removing antibiotic resistance genes (ARGs) are very important to prevent their spread into the environment. Previous efforts have been taken to eliminate ARB and ARGs from aqueous solution and sludges, however, few satisfying results have been obtained. This study investigated whether photocatalysis by TiO2 was able to reduce the two ARGs, mecA and ampC, within the host ARB, methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, respectively. The addition of H2O2 and matrix effect on the removal of ARB and ARGs were also studied. TiO2 thin films showed great effect on both ARB inactivation and ARGs removal. Approximately 4.5-5.0 and 5.5–5.8 log ARB reductions were achieved by TiO2 under 6 and 12mJ/cm2 UV254 fluence dose, respectively. For ARGs, 5.8 log mecA reduction and 4.7 log ampC reduction were achieved under 120mJ/cm2 UV254 fluence dose in the presence of TiO2. Increasing dosage of H2O2 enhanced the removal efficiencies of ARB and ARGs. The results also demonstrated that photocatalysis by TiO2 was capable of removing both intracellular and extracellular forms of ARGs. This study provided a potential alternative method for the removal of ARB and ARGs from aqueous solution.

A review of the influence of treatment strategies on antibiotic resistant bacteria and antibiotic resistance genes

Antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARG) in the aquatic environment have become an emerging contaminant issue, which has implications for human and ecological health. This review begins with an introduction to the occurrence of ARB and ARG in different environmental systems such as natural environments and drinking water resources. For example, ARG or ARB with resistance to ciprofloxacin, sulfamethoxazole, trimethoprim, quinolone, vancomycin, or tetracycline (e.g., tet(A), tet(B), tet(C), tet(G), tet(O), tet(M), tet(W), sul I, and sul II) have been detected in the environment. The development of resistance may be intrinsic, may be acquired through spontaneous mutations (de novo), or may occur due to horizontal gene transfer from donor bacteria, phages, or free DNA to recipient bacteria. An overview is also provided of the current knowledge regarding inactivation of ARB and ARG, and the mechanism of the effects of different disinfection processes in water and wastewater (chlorination, UV irradiation, Fenton reaction, ozonation, and photocatalytic oxidation). The effects of constructed wetlands and nanotechnology on ARB and ARG are also summarized.

Modulation of inflammatory response of wounds by antimicrobial photodynamic therapy.

Management of infections caused by Pseudomonas aeruginosa is becoming difficult due to the rapid emergence of multi-antibiotic resistant strains. Antimicrobial photodynamic therapy (APDT) has a lot of potential as an alternative approach for inactivation of antibiotic resistant bacteria. In this study we report results of our investigations on the effect of poly-L-lysine conjugate of chlorine p6 (pl-cp6) mediated APDT on the healing of P.aeruginosa infected wounds and the role of Nuclear Factor kappa B (NF-kB) induced inflammatory response in this process. Excisional wounds created in Swiss albino mice were infected with ∼10(7) colony forming units of P.aeruginosa. Mice with wounds were divided into three groups: 1) Uninfected, 2) Infected, untreated control (no light, no pl-cp6), 3) Infected, APDT. After 24 h of infection (day 1 post wounding), the wounds were subjected to APDT [pl-cp6 applied topically and exposed to red light (660 ± 25 nm) fluence of ∼ 60 J/cm(2)]. Subsequent to APDT, on day 2 and 5 post wounding (p.w), measurements were made on biochemical parameters of inflammation [toll like receptor-4 (TLR-4), NF-kB, Inteleukin (IL)-[1α, IL-β, and IL-2)] and cell proliferation [(fibroblast growth factor-2 (FGF-2), alkaline phosphatase (ALP)]. In comparison with untreated control, while expression of TLR-4, NF-kB (p105 and p50), and proinflammatory interleukins (IL-1α, IL-1β,IL-2) were reduced in the infected wounds subjected to APDT, the levels of FGF-2 and ALP increased, on day 5 p.w. The measurements made on the inflammatory markers and cell proliferation markers suggest that APDT reduces inflammation caused by P.aeruginosa and promotes cell proliferation in wounds.

Advanced treatment of urban wastewater by UV radiation: Effect on antibiotics and antibiotic-resistant E. coli strains

Urban wastewater treatment plant (UWWTP) effluents are among the possible sources of antibiotics and antibiotic-resistant bacteria (ARB) spread into the environment. In this work, the effect of UV radiation on antibiotic-resistant Escherichia coli (E. coli) strains was compared with that of chlorination process. Under the investigated conditions, UV disinfection process resulted in a total inactivation after 60min of irradiation (1.25×104μWscm−2) compared to 120min chlorine contact time (initial chlorine dose of 2mgL−1). Moreover, no change in E. coli strains’ resistance to amoxicillin (AMX) (minimum inhibiting concentration (MIC)>256mgL−1) and sulfamethoxazole (SMZ) (MIC>1024mgL−1) could be observed after UV treatment, while the treatment affected resistance of the lower resistance strain to ciprofloxacin (CPX) (MIC decreased by 33% and 50% after 60 and 120min, respectively). Contrarily, chlorination process did not affect antibiotic resistance of the investigated E. coli strains. Finally, the effect of UV radiation on the mixture of three antibiotics was also investigated and photodegradation data fit quite well pseudo first order kinetic models with t1/2 values of 14, 20 and 25min for CPX, AMX and SMZ, respectively. According to these results, conventional disinfection processes may not be effective in the inactivation of ARB, and the simultaneous release of ARB and antibiotics at sub-lethal concentrations into UWWTP effluent may promote the development of resistance among bacteria in receiving water.

Comparative photodynamic inactivation of antibiotic resistant bacteria by first and second generation cationic photosensitizers

Photodynamic inactivation (PDI) is an efficient approach against a wide range of microorganisms and can be viewed as an alternative for the treatment of microbial infections. In this work we synthesized "first" and "second" generation photosensitizers (PSs), the tetra-cationic porphyrin and the new penta-cationic chlorin , respectively, and evaluated their efficiency against two antibiotic resistant bacterial strains, Staphylococcus aureus and Pseudomonas aeruginosa. The PS was obtained in very good yield by an easy synthesis method. The PDI studies were performed in parallel with 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (), a widely studied PS in PDI, and the obtained results were compared. Two different light ranges were used: white light (400-800 nm) and red light (530-800 nm) delivered at a fluence rate of 150 mW cm(-2). The results show that both strains, even though antibiotic resistant, were efficiently inactivated by the three PSs, chlorin being the most effective. For the Gram positive bacterium S. aureus a 7.0 log reduction was observed after 5-10 min of irradiation, at a concentration of 0.5 μM, whereas for the Gram negative P. aeruginosa, similar photoinactivation occurred at a higher PS concentration (10 μM) and after a longer irradiation period (30 min). The synthetic chlorin can be regarded as promising for the treatment of bacterial infections under red light, which penetrates deeper in living tissues. The results of this study open the possibility to prepare a new series of chlorin-type derivatives to efficiently photoinactivate Gram (+) and (-) antibiotic resistant bacteria. The efficient PDI with the chlorin indicates high potential for the use of a scaffold in the preparation of new generation PSs based on cationic chlorin derivatives.

Ultraviolet Disinfection of Antibiotic Resistant Bacteria and Their Antibiotic Resistance Genes in Water and Wastewater

Disinfection of wastewater treatment plant effluent may be an important barrier for limiting the spread of antibiotic-resistant bacteria (ARBs) and antibiotic resistance genes (ARGs). While ideally disinfection should destroy ARGs, to prevent horizontal gene transfer to downstream bacteria, little is known about the effect of conventional water disinfection technologies on ARGs. This study examined the potential of UV disinfection to damage four ARGs, mec(A), van(A), tet(A), and amp(C), both in extracellular form and present within a host ARBs: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium (VRE), Escherichia coli SMS-3-5, and Pseudomonas aeruginosa 01, respectively. An extended amplicon-length quantitative polymerase chain reaction assay was developed to enhance capture of ARG damage events and also to normalize to an equivalent length of target DNA (∼1000 bp) for comparison. It was found that the two Gram-positive ARBs (MRSA and VRE) were more resistant to UV disinfection than the two Gram-negative ARBs (E. coli and P. aeruginosa). The two Gram-positive organisms also possessed smaller total genome sizes, which could also have reduced their susceptibility to UV because of fewer potential pyrimidine dimer targets. An effect of cell type on damage to ARGs was only observed in VRE and P. aeruginosa, the latter potentially because of extracellular polymeric substances. In general, damage of ARGs required much greater UV doses (200-400 mJ/cm² for 3- to 4-log reduction) than ARB inactivation (10-20 mJ/cm² for 4- to 5-log reduction). The proportion of amplifiable ARGs following UV treatment exhibited a strong negative correlation with the number of adjacent thymines (Pearson r < -0.9; p < 0.0001). ARBs surviving UV treatment were negatively correlated with total genome size (Pearson r < -0.9; p < 0.0001) and adjacent cytosines (Pearson r < -0.88; p < 0.0001) but positively correlated with adjacent thymines (Pearson r > 0.85; p < 0.0001). This suggests that formation of thymine dimers is not the sole mechanism of ARB inactivation. Overall, the results indicate that UV is limited in its potential to damage ARGs and other disinfection technologies should be explored.

Novel fluorescent cephalosporins: Synthesis, antimicrobial activity and photodynamic inactivation of antibiotic resistant bacteria

Two novel fluorescent cephalosporins, TCA and TBCA, were synthesized and characterized by 1H NMR, 13C NMR, UV–vis, and fluorescence spectroscopies. Biological activity assays demonstrated that TCA inactivated a Klebsiella pneumonia strain that expressed extended-spectrum β-lactamases. Incubation of 6 μM TCA with K. pneumonia cultures resulted in cell death for 84% of the cells after 126 J/cm2 of light irradiation. In vitro, TCA exhibited a MIC = 0.5 μg/mL with Staphylococcus aureus. Kinetic evaluation revealed that TCA and TBCA were substrates for B1 and B3 subclass metallo-β-lactamases. TBCA exhibited stronger binding affinities to the Gram-positive bacterial strains MRSA1, MRSA2, and S. aureus with value of 2.95–6.59 μM per 108 cells/mL.