A new modelling framework for assessing the relative burden of antimicrobial resistance in aquatic environments.

The infections caused by antibiotic resistant bacteria (ARB) can lead to higher medical costs, prolonged hospital stays, and increased mortality compared to bacteria that are susceptible to antibiotics. Challenges exist in quantifying the potential risk/burden associated with antimicrobial resistance (AMR) as there is a lack of dose-response models available for pathogens which are resistant to antibiotics, in addition to the fact that very little is known regarding the health risks posed by antibiotic resistant genes (ARG). In this paper, we proposed a new modelling framework to evaluate the relative burden of AMR in natural aquatic environments. With this framework, an AMR burden score for each sample was calculated based on burden coefficients assigned for each ARB and ARG, as well as weighted burdens for the separate ARBs and ARGs components. The method developed in this study was applied to assess the relative burden of AMR in local aquatic environments with different land uses at different seasons. The collected filed data were used to verify the applicability of the proposed relative burden assessment method. Through the established method, the spatial and temporal hotspots of AMR were identified, which could provide useful information to agencies for better control and management of AMR emergence in natural aquatic environments.

Removal of antibiotic residues, antibiotic resistant bacteria and antibiotic resistance genes in municipal wastewater by membrane bioreactor systems.

Antibiotic residues, antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are considered new classes of water contaminants due to their potential adverse effects on aquatic ecosystems and human health. This paper provides comprehensive data on the occurrences of 19 antibiotics, bacteria resistant to 10 antibiotics, and 15 ARGs in raw influent and different treatment stages of conventional activated sludge (CAS) and membrane bioreactor (MBR) systems. Seventeen out of the 19 target antibiotics were detected in raw influent with concentrations of up to ten micrograms per liter. Concentrations of antibiotics measured in the secondary effluent were much lower compared to those in the raw influent. Among the antibiotics, amoxicillin, azithromycin, ciprofloxacin, chloramphenicol, meropenem, minocycline, oxytetracycline, sulfamethazine and vancomycin had highest removal by CAS or MBR systems with median removal efficiency (RE) > 70%, while trimethoprim and lincomycin were recalcitrant in the CAS system with median RE <50%. Similarly, the target ARB and ARGs were omnipresent in the raw influent samples with average concentrations as high as 2.6 × 106 CFU/mL and 2.0 × 107 gene copies/mL, respectively. The concentrations of ARB in secondary effluent of the CAS system declined relative to the raw influent (i.e. lower than raw influent by 2-3 orders of magnitude) and no ARB were detected in the MF permeate of the MBR system. For ARGs, their concentrations in secondary effluent/MF permeate ranged from below method quantification limit (

Occurrence and characteristics of extended-spectrum ß-lactamase- and carbapenemase- producing bacteria from hospital effluents in Singapore.

One of the most important resistance mechanisms in Gram-negative bacteria today is the production of enzymes causing resistance to cephalosporin and carbapenem antibiotics. The spread of extended-spectrum ß-lactamases (ESBL)- and carbapenemase- producing Gram-negative bacteria is an emerging global public health problem. The aim of the present study was to (i) assess the prevalence of carbapenem-resistant bacteria (CRB) and ESBL-producing strains in sewage effluents from two major hospitals in Singapore, (ii) characterize the isolated strains and (iii) identify some of the ESBL and carbapenemase genes responsible for the resistance. CHROMagar ESBL and KPC plates were used to rapidly screen for ESBL-producing bacteria and those expressing reduced susceptibility to carbapenems, respectively. The abundance of ESBL-producers and CRB in hospital wastewater ranged between 103 and 106CFU/mL. Out of the 66 isolates picked from ESBL and KPC plates, 95%, 82%, 82% and 76% were resistant to ceftriaxone, ceftazidime (3rd generation cephalosporin family), ertapenem and meropenem (carbapenem family), respectively. Among the resistant isolates, the most predominant taxa identified were Pseudomonas spp. (28.2%), Klebsiella spp. (28.2%), Enterobacter spp. (18.3%) and Citrobacter spp. (11.3%). PCR and sequencing analysis showed that the predominant ß-lactamase genes were blaSHV (41.1%) followed by blaNDM-1 (35.6%), blaCTX (35.6%) and blaKPC (28.8%). The results of this study show a high prevalence of bacteria resistant to modern extended-spectrum cephalosporins and carbapenems and the presence of ESBL- and carbapenemase producers in hospital effluents. These findings support the need to improve management of hospital wastewater in order to minimize the spread of antimicrobial resistant microorganisms from this source.

Corrigendum: Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters.

[This corrects the article on p. 2200 in vol. 8, PMID: 29201017.].

Characterization of Metagenomes in Urban Aquatic Compartments Reveals High Prevalence of Clinically Relevant Antibiotic Resistance Genes in Wastewaters.

The dissemination of antimicrobial resistance (AMR) is an escalating problem and a threat to public health. Comparative metagenomics was used to investigate the occurrence of antibiotic resistant genes (ARGs) in wastewater and urban surface water environments in Singapore. Hospital and municipal wastewater (n = 6) were found to have higher diversity and average abundance of ARGs (303 ARG subtypes, 197,816 x/Gb) compared to treated wastewater effluent (n = 2, 58 ARG subtypes, 2,692 x/Gb) and surface water (n = 5, 35 subtypes, 7,985 x/Gb). A cluster analysis showed that the taxonomic composition of wastewaters was highly similar and had a bacterial community composition enriched in gut bacteria (Bacteroides, Faecalibacterium, Bifidobacterium, Blautia, Roseburia, Ruminococcus), the Enterobacteriaceae group (Klebsiella, Aeromonas, Enterobacter) and opportunistic pathogens (Prevotella, Comamonas, Neisseria). Wastewater, treated effluents and surface waters had a shared resistome of 21 ARGs encoding multidrug resistant efflux pumps or resistance to aminoglycoside, macrolide-lincosamide-streptogramins (MLS), quinolones, sulfonamide, and tetracycline resistance which suggests that these genes are wide spread across different environments. Wastewater had a distinctively higher average abundance of clinically relevant, class A beta-lactamase resistant genes (i.e., blaKPC, blaCTX-M, blaSHV, blaTEM). The wastewaters from clinical isolation wards, in particular, had a exceedingly high levels of blaKPC-2 genes (142,200 x/Gb), encoding for carbapenem resistance. Assembled scaffolds (16 and 30 kbp) from isolation ward wastewater samples indicated this gene was located on a Tn3-based transposon (Tn4401), a mobilization element found in Klebsiella pneumonia plasmids. In the longer scaffold, transposable elements were flanked by a toxin-antitoxin (TA) system and other metal resistant genes that likely increase the persistence, fitness and propagation of the plasmid in the bacterial host under conditions of stress. A few bacterial species (Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii, Pseudomonas aeruginosa) that were cultured from the isolation ward wastewaters on CHROMagar media harbored the blaKPC-2 gene. This suggests that hospital wastewaters derived from clinical specialty wards are hotspots for the spread of AMR. Assembled scaffolds of other mobile genetic elements such as IncQ and IncF plasmids bearing quinolone resistance genes (qnrS1, qnrS2) and the class A beta-lactamase gene (blaTEM-1) were recovered in wastewater samples which may aid the transfer of AMR.

Multimodal Personal Verification Using Likelihood Ratio for the Match Score Fusion.

In this paper, the authors present a novel personal verification system based on the likelihood ratio test for fusion of match scores from multiple biometric matchers (face, fingerprint, hand shape, and palm print). In the proposed system, multimodal features are extracted by Zernike Moment (ZM). After matching, the match scores from multiple biometric matchers are fused based on the likelihood ratio test. A finite Gaussian mixture model (GMM) is used for estimating the genuine and impostor densities of match scores for personal verification. Our approach is also compared to some different famous approaches such as the support vector machine and the sum rule with min-max. The experimental results have confirmed that the proposed system can achieve excellent identification performance for its higher level in accuracy than different famous approaches and thus can be utilized for more application related to person verification.

Occurrences and Characterization of Antibiotic-Resistant Bacteria and Genetic Determinants of Hospital Wastewater in a Tropical Country.

Wastewater discharged from clinical isolation and general wards at two hospitals in Singapore was examined to determine the emerging trends of antibiotic resistance (AR). We quantified the concentrations of 12 antibiotic compounds by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibiotic-resistant bacteria (ARB), the class 1 integrase gene (intI1), and 16 antibiotic resistance genes (ARGs) that confer resistance to 10 different clinically relevant antibiotics. A subset of 119 antibiotic-resistant isolates were phylogenetically classified and tested for the presence of ARGs encoding resistance to ß-lactam antibiotics (blaNDM, blaKPC, blaSHV, blaCTX-M), amikacin [aac(6')-Ib], co-trimoxazole (sul1, sul2, dfrA), ciprofloxacin (qnrA, qnrB), and the intI1 gene. Among these resistant isolates, 80.7% were detected with intI1 and 66.4% were found to carry at least 1 of the tested ARGs. Among 3 sampled locations, the clinical isolation ward had the highest concentrations of ARB and the highest levels of ARGs linked to resistance to ß-lactam (blaKPC), co-trimoxazole (sul1, sul2, dfrA), amikacin [aac(6')-Ib], ciprofloxacin (qnrA), and intI1 We found strong positive correlations (P < 0.05) between concentrations of bacteria resistant to meropenem, ceftazidime, amikacin, co-trimoxazole, and ciprofloxacin and abundances of blaKPC, aac(6')-Ib, sul1, sul2, dfrA, qnrA, and intI1 genes.

Antimicrobial Properties of Lysosomal Enzymes Immobilized on NH2Functionalized Silica-Encapsulated Magnetite Nanoparticles.

The immobilization efficiency, antimicrobial activity and recovery of lysosomal enzymes on NH2 functionalized magnetite nanoparticles have been studied under various conditions. The immobi- lization efficiency depends upon the ratio of the amount of enzyme and magnetite and it shows an increase with magnetite concentration which is due to the presence of amine group at the magnetite surface that leads to a strong attraction. The optimized reaction time to immobilize the lysosomal enzymes on magnetite was determined by using a rolling method. The immobilization efficiency increases with reaction time and reached a plateau after 5 minutes and then remained constant for 10 minutes. However, after 30 minutes the immobilization efficiency decreased to 85%, which is due to the weaker electrostatic interactions between magnetite and detached lysosomal enzymes. The recovery and stability of immobilized lysosomal enzymes has also been studied. The antimicrobial activity was almost 100% but it decreased upon reuse and no activity was observed after its reuse for seven times. The storage stability of lysosomal enzymes as an antimicrobial agent was about 88%, which decreased to 53% after one day and all activity of immobilized lysosomal enzymes was maintained after five days. Thus, the lysosomal enzymes immobilized on magnetite nanoparticles could potentially be used as antimicrobial agents to remove bacteria.

Correction: A Comparison of Microbial Water Quality and Diversity for Ballast and Tropical Harbor Waters.

[This corrects the article DOI: 10.1371/journal.pone.0143123.].

A Comparison of Microbial Water Quality and Diversity for Ballast and Tropical Harbor Waters.

Indicator organisms and antibiotic resistance were used as a proxy to measure microbial water quality of ballast tanks of ships, and surface waters in a tropical harbor. The survival of marine bacteria in ballast tanks appeared to diminish over longer water retention time, with a reduction of cell viability observed after a week based on heterotrophic plate counts. Pyrosequencing of 16S rRNA genes showed distinct differences in microbial composition of ballast and harbor waters. The harbor waters had a higher abundance of operational taxonomic units (OTUs) assigned to Cyanobacteria (Synechococcus spp.) and α-proteobacteria (SAR11 members), while marine hydrocarbon degraders such as γ-proteobacteria (Ocenspirillaes spp., Thiotrchales spp.) and Bacteroidetes (Flavobacteriales spp.) dominated the ballast water samples. Screening of indicator organisms found Escherichia coli (E. coli), Enterococcus and Pseudomonas aeruginosa (P. aeruginosa) in two or more of the ballast and harbor water samples tested. Vibrio spp. and Salmonella spp. were detected exclusively in harbor water samples. Using quantitative PCR (qPCR), we screened for 13 antibiotic resistant gene (ARG) targets and found higher abundances of sul1 (4.13-3.44 x 102 copies/mL), dfrA (0.77-1.80 x10 copies/mL) and cfr (2.00-5.21 copies/mL) genes compared to the other ARG targets selected for this survey. These genes encode for resistance to sulfonamides, trimethoprim and chloramphenicol-florfenicol antibiotics, which are also known to persist in sediments of aquaculture farms and coastal environments. Among the ARGs screened, we found significant correlations (P<0.05) between ereA, ermG, cfr and tetO genes to one or more of the indicator organisms detected in this study, which may suggest that these members contribute to the environmental resistome. This study provides a baseline water quality survey, quantitatively assessing indicators of antibiotic resistance, potentially pathogenic organisms and a broad-brush description of difference in microbial composition and diversity between open oceans and tropical coastal environments through the use of next generation sequencing technology.

Quantification of polyketide synthase genes in tropical urban soils using real-time PCR.

Polyketide synthases (PKSs) catalyze the biosynthesis of polyketides and may contribute to the natural production of antibiotics and pose selective pressure for the development of antibiotic resistant bacteria in the environment. Although conventional PCR have been developed to detect the presence of PKS genes, no previous studies have been done to quantify the abundance of PKS genes in environmental samples. In this study, two sets of degenerate real-time PCR (qPCR) primers (PKS1-F/PKS1-R, PKS2-F/PKS2-R) with high specificity and sensitivity were developed to quantify PKS type I and type II genes. These primers were subsequently used to quantify PKS genes in tropical urban soils, and both PKS genes were widely detected in all soil samples. The absolute abundance of PKS type I ranged from 1.7×10(6) to 4.7×10(6) copies per gram of soil and the absolute abundance of PKS type II genes ranged from 2.4×10(5) to 1.5×10(6) per gram of soil, and the abundance of PKS type I gene was consistently higher than that of PKS type II gene. The relative abundance of PKS type I gene was positively correlated with that of PKS type II gene (p<0.01). Regression analyses indicate that PKS gene abundance was negatively correlated with environmental factors, such as selected antibiotics, sulfate, and metals (p<0.05), but was not correlated with land use type. The studies on the correlation between environmental factors and PKS genes could provide useful information to understand natural production of antibiotics and its associated environmental risks.

Proteomic analysis in Daphnia magna exposed to As(III), As(V) and Cd heavy metals and their binary mixtures for screening potential biomarkers.

In this study, the effects of three widespread heavy metals, As(III), As(V) and Cd, and their binary mixtures on the proteomic profile in D. magna were examined to screen novel protein biomarkers using the two-dimensional gel electrophoresis method (2DE). Ten 20d daphnia were exposed to the LC20 concentrations for each of a total of 8 treatments, including the control, As(III), As(V), Cd, [As(III)+As(V)], [As(III)+Cd], [As(V)+Cd], and [As(III), As(V), Cd], for 24h before protein isolation. Three replicates were performed for each treatment. These protein samples were employed for 2DE experiments with a pH gradient gel strip from pH 3 to pH 10. The protein spots were detected by a silver staining process and their intensities were analyzed by Progenesis software to discover the differentially expressed proteins (DEPs) in response to each heavy metal. A total of 117 differentially expressed proteins (DEPs) were found in daphnia responding to the 8 treatments and mapped onto a 2D proteome map, which provides some information of the molecular weight (MW) and pI value for each protein. All of these DEPs are considered as potential candidates for protein biomarkers in D. magna for detecting heavy metals in the aquatic ecosystem. Comparing the proteomic results among these treatments suggested that exposing D. magna to binary mixtures of heavy metals may result in some complex interactive molecular responses within them, rather than just the simple sum of the proteomic profiles of the individual chemicals, (As(III), As(V), and Cd).

Phenol degradation activity and reusability of Corynebacterium glutamicum coated with NH(2)-functionalized silica-encapsulated Fe3O4 nanoparticles.

In this study, a novel method to immobilize and separate Corynebacterium glutamicum for phenol degradation was developed using Fe(3)O(4) nanoparticles (NPs). The Fe(3)O(4) NPs were encapsulated with silica and functionalized with NH(2) groups to enhance their capacity to adsorb on the cell surface. The results showed that the NH(2)-functionalized silica-encapsulated Fe(3)O(4) NPs strongly adsorbed on the cell surface of C. glutamicum during 32 d culture without any interruptions of their normal cell growth. The coated C. glutamicum were easily separated from the culture broth within 2 min by applying an external magnetic field Also, the coated C.glutamicum were able to completely degrade 50 ppm phenol in the culture broth after 8d culture at 30 °C. Concerning reusability, the coated cells could completely degrade phenol during the first 2 cycles, and retain ~60% activity of phenol degradation for the third and four cycles.

Toxicity Assessment of Titanium (IV) Oxide Nanoparticles Using Daphnia magna (Water Flea).

OBJECTIVES: Titanium dioxide (TiO(2)), a common nanoparticle widely used in industrial production, is one of nano-sized materials. The purpose of this study was to determine the acute and chronic toxicity of TiO(2) using different size and various concentrations on Daphnia magna. METHODS: In the acute toxicity test, four concentrations (0, 0.5, 4, and 8 mM) for TiO(2) with 250 or 500 nm and five concentrations (0, 0.25, 0.5, 0.75, and 1 mM) for TiO(2) with 21 nm were selected to analyze the toxic effect to three groups of ten daphnia neonates over 96 hours. In addition, to better understand their toxicity, chronic toxicity was examined over 21 days using 0, 1, and 10 mM for each type of TiO(2). RESULTS: Our results showed that all organisms died before the reproduction time at a concentration of 10 mM of TiO(2). In addition, the exposure of anatase (21 nm) particles were more toxic to D. magna, comparing with that of anatase (250 nm) and rutile (500 nm) particles. CONCLUSIONS: This study indicated that TiO(2) had adverse impacts on the survival, growth and reproduction of D. magna after the 21days exposure. In addition, the number of test organisms that were able to reproduce neonates gradually were reduced as the size of TiO(2) tested was decreased.

Toxicity evaluation of verapamil and tramadol based on toxicity assay and expression patterns of Dhb, Vtg, Arnt, CYP4, and CYP314 in Daphnia magna.

In this study, the toxicities of two pharmaceuticals, verapamil and tramadol were evaluated in Daphnia magna using the conventional toxicity tests (acute and chronic test) and the expression patterns of five stress responsive genes. In the chronic toxicity test, several parameters, such as the survival percentage, the body length of D. magna, the time of first reproduction, and the number of offspring per female, were adversely affected during the exposure to 4.2 mg L(-1) verapamil and 34 mg L(-1) tramadol. During the 24-h short-term exposure, verapamil particularly caused a downregulated expression of the CYP4 and CYP314 genes, whereas tramadol upregulated the expression of the CYP314 gene. Neither pharmaceutical affected the expression of Dhb, Arnt, and Vtg. However, during the 21-day long-term exposure, both verapamil and tramadol significantly reduced the expression level of the Vtg gene, a biomarker of the reproduction ability in an oviparous animal, whereas neither affected the other genes.

Utilization of phenol and naphthalene affects synthesis of various amino acids in Corynebacterium glutamicum.

This article reports multiple metabolic pathways of amino acid production via phenol and naphthalene use by Corynebacterium glutamicum. Biodegradation of phenol and naphthalene by C. glutamicum occurred in a mineral salt medium containing 1% yeast extract without any additional carbon sources. Among the amino acids synthesized via the TCA-cycle, glutamate synthesis increased in C. glutamicum supplemented with 8.5 mM phenol or with 4.2 mM naphthalene. Aspartate synthesis significantly increased when cultured with 4.2 mM naphthalene, and increased synthesis of threonine and histidine was observed only with the addition of phenol. In addition, synthesis of valine and leucine decreased considerably under both conditions. Moreover, the bioconversion of glutamate from phenol and naphthalene is regulated by a transcriptional regulator, FarR, at the transcription level of the gltBD and gdh genes. In this study, we found that the utilization of phenol and naphthalene enhances biosynthesis of several amino acids and that this mechanism is controlled by a transcriptional regulator.

Effects of glyphosate and methidathion on the expression of the Dhb, Vtg, Arnt, CYP4 and CYP314 in Daphnia magna.

In this study, the expression of five stress responsive genes was quantified and analyzed using a semi-quantitative RT-PCR to study the changes in their expression in Daphnia magna after exposure to known pesticides, glyphosate and methidathion. Hemoglobin (Dhb), which was used to show the effect of the oxygen level in the aquatic system, was significantly expressed in D. magna after exposure to glyphosate and methidathion. Additionally, aryl hydrocarbon receptor nuclear translocator (Arnt), a gene related to the metabolism of aryl hydrocarbons, had lower expression levels in D. magna than within the control. CYP4, which was used among cytochrome P450s (CYPs) to show the effects on the fatty acid and steroids metabolisms, was down-regulated in D. magna exposed to glyphosate. However, methidathion affected the expression of CYP314, which was used to show effects of ecdysis, not CYP4 in D. magna. Therefore, glyphosate and methidathion probably caused physiological effects with different patterns in D. magna, especially metabolisms related to CYPs. On the other hand, only vitellogenin (Vtg), which was responsive to the estrogenic potency, did not show any differences in D. magna after exposure to glyphosate or methidathion.

Separation of D-psicose and D-fructose using simulated moving bed chromatography.

Simulated moving bed (SMB) processes have been widely used in the sugar industries with ion-exchange resin as a stationary phase. D-psicose, a rare monosaccharide known as a valuable pharmaceutical substrate, was synthesized by the enzymatic conversion from D-fructose. The SMB process was adopted to separate D-psicose from D-fructose. Before the SMB experiment, the reaction mixture including D-psicose and D-fructose was treated by a deashing process to remove contaminants, such as buffers, proteins, and other organic materials. Four columns packed with Dowex 50WX4-Ca2+ (200-400 mesh) ion-exchange resins were used in the four-zone SMB. Single-step frontal analysis was performed to estimate the isotherm parameters of each monosaccharide. The operating conditions of the SMB process were determined based on the Equilibrium Theory. According to the simulation of the SMB process, the purity and yield of extract product (D-psicose) achieved were 99.04 and 97.46%, respectively and those of raffinate product (D-fructose) were 99.06 and 99.53%, respectively. Under the optimized operating condition, complete separation (extract purity = 99.36%, raffinate purity = 99.67%) was achieved experimentally.