Fate of Extracellular DNA in the Production of Fertilizers from Source-Separated Urine. - Related Documents




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778501.0000Fate of Extracellular DNA in the Production of Fertilizers from Source-Separated Urine. The practice of urine source-separation for fertilizer production necessitates an understanding of the presence and impact of extracellular DNA in the urine. This study examines the fate of plasmid DNA carrying ampicillin and tetracycline resistance genes in aged urine, including its ability to be taken up and expressed by competent bacteria. Plasmid DNA incubated in aged urine resulted in a >2 log loss of bacterial transformation efficiency in Acinetobacter baylyi within 24 h. The concentration of ampicillin and tetracycline resistance genes, as measured with quantitative polymerase chain reaction, did not correspond with the observed transformation loss. When the plasmid DNA was incubated in aged urine that had been filtered (0.22 μm) or heated (75 °C), the transformation efficiencies were more stable than when the plasmids were incubated in unfiltered and unheated aged urine. Gel electrophoresis results indicated that plasmid linearization by materials larger than 100 kDa in the aged urine caused the observed transformation efficiency decreases. The results of this study suggest that extracellular DNA released into aged urine poses a low potential for the spread of antibiotic resistance genes to bacteria once it is released to the environment.202031965791
778410.9998No evidential correlation between veterinary antibiotic degradation ability and resistance genes in microorganisms during the biodegradation of doxycycline. Biodegradation of antibiotic residues in the environment by microorganisms may lead to the generation of antibiotic resistance genes (ARGs), which are of great concern to human health. The aim of this study was to determine whether there is a relationship between the ability to degrade antibiotic doxycycline (DOX) and the development of resistance genes in microorganisms. We isolated and identified ten bacterial strains from a vegetable field that had received long-term manure application as fertilizer and were capable of surviving in a series of DOX concentrations (25, 50, 80, and 100mg/L). Our results showed no evidential correlation between DOX degradation ability and the development of resistance genes among the isolated microorganisms that had high DOX degradation capability (P > 0.05). This was based on the fact that Escherichia sp. and Candida sp. were the most efficient bacterial strains to degrade DOX (92.52% and 91.63%, respectively), but their tetracycline resistance genes showed a relatively low risk of antibiotic resistance in a 7-day experiment. Moreover, the tetM of the ribosomal protection protein genes carried by these two preponderant bacteria was five-fold higher than that carried by other isolates (P < 0.05). Pearson correlations between the C(t)/C(0) of DOX and tet resistance genes of three isolates, except for Escherichia sp. and Candida sp., showed remarkable negative correlations (P < 0.05), mainly because tetG markedly increased during the DOX degradation process. Our results concluded that the biodegradation of antibiotic residues may not necessarily lead to the development of ARGs in the environment. In addition, the two bacteria that we isolated, namely, Escherichia sp. and Candida sp., are potential candidates for the engineering of environmentally friendly bacteria.201828942279
353120.9998Commensal E. coli rapidly transfer antibiotic resistance genes to human intestinal microbiota in the Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). Food-producing animals are indicated as a reservoir of antibiotic resistance genes and a potential vector for transmission of plasmid-encoded antibiotic resistance genes by conjugation to the human intestinal microbiota. In this study, transfer of an antibiotic resistance plasmid from a commensal E. coli originating from a broiler chicken towards the human intestinal microbiota was assessed by using a Mucosal Simulator of the Human Intestinal Microbial Ecosystem (M-SHIME). This in vitro model mimics the human intestinal ecosystem and received a single dose of 10(9)E. coli MB6212, which harbors a plasmid known to confer resistance towards several antibiotics including tetracycline, sulfamethoxazole and cefotaxime. Since the degree of stress imposed by stomach pH and bile acids vary with the consumed meal size, the effect of meal size on E. coli donor survival and on plasmid transfer towards lumen and mucosal coliforms and anaerobes was determined. The administered commensal E. coli strain survived stomach acid and bile salt stress and was able to grow in the colon environment during the timeframe of the experiment (72 h). Transfer of antibiotic resistance was observed rapidly since cultivable transconjugant coliforms and anaerobes were already detected in the lumen and mucosa after 2 h in the simulated proximal colon. The presence of the resistance plasmid in the transconjugants was confirmed by PCR. Differences in meal size and adapted digestion had neither a detectable impact on antibiotic resistance transfer, nor on the survival of the E. coli donor strain, nor on short chain fatty acid profiles. The median number of resistant indigenous coliforms in the lumen of the inoculated colon vessels was 5.00 × 10(5) cfu/ml [min - max: 3.47 × 10(4)-3.70 × 10(8) cfu/ml], and on the mucosa 1.44 × 10(7) cfu/g [min-max: 4.00 × 10(3)-4.00 × 10(8) cfu/g]. Exact quantification of the anaerobic transconjugants was difficult, as (intrinsic) resistant anaerobic background microbiota were present. QPCR data supported the observation of plasmid transfer in the simulated colon. Moreover, inoculation of E. coli MB6212 had no significant impact on the microbial diversity in the lumen as determined by 16 S ribosomal gene based next generation sequencing on lumen samples. This study demonstrates that a commensal, antibiotic resistant E. coli strain present in food can transfer its antibiotic resistance plasmid relatively quickly to intestinal microbiota in the M-SHIME. The spread and persistence of antibiotic resistance genes and resistant bacteria in our intestinal system is an alarming scenario which might present clinical challenges, since it implies a potential reservoir for dissemination to pathogenic bacteria.201931536878
342130.9998Survival of antibiotic resistant bacteria following artificial solar radiation of secondary wastewater effluent. Urban wastewater treatment plant effluents represent one of the major emission sources of antibiotic-resistant bacteria (ARB) in natural aquatic environments. In this study, the effect of artificial solar radiation on total culturable heterotrophic bacteria and ARB (including amoxicillin-resistant, ciprofloxacin-resistant, rifampicin-resistant, sulfamethoxazole-resistant, and tetracycline-resistant bacteria) present in secondary effluent was investigated. Artificial solar radiation was effective in inactivating the majority of environmental bacteria, however, the proportion of strains with ciprofloxacin-resistance and rifampicin-resistance increased in the surviving populations. Isolates of Pseudomonas putida, Serratia marcescens, and Stenotrophomonas maltophilia nosocomial pathogens were identified as resistant to solar radiation and to at least three antibiotics. Draft genome sequencing and typing revealed isolates carrying multiple resistance genes; where S. maltophilia (resistant to all studied antibiotics) sequence type was similar to strains isolated in blood infections. Results from this study confirm that solar radiation reduces total bacterial load in secondary effluent, but may indirectly increase the relative abundance of ARB.201829898509
342240.9998Exploring the Role of Coliform Bacteria in Class 1 Integron Carriage and Biofilm Formation During Drinking Water Treatment. This study investigates the role of coliforms in the carriage of class 1 integron and biocide resistance genes in a drinking water treatment plant and explores the relationship between the carriage of such genes and the biofouling abilities of the strain. The high incidence of class 1 integron and biocide resistance genes (33.3 % of the isolates) highlights the inherent risk of genetic contamination posed by coliform populations during drinking water treatment. The association between the presence of intI1 gene and qac gene cassettes, especially qacH, was greater in biofilm cells. In coliforms recovered from biofilms, a higher frequency of class 1 integron elements and higher diversity of genetic patterns occurred, compared to planktonic cells. The coliform isolates under the study proved to mostly carry non-classical class 1 integrons lacking the typical qacEΔ1/sul1 genes or a complete tni module, but bearing the qacH gene. No link was found between the carriage of integron genes and the biofouling degree of the strain, neither in aerobic or in anaerobic conditions. Coliform bacteria isolated from established biofilms rather adhere in oxygen depleted environments, while the colonization ability of planktonic cells is not significantly affected by oxygen availability.201627079455
575750.9998The expression regulation of recA gene and bacterial class 2 integron-associated genes induced by antibiotics. OBJECTIVE: To investigate the effects and mechanisms of common antibiotics induction on the expression of class 2 integron integrase and variable region resistance genes in bacteria, as well as potential structural mutations. METHODS: Clinical isolates containing non-functional class 2 integrons and functional class 2 integrons were selected. Strains containing non-functional class 2 integrons or functional class 2 integrons were constructed using isolated DNA templates. These strains were subjected to continuous induction with drug concentrations of 1/2 MIC and 1/4 MIC (ciprofloxacin, ampicillin, and kanamycin) and a concentration of 0.2 μg/ml (mitomycin C) over 8 days. The relative expression levels of relevant genes were measured on days 1, 3, and 8. Drug resistance in the experimental strains was assessed before and after induction to identify any differences. Finally, the sequence of the non-functional class 2 integron integrase gene was analyzed for structural changes that occurred as a result of induction. RESULTS: All drugs selected in this study increased the relative expression levels of recA, intI2, dfrA1, sat2, and aadA1. Significant differences in inductive abilities were observed among the drugs. The 1/2 MIC concentrations were more effective than 1/4 MIC concentrations in increasing the relative expression levels of target genes and enhancing the resistance of the experimental strains. The relative expression levels of recA, intI2, and dfrA1 rose on day 1, peaked on day 3, and slightly declined by day 8. Induced strains exhibited increased resistance to the drugs, with the most significant changes observed in the clinical isolates, particularly concerning CIP resistance. Notably, clinical isolate 7b induced with 1/2 MIC KAN exhibited the loss of one base at position 12bp in the integrase sequence. However, none of the four drugs induced mutations at the 444 bp position of class 2 integrons. CONCLUSION: Sub-MIC concentrations of drugs have been shown to induce an increase in the relative expression level of the SOS response-related gene recA, as well as the integrase and resistance genes of class 2 integrons. Continuous induction leads to sustained upregulation of these genes, which stabilizes or slightly decreases upon reaching a plateau. However, the capacity of different drugs to induce expression varies significantly. Short-term antibiotic exposure did not result in critical mutations that convert class 2 integrons into functional forms.202540950603
528960.9998Examination of the Aerobic Microflora of Swine Feces and Stored Swine Manure. Understanding antibiotic resistance in agricultural ecosystems is critical for determining the effects of subtherapeutic and therapeutic uses of antibiotics for domestic animals. This study was conducted to ascertain the relative levels of antibiotic resistance in the aerobic bacterial population to tetracycline, tylosin, and erythromycin. Swine feces and manure samples were plated onto various agar media with and without antibiotics and incubated at 37°C. Colonies were counted daily. Randomly selected colonies were isolated and characterized by 16S rRNA sequence analyses and additional antibiotic resistance and biochemical analyses. Colonies were recovered at levels of 10 to 10 CFU mL for swine slurry and 10 to 10 CFU g swine feces, approximately 100-fold lower than numbers obtained under anaerobic conditions. Addition of antibiotics to the media resulted in counts that were 60 to 80% of those in control media without added antibiotics. Polymerase chain reaction analyses for antibiotic resistance genes demonstrated the presence of a number of different resistance genes from the isolates. The recoverable aerobic microflora of swine feces and manure contain high percentages of antibiotic-resistant bacteria, which include both known and novel genera and species, and a variety of antibiotic resistance genes. Further analyses of these and additional isolates should provide additional information on these organisms as potential reservoirs of antibiotic resistance genes in these ecosystems.201627065407
336870.9997Horizontal Transfer of Plasmid-Mediated Cephalosporin Resistance Genes in the Intestine of Houseflies (Musca domestica). Houseflies are a mechanical vector for various types of bacteria, including antimicrobial-resistant bacteria (ARB). If the intestine of houseflies is a suitable site for the transfer of antimicrobial resistance genes (ARGs), houseflies could also serve as a biological vector for ARB. To clarify whether cephalosporin resistance genes are transferred efficiently in the housefly intestine, we compared with conjugation experiments in vivo (in the intestine) and in vitro by using Escherichia coli with eight combinations of four donor and two recipient strains harboring plasmid-mediated cephalosporin resistance genes and chromosomal-encoded rifampicin resistance genes, respectively. In the in vivo conjugation experiment, houseflies ingested donor strains for 6 hr and then recipient strains for 3 hr, and 24 hr later, the houseflies were surface sterilized and analyzed. In vitro conjugation experiments were conducted using the broth-mating method. In 3/8 combinations, the in vitro transfer frequency (Transconjugants/Donor) was ≥1.3 × 10(-4); the in vivo transfer rates of cephalosporin resistance genes ranged from 2.0 × 10(-4) to 5.7 × 10(-5). Moreover, cephalosporin resistance genes were transferred to other species of enteric bacteria of houseflies such as Achromobacter sp. and Pseudomonas fluorescens. These results suggest that houseflies are not only a mechanical vector for ARB but also a biological vector for the occurrence of new ARB through the horizontal transfer of ARGs in their intestine.201626683492
780080.9997Effects of ultraviolet disinfection on antibiotic-resistant Escherichia coli from wastewater: inactivation, antibiotic resistance profiles and antibiotic resistance genes. AIMS: To evaluate the effect of ultraviolet (UV) disinfection on antibiotic-resistant Escherichia coli. METHODS AND RESULTS: Antibiotic-resistant E. coli strains were isolated from a wastewater treatment plant and subjected to UV disinfection. The effect of UV disinfection on the antibiotic resistance profiles and the antibiotic resistance genes (ARGs) of antibiotic-resistant E. coli was evaluated by a combination of antibiotic susceptibility analysis and molecular methods. Results indicated that multiple-antibiotic-resistant (MAR) E. coli were more resistant at low UV doses and required a higher UV dose (20 mJ cm(-2) ) to enter the tailing phase compared with those of antibiotic-sensitive E. coli (8 mJ cm(-2) ). UV disinfection caused a selective change in the inhibition zone diameters of surviving antibiotic-resistant E. coli and a slight damage to ARGs. The inhibition zone diameters of the strains resistant to antibiotics were more difficult to alter than those susceptible to antibiotics because of the existence and persistence of corresponding ARGs. CONCLUSIONS: The resistance of MAR bacteria to UV disinfection at low UV doses and the changes in inhibition zone diameters could potentially contribute to the selection of antibiotic-resistant bacteria in wastewater treatment after UV disinfection. The risk of spread of antibiotic resistance still exists owing to the persistence of ARGs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study highlights the acquisition of other methods to control the spread of ARGs.201728459506
343390.9997Effect of subinhibitory concentrations on the spreading of the ampicillin resistance gene bla(CMY-2) in an activated sludge microcosm. As the problem of multi-resistant bacteria grows a better understanding of the spread of antibiotic resistance genes is of utmost importance for society. Wastewater treatment plants contain subinhibitory concentrations of antibiotics and are thought to be hotspots for antibiotic resistance gene propagation. Here we evaluate the influence of sub-minimum inhibitory concentrations of antibiotics on the spread of resistance genes within the bacterial community in activated sludge laboratory-scale sequencing batch reactors. The mixed communities were fed two different ampicillin concentrations (500 and 5000 µg/L) and the reactors were run and monitored for 30 days. During the experiment the β-lactamase resistance gene bla(CMY-2) was monitored via qPCR and DNA samples were taken to monitor the effect of ampicillin on the microbial community. The relative copy number of bla(CMY-2) in the reactor fed with the sub-minimum inhibitory concentration of 500 µg/L ampicillin was spread out over a wider range of values than the control and 5000 µg/L ampicillin reactors indicating more variability of gene number in the 500 µg/L reactor. This result emphasises the problem of sub-minimum inhibitory concentrations of antibiotics in wastewater. High-throughput sequencing showed that continuous exposure to ampicillin caused a shift from a Bacteroidetes to Proteobacteria in the bacterial community. The combined use of qPCR and high-throughput sequencing showed that ampicillin stimulates the spread of resistance genes and leads to the propagation of microbial populations which are resistant to it.202539215485
6760100.9997Mechanism of antibiotic resistance spread during sub-lethal ozonation of antibiotic-resistant bacteria with different resistance targets. The increase and spread of antibiotic-resistant bacteria (ARB) in aquatic environments and the dissemination of antibiotic resistance genes (ARGs) greatly impact environmental and human health. It is necessary to understand the mechanism of action of ARB and ARGs to formulate measures to solve this problem. This study aimed to determine the mechanism of antibiotic resistance spread during sub-lethal ozonation of ARB with different antibiotic resistance targets, including proteins, cell walls, and cell membranes. ARB conjugation and transformation frequencies increased after exposure to 0-1.0 mg/L ozone for 10 min. During sub-lethal ozonation, compared with control groups not stimulated by ozone, the conjugative transfer frequencies of E. coli DH5α (CTX), E. coli DH5α (MCR), and E. coli DH5α (GEN) increased by 1.35-2.02, 1.13-1.58, and 1.32-2.12 times, respectively; the transformation frequencies of E. coli DH5α (MCR) and E. coli DH5α (GEN) increased by 1.49-3.02 and 1.45-1.92 times, respectively. When target inhibitors were added, the conjugative transfer frequencies of antibiotics targeting cell wall and membrane synthesis decreased 0.59-0.75 and 0.43-0.76 times, respectively, while that for those targeting protein synthesis increased by 1-1.38 times. After inhibitor addition, the transformation frequencies of bacteria resistant to antibiotics targeting the cell membrane and proteins decreased by 0.76-0.89 and 0.69-0.78 times, respectively. Cell morphology, cell membrane permeability, reactive oxygen species, and antioxidant enzymes changed with different ozone concentrations. Expression of most genes related to regulating different antibiotic resistance targets was up-regulated when bacteria were exposed to sub-lethal ozonation, further confirming the target genes playing a crucial role in the inactivation of different target bacteria. These results will help guide the careful utilization of ozonation for bacterial inactivation, providing more detailed reference information for ozonation oxidation treatment of ARB and ARGs in aquatic environments.202438810347
4648110.9997Potential of phage cocktails in the inactivation of Enterobacter cloacae--An in vitro study in a buffer solution and in urine samples. The objective of this study was to compare the dynamics of three previously isolated phages for Enterobacter cloacae in order to evaluate their ability to treat urinary tract infections (UTI). The phages genomes, survival, host range, were characterized, and the host-phage dynamics was determined in culture medium and urine samples. The presence of prophages in bacteria, host recovery and development of resistance to phage after treatment was also evaluated. The growth of the E. cloacae was inhibited by the three phages, resulting in a decrease of ≈3 log. The use of cocktails with two or three phages was significantly more effective (decrease of ≈4 log). In urine, the inactivation was still effective (≈2 log). Both phages were considered safe to inactivate the bacteria (no integrase and toxin codifying genes). Some bacteria remained viable in the presence of the phages, but their colonies were smaller than those of the non-treated control and were visible only after 5 days of incubation (visible after 24h in the control). A high bacterial inactivation efficiency with phage cocktails combined with the safety of the phages and their long periods of survival, even in urine samples, paves the way for depth studies, especially in vivo studies, to control urinary tract infection and to overcome the development of resistances by the nosocomial bacterium E. cloacae.201626541317
3425120.9997Horizontal transfer of antibiotic resistance genes among gram negative bacteria in sewage and lake water and influence of some physico-chemical parameters of water on conjugation process. Transfer of antibiotic resistance genes among gram negative bacteria in sewage and lake water and easy access of these bacteria to the community are major environmental and public health concern. The aim of this study was to determine transfer of the antimicrobial resistance genes from resistant to susceptible gram negative bacteria in the sewage and lake water by conjugation process and to determine the influence of some physico-chemical parameters of sewage and lake water on the transfer of these resistance genes. For this reason, we isolated 20 liter of each sewage and lake water from coconut area within university campus and Lingambudi lake respectively in Mysore city, India, during monsoon season and studied different physical parameters of the water samples like pH, temperature, conductivity turbidity and color as well as chemical parameters like BOD, COD, field DO and total chloride ion. The gram negative bacteria were isolated and identified from the above water samples using microbiological and biochemical methods and their sensitivity to different antibiotics was determined by disc diffusion break point assay. Conjugation between two multiple antibiotic resistant isolates Pseudomonas aeuginosa and E. coli as donor and E. coli Rif(r) (sensitive to antibiotics) as recipient were carried out in 5ml sterile sewage and lake water. All isolates were resistant to Am, moderately resistant to Te and E, while majority were sensitive to Cip, Gm and CAZ antibiotics. Horizontal transfer of antibiotic resistance genes by conjugation process revealed transfer of Gm, Te and E resistant genes from Ps. aeruginosa to E. coli Rif(r) recipient with mean frequency of +/- 2.3 x 10(-4) in sewage and +/- 2.6 x 10(-6) in lake water respectively Frequency of conjugation in sewage was two fold more as compared to lake water (p< or =0.05). Co- transfer study revealed simultaneous transfer of above resistant markers together to the recipient cells. As the above results indicate, due to selective pressure in sewage (presence of antibiotics), the isolates from sewage were more resistant to different antibiotics as compared to those from lake water. Furthermore, these resistance genes can transfer to sensitive bacteria by conjugation. Physico-chemical parameters of water may play role in this process.200920112862
3520130.9997Influence of tetracycline on tetracycline-resistant heterotrophs and tet genes in activated sludge process. The concentrations of tetracycline-intermediate resistant, tetracycline-resistant heterotrophic bacteria, and total heterotrophic bacteria were examined to assess the influence of tetracycline on tetracycline-resistant heterotrophs by the R2A agar cultivation method in the tetracycline fortified activated sludge process and in the natural background. Results showed that the percentages of both tetracycline-intermediate resistant and tetracycline-resistant heterotrophic bacteria in total heterotrophic bacteria were significantly increased, after tetracycline was fed to activated sludge for a 3 months period under four different operating conditions, as compared with the background. In order to investigate the mechanism of activated sludge resistance to tetracycline, polymerase chain reaction experiments were carried out to analyze the existence and evolution of tet genes in the presence of tetracycline. Results revealed that only tet A and tet B genes out of the 11 target tet genes were observed in tetracycline treated activated sludge while no tet gene was detected in background. This indicated that tet A gene could accumulate in activated sludge with slower and continuous influent, while the accumulation of tet B gene could be attributed to shorter hydraulic retention time. Therefore, it was proposed in this study that tetracycline-resistant genes created by efflux pumps spread earlier and quicker to encode resistance to tetracycline, which facilitated the increase in tetracycline-resistance.201525424345
3532140.9997Transfer of Antibiotic Resistance Plasmid from Commensal E. coli Towards Human Intestinal Microbiota in the M-SHIME: Effect of E. coli dosis, Human Individual and Antibiotic Use. Along with (in) direct contact with animals and a contaminated environment, humans are exposed to antibiotic-resistant bacteria by consumption of food. The implications of ingesting antibiotic-resistant commensal bacteria are unknown, as dose-response data on resistance transfer and spreading in our gut is lacking. In this study, transfer of a resistance plasmid (IncF), harbouring several antibiotic resistance genes, from a commensal E. coli strain towards human intestinal microbiota was assessed using a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME). More specifically, the effect of the initial E. coli plasmid donor concentration (10(5) and 10(7) CFU/meal), antibiotic treatment (cefotaxime) and human individual (n = 6) on plasmid transfer towards lumen coliforms and anaerobes was determined. Transfer of the resistance plasmid to luminal coliforms and anaerobes was observed shortly after the donor strain arrived in the colon and was independent of the ingested dose. Transfer occurred in all six simulated colons and despite their unique microbial community composition, no differences could be detected in antibiotic resistance transfer rates between the simulated human colons. After 72 h, resistant coliform transconjugants levels ranged from 7.6 × 10(4) to 7.9 × 10(6) CFU(cefotaxime resistant)/Ml colon lumen. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime treatment led to a significant reduction (85%) in resistant coliforms, however no significant effect on the total number of cultivable coliforms and anaerobes was observed.202133670965
2801150.9997Principal component analysis exploring the association between antibiotic resistance and heavy metal tolerance of plasmid-bearing sewage wastewater bacteria of clinical relevance. This paper unravels the occurrence of plasmid-mediated antibiotic resistance in association with tolerance to heavy metals among clinically relevant bacteria isolated from sewage wastewater. The bacteria isolated were identified following conventional phenotypic and/or molecular methods, and were subjected to multiple-antibiotic resistance (MAR) profiling. The isolates were tested against the heavy metals Hg(2+), Cd(2+), Cr(2+) and Cu(2+). SDS-PAGE and agarose gel electrophoretic analyses were performed, respectively, for the characterization of heavy metal stress protein and R-plasmid among the isolated bacteria. Principal component analysis was applied in determining bacterial resistance to antibiotics and heavy metals. Both lactose-fermenting ( Escherichia coli ) and non-fermenting ( Acinetobacter baumannii and Pseudomonas putida ) Gram-negative bacterial strains were procured, and showed MAR phenotypes with respect to three or more antibiotics, along with resistance to the heavy metals Hg(2+), Cd(2+), Cr(2+) and Cu(2+). The Gram-positive bacteria, Enterococcus faecalis , isolated had 'ampicillin-kanamycin-nalidixic acid' resistance. The bacterial isolates had MAR indices of 0.3-0.9, indicating their ( E. faecalis , E. coli , A. baumannii and P. putida ) origin from niches with high antibiotic pollution and human faecal contamination. The Gram-negative bacteria isolated contained a single plasmid (≈54 kb) conferring multiple antibiotic resistance, which was linked to heavy metal tolerance; the SDS-PAGE analysis demonstrated the expression of heavy metal stress proteins (≈59 and ≈10 kDa) in wastewater bacteria with a Cd(2+) stressor. The study results grant an insight into the co-occurrence of antibiotic resistance and heavy metal tolerance among clinically relevant bacteria in sewage wastewater, prompting an intense health impact over antibiotic usage.202032974572
3391160.9997Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline-amended and ciprofloxacin-amended growth media. AIMS: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. METHODS AND RESULTS: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline- (n=164) and ciprofloxacin-amended (n=65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin-amended growth media (62%) compared to the bacteria isolated on tetracycline-amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline-resistant bacteria and almost half of the ciprofloxacin-resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline-resistant bacteria was capable of lateral gene transfer. CONCLUSIONS: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline-resistant and ciprofloxacin-resistant bacteria in municipal wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.201020629799
3844170.9997Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures. Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h(-1) versus 0.15 h(-1)) led to higher conjugation frequencies (10(-4) versus 10(-6) transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.202236094214
7782180.9997Degradation of Extracellular Antibiotic Resistance Genes with UV(254) Treatment. Disinfected wastewater effluent contains a complex mixture of biomolecules including DNA. If intact genes conveying antibiotic resistance survive the disinfection process, environmental bacteria may take them up. We treated plasmid pWH1266, which contains ampicillin resistance gene bla(TEM-1) and tetracycline resistance gene tetA, with UV(254) doses up to 430 mJ/cm(2) and studied the ability of those genes to be acquired by Acinetobacter baylyi. The plasmids required approximately 20-25 mJ/cm(2) per log(10) loss of transformation efficiency. We monitored plasmid DNA degradation using gel electrophoresis and qPCR with both short amplicons (∼200 bps, representative of ARG amplicon lengths commonly used for environmental monitoring) and long amplicons (800-1200 bps, designed to cover the entire resistance genes). The rate of transformability loss due to UV(254) treatment was approximately 20× and 2× larger than the rate of gene degradation measured with the short and long amplicons qPCR, respectively. When extrapolated to account for the length of the entire pWH1266 plasmid, the qPCR rate constants were 2-7× larger than the rate constants measured with transformation assays. Gel electrophoresis results confirmed that DNA cleavage was not a major inactivating mechanism. Overall, our results demonstrate that qPCR conservatively measures the potential for a gene to be transformed by environmental bacteria following UV(254) treatment.201728475324
3423190.9997bla(TEM) and vanA as indicator genes of antibiotic resistance contamination in a hospital-urban wastewater treatment plant system. Four indicator genes were monitored by quantitative PCR in hospital effluent (HE) and in the raw and treated wastewater of the municipal wastewater treatment plant receiving the hospital discharge. The indicator genes were the class 1 integrase gene intI1, to assess the capacity of bacteria to be involved in horizontal gene transfer processes; bla(TEM), one of the most widespread antibiotic resistance genes in the environment, associated with Enterobacteriaceae; vanA, an antibiotic resistance gene uncommon in the environment and frequent in clinical isolates; and marA, part of a locus related to the stress response in Enterobacteriaceae. Variation in the abundance of these genes was analysed as a function of the type of water, and possible correlations with cultivable bacteria, antimicrobial residue concentrations, and bacterial community composition and structure were analysed. HE was confirmed as an important source of bla(TEM) and vanA genes, and wastewater treatment showed a limited capacity to remove these resistance genes. The genes bla(TEM) and vanA presented the strongest correlations with culturable bacteria, antimicrobial residues and some bacterial populations, representing interesting candidates as indicator genes to monitor resistance in environmental samples. The intI1 gene was the most abundant in all samples, demonstrating that wastewater bacterial populations hold a high potential for gene acquisition.201427873693