Antibiotic Resistance Genes Markers Research Articles

Optimized Antibiotic Resistance Genes Monitoring Scenarios Promote Sustainability of Urban Water Cycle.

Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

Elevated levels of anthropogenic antibiotic resistance gene marker, sul1, linked with extreme fecal contamination and poor water quality in wastewater-receiving ponds.

In several low- and middle-income countries, such as India, the rapid construction of toilets to combat open defecation has not been matched with adequate wastewater treatment, resulting in extreme fecal contamination of the receiving environments. The sewage-receiving surface water bodies, typically close to the residences, are a potential hotspot for disease transmission and antibiotic resistance. Water, soil, and sediment samples from seven wastewater-receiving ponds (WRPs) were analyzed for water quality, chlorophyll-a, fecal contamination (yccT for Escherichia coli), 16S rRNA gene copies, and anthropogenic antibiotic resistance gene markers-sul1 and intI1. These WRPs were contrasted with two ponds that did not directly receive sewage. The water quality in the WRPs was comparable to raw sewage (BOD: 210-380mg/L; COD: 350-630mg/L; total-N: 100-190mg/L; and total-P: 6-21mg/L), and the relative levels of the DNA marker of E. coli were very high (yccT: 0.1% to ∼100% of total bacterial count) indicating extreme fecal contamination. The relative levels of sul1 and intI1 were 1-3 orders of magnitude higher in WRPs (sul1: 0.32%-10% of total bacterial count; and intI1: 0.2%-5% of total bacterial count) compared to the ponds that did not receive sewage directly. The relative levels of sul1 correlated with the DNA marker for the fecal indicator, E. coli (p-value<0.05; r=0.50; Spearman's rank correlation), and poor water quality.

Metagenomic Profiling of Pathogens in Simulated Reclaimed Water Distribution Systems Operated at Elevated Temperature Reveals Distinct Response of Mycobacteria to Filtration and Disinfection Conditions

Distribution of reclaimed water presents distinct challenges for pathogen control relative to the distribution of conventional potable water. The elevated temperatures common in arid regions can exacerbate such challenges by accelerating disinfectant decay and increasing microbial growth rates. Here, we carried out a controlled, continuous-flow, head-to-head study of six simulated reclaimed water distribution systems (RWDSs) in order to assess whether treatments identified to be effective for microbiological control at a lower temperature (22 °C) were also effective at 30 °C. Shotgun metagenomic sequencing was carried out to comprehensively profile fecal pathogens (FPs) (e.g., Escherichia and Klebsiella spp.), non-FPs (non-FPs) (e.g., Legionella and Mycobacterium spp.), and antibiotic-resistance genes (ARGs). Consistent with prior studies, generally, all FP, non-FP, and ARG markers were lowest when the influent water was biologically active carbon (BAC) filtered and disinfected. However, in contrast to observations at the lower temperature, where BAC filtration combined with chlorine disinfection effectively controlled Mycobacterium spp., they were markedly elevated in BAC-filtered and chlorine- or chloramine-disinfected conditions. This study highlights the need for further research to improve the understanding of conditions that select for different mycobacterial species and identify strategies to effectively control them in RWDSs under elevated temperature conditions.

High-sugar, high-fat, and high-protein diets promote antibiotic resistance gene spreading in the mouse intestinal microbiota

ABSTRACT Diet can not only provide nutrition for intestinal microbiota, it can also remodel them. However, is unclear whether and how diet affects the spread of antibiotic resistance genes (ARGs) in the intestinal microbiota. Therefore, we employed selected high-sugar, high-fat, high-protein, and normal diets to explore the effect. The results showed that high-sugar, high-fat, and high-protein diets promoted the amplification and transfer of exogenous ARGs among intestinal microbiota, and up-regulated the expression of trfAp and trbBp while significantly altered the intestinal microbiota and its metabolites. Inflammation-related products were strongly correlated with the spread of ARGs, suggesting the intestinal microenvironment after diet remodeling might be conducive to the spreading of ARGs. This may be attributed to changes in bacterial membrane permeability, the SOS response, and bacterial composition and diversity caused by diet-induced inflammation. In addition, acceptor bacteria (zygotes) screened by flow cytometry were mostly Proteobacteria, Firmicutes and Actinobacteria, and most were derived from dominant intestinal bacteria remodeled by diet, indicating that the transfer of ARGs was closely linked to diet, and had some selectivity. Metagenomic results showed that the gut resistance genome could be affected not only by diet, but by exogenous antibiotic resistant bacteria (ARB). Many ARG markers coincided with bacterial markers in diet groups. Therefore, dominant bacteria in different diets are important hosts of ARGs in specific dietary environments, but the many pathogenic bacteria present may cause serious harm to human health.

Molecular Characterization of Staphylococcus aureus Isolated from Human and Food Samples in Northern Algeria.

Staphylococcus aureus is a commensal resident of the skin and nasal cavities of humans and can cause various infections. Some toxigenic strains can contaminate food matrices and cause foodborne intoxications. The present study aimed to provide relevant information (clonal complex lineages, agr types, virulence and antimicrobial resistance-associated genes) based on DNA microarray analyses as well as the origins and dissemination of several circulating clones of 60 Staphylococcus aureus isolated from food matrices (n = 24), clinical samples (n = 20), and nasal carriers (n = 16) in northern Algeria. Staphylococcus aureus were genotyped into 14 different clonal complexes. Out of 60 S. aureus, 13 and 10 isolates belonged to CC1-MSSA and CC97-MSSA, respectively. The CC 80-MRSA-IV was the predominant S. aureus strain in clinical isolates. The accessory gene regulator allele agr group III was mainly found among clinical isolates (70.4%). Panton–Valentine leukocidin genes lukF/lukS-PV were detected in 13.3% of isolates that all belonged to CC80-MRSA. The lukF/S-hlg, hlgA, and hla genes encoding for hemolysins and leucocidin components were detected in all Staphylococcus aureus isolates. Clinical and food isolates harbored more often the antibiotic resistance genes markers. Seventeen (28.3%) methicillin-resistant Staphylococcus aureus carrying the mecA gene localized on a SCCmec type IV element were identified. The penicillinase operon (blaZ/I/R) was found in 71.7% (43/60) of isolates. Food isolates belonging to CC97-MSSA carried several antibiotic resistance genes (blaZ, ermB, aphA3, sat, tetM, and tetK). The results of this study showed that all clones were found in their typical host, but interestingly, some nasal carriers had isolates assigned to CC705 thought to be absent in humans. The detection of MRSA strains among food isolates should be considered as a potential public health risk. Therefore, controlling the antibiotics prescription for a rational use in human and animal infections is mandatory.

Increase in anthropogenic antibiotic resistance markers in water supplied by an overhead tank based-water distribution system

The levels of anthropogenic antibiotic resistance gene markers increased in tap water supplied by an overhead tank-based water distribution system, which are popular in many low and middle-income countries.

Cross-comparison of methods for quantifying antibiotic resistance in agricultural soils amended with dairy manure and compost

Agricultural soils are often amended with livestock manure, making them a key reservoir of antibiotic resistance genes (ARGs). Given that soils are among the most microbially-diverse environments on the planet; effective characterization and quantification of the effects of manure-derived amendments on soil resistomes is a major challenge. This study examined the effects of dairy manure-derived amendments on agricultural soils via two strategies: quantification of anthropogenic ARG markers via qPCR and shotgun metagenomic resistome profiling; and these strategies were compared to a previously published antibiotic resistant fecal coliform dataset. Soil samples were collected throughout a 120 day complete block field experiment to compare the effects of amendment type on antibiotic resistance. Results of all three measurements were consistent with the hypothesis that the application of composted manure reduced antibiotic resistance in soil relative to the application of raw manure, although some differences were noted in comparing the patterns of the three measurements with time. Raw dairy manure-amended soils yielded high sul1 and tet(W) relative abundances on Day 0 (following amendment application), but significantly decreased to background levels by Day 67 (harvest) and Day 120 (study completion). Shotgun metagenomics similarly detected a decrease in the relative abundances of sulfonamide and tetracycline-associated ARGs over time in the raw manure- and compost-amended soils; however, these levels were significantly lower than those estimated by qPCR. Interestingly, although patterns of sulfonamide and tetracycline resistance among culturable fecal coliforms echoed those observed via qPCR and metagenomics; erythromycin resistant coliforms were directly recovered by culture in amended soils, but corresponding ARGs were not detected by qPCR or metagenomics. This study supports both composting and time restrictions as means of reducing the potential for antibiotic resistance in manure to spread via soil application. Results suggest some differences in finer conclusions drawn depending on which antibiotic resistance monitoring target is selected.

Propagation of antibiotic resistance genes in an industrial recirculating aquaculture system located at northern China

The increasing prevalence and spread of antibiotic resistance genes (ARGs) in intensive aquaculture environments are of great concern to food safety and public health. However, the level of ARGs and their potential propagation factors in an industrial recirculating aquaculture system (RAS) have not previously been comprehensive explored. In this study, the levels of 14 different ARG markers and 2 kinds of mobile genetic elements (MGEs) were investigated in a RAS (including water, fish, feces, pellet feed meal, and biofilm samples) located northern China. qnrA, qnrB, qnrS, qepA, aac(6′)-Ib, and floR were dominant ARGs, which average concentration levels were presented at 4.51–7.74 copies/L and 5.36–13.07 copies/g, respectively, suggesting that ARGs were prevalent in RAS with no recorded history of antibiotic use. Elevated level of ARGs was found in water of RAS even after the final UV treatment compared with its influent. In RAS, Proteobacteria, Verrucomicrobia, Bacteroidetes, and Planctomycetes were the predominant phyla. Notably, elevated levels of potential opportunistic pathogens were observed along with abundant ARGs suggesting an increasing risk of capturing ARGs and MGEs for human pathogens. This study has revealed for the first time that reared fish, their feces, pellet feed meal as the introduction sources and the selection roles of treatment units co-driven the ARG profile, and the co-selection of water environmental factors and their consequently induced bacterial community shifts formed by their influence are the determining drivers for the ARG propagation in RAS.

Removal of antibiotic resistance genes in two tertiary level municipal wastewater treatment plants

Raw wastewater can contain high levels of antibiotic resistance genes (ARGs), making municipal wastewater treatment plants (WWTPs) critical for the control of the release of ARGs into the environment. The objective of this study was to investigate how individual treatment steps in two tertiary WWTPs affected the removal (copies/mL) and relative abundance of ARGs (copies/copies 16S rRNA genes). Nine ARG markers, representing resistance to commonly used antibiotics, as well as one integron gene (intl1) to assess ARG mobility potential, were quantified using quantitative real-time PCR (qPCR). Both WWTPs met provincial effluent regulations for removal of carbonaceous oxygen demand (CBOD5) and total suspended solids. Eight of the ten ARG markers (intl1, sul1, sul2, tet(O), ermB, blaCTX-M, blaTEM, qnrS) were detected in all samples. In contrast, mecA was detected intermittently and vanA remained below the detection limit in all samples. The total ARG marker abundances decreased by log 1.77 (p < 0.05) in the plant using an aerated lagoon (AL), and by 2.69 logs (p < 0.05) through treatment in the plant employing a biological nutrient removal (BNR) system. The BNR and secondary clarifier steps in both plants afforded the most removal of ARGs. The relative abundance of ARGs remained unchanged at the AL plant and showed a decreasing trend at the BNR plant. Levels of CBOD5, nitrate and the human Bacteroides fecal marker correlated with ARG concentrations, suggesting these variables may be useful in predicting ARG removal. In conclusion, the effluent coming from the WWTPs contained eight of the studied ARG markers in concentrations ranging from 0.01 to 3.6 log copies/mL, indicating their release into the environment, however, the relative abundance of ARGs was not enriched during treatment in the two WWTPs.

Antibiotic Susceptibility of Non-Cholera Vibrios Isolated from Farmed and Wild Marine Fish (Argyrosomus japonicus), Implications for Public Health.

This study aimed to evaluate the antibiogram and antibiotic resistance genes (ARGs) of Vibrio isolates recovered from a marine fish (Argyrosomus japonicus) and water samples from two commercial dusky kob aquaculture farms and the Kariega estuary, South Africa, and to evaluate these findings for their public health implications. A total of 277 molecularly confirmed Vibrio isolates consisting of 126 Vibrio fluvialis, 45 Vibrio vulnificus, 30 Vibrio Parahaemolyticus, and 76 vibrios belonging to species of the genus other than Vibrio cholerae were subjected to susceptibility testing to 15 antibiotics by the disc diffusion method. Multiple antibiotic resistance index (MARI) was used to determine the antibiotic resistance-associated health risk, while polymerase chain reaction was used to evaluate the presence of 14 ARGs for nonsusceptible strains. Highest resistances were recorded to amoxicillin (76.2%), ampicillin (67.5%), erythromycin (38.3%), and doxycycline (35.0%), while susceptibilities were highest to gentamicin (100%), followed by norfloxacin (97.8%), florfenicol (90.3%), tetracycline (87.7%), and chloramphenicol (87.4%). We recorded a 58.5% multidrug resistance (resistance to ≥2 antimicrobial classes). MARI did not vary significantly between sites (p > 0.05); however, values of >0.2 were recorded in 40% (108/277) of all strains tested. ARG markers, ampC, blaOXA, tetA, tetM, dfr1, sul1, sul2, ermB, nptII, strA, and SXT integrase, were detected in one or more strains with ermB (82.5%), sul2 (53.8%), strA (44%), dfr1 (42.3%), and tetM (38.3%) being the most abundant. Healthy marine finfish (dusky kob) and their environment can serve as reservoirs for antibiotic resistant vibrios and ARGs, which could be disseminated to humans and other susceptible bacteria and this therefore becomes a public health concern.

Antibiotic resistance genes in municipal wastewater treatment systems and receiving waters in Arctic Canada

Domestic wastewater discharges may adversely impact arctic ecosystems and local indigenous people, who rely on being able to hunt and harvest food from their local environment. Therefore, there is a need to develop efficient wastewater treatment plants (WWTPs), which can be operated in remote communities under extreme climatic conditions. WWTPs have been identified as reservoirs of antibiotic resistance genes (ARGs). The objective of this work was to quantify the presence of nine different ARG markers (int1, sul1, sul2, tet(O), erm(B), mecA, blaCTX-M, blaTEM, and qnr(S)) in two passive systems (waste stabilization ponds [WSPs]) and one mechanical filtration plant operating in two smaller and one large community, respectively, in Nunavut, Canada. Measurement of water quality parameters (carbonaceous oxygen demand, ammonia, total suspended solids, Escherichia coli and total coliforms) showed that the WWTPs provided only primary treatment. Low levels of the ARGs (2logcopies/mL) were observed in the effluent, demonstrating that bacteria residing in three northern WWTPs harbour ARGs conferring resistance to multiple clinically-relevant classes of antibiotics. Our results indicate that long-term storage in WSPs benefitted removal of organic material and some ARGs. However, one WSP system showed evidence of the enrichment of sul1, sul2, mecA, tet(O) and qnr(S). Further research is needed to fully understand if these ARG releases pose a risk to human health, especially in the context of traditional hunting and fishing activities.

Development of a Biosafety Enhanced and Immunogenic Salmonella Enteritidis Ghost Using an Antibiotic Resistance Gene Free Plasmid Carrying a Bacteriophage Lysis System

In the development of genetically inactivated bacterial vaccines, plasmid retention often requires the antibiotic resistance gene markers, the presence of which can cause the potential biosafety hazards such as the horizontal spread of resistance genes. The new lysis plasmid was constructed by utilizing the approach of balanced-lethal systems based on auxotrophic gene Aspartate semialdehyde dehydrogenase (asd). The PhiX174 lysis gene E and λPR37-cI857 temperature-sensitive regulatory system was cloned in the asd gene positive plasmid and this novel approach allowed the production of antibiotic resistance marker free Salmonella Enteritidis (S. Enteritidis) ghost. The immunogenic potential of the biosafety enhanced antibiotic resistance gene free S. Enteritidis ghost was evaluated in chickens by employing the prime-boost vaccination strategy using a combination of oral and intramuscular routes. A total of 75 two-week-old chickens were equally divided into five groups: group A (non-immunized control), group B (intramuscularly primed and boosted), group C (primed intramuscularly and boosted orally), group D (primed and boosted orally), and group E (primed orally and boosted intramuscularly). Chickens from all immunized groups demonstrated significant increases in plasma IgG, intestinal secretory IgA levels, and antigen-specific lymphocyte proliferative response. After a virulent S. Enteritidis challenge, all immunized groups showed fewer gross lesions and decreased bacterial recovery from organs in comparison with the non-immunized control group. Among the immunized chickens, groups B and D chickens showed optimized protection, indicating that the prime-booster immunization with the ghost via intramuscular or oral route is efficient. Taken together, our results demonstrate that an antibiotic resistance gene free lysis plasmid was successfully constructed and utilized for production of safety enhanced S. Enteritidis ghost, which can be used as a safe and effective vaccine against virulent S. Enteritidis infections.

Construction of an engineered strain free of antibiotic resistance gene markers for simultaneous mineralization of methyl parathion and ortho-nitrophenol

Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP-0.1 mM ONP simultaneously. This method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental pollutants.

Comparative analysis of antibiotic resistance gene markers in Mycoplasma genitalium: application to studies of the minimal gene complement

Mycoplasma genitalium has been proposed as a suitable model for an in-depth understanding of the biology of a free-living organism. This paper reports that the expression of the aminoglycoside resistance gene aac(6')-aph(2''), the only selectable marker hitherto available for M. genitalium genetic studies, correlates with a growth impairment of the resistant strains. In light of this finding, a tetM438 construction based on the tetracycline resistance gene tetM was developed; it can be used efficiently in M. genitalium and confers multiple advantages when compared to aac(6')-aph(2''). The use of tetM438 significantly improves transformation efficiency and generates visible colonies faster. Finally, the improvements in the pMTnTetM438 construction made it possible to obtain insertions in genes which have not been previously considered to be dispensable under laboratory growth conditions.

A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains.

A TnpI-mediated site-specific recombination system to construct genetically modified Bacillus thuringiensis strains was developed. Recombinant B. thuringiensis strains from which antibiotic resistance genes can be selectively eliminated were obtained in vivo with a new vector based on the specific resolution site of transposon Tn4430. For example, a cryIC gene, whose product is active against Spodoptera littoralis, was introduced into B. thuringiensis Kto harboring a cryIA(c) gene active against Ostrinia nubilalis. The resulting strain had a broader activity spectrum than that of the parental strain. It contained only B. thuringiensis DNA and was free of antibiotic resistance genes. This should facilitate regulatory approval for its development as a commercial biopesticide.