Co-selection Of Antibiotic Resistance Genes Research Articles

Emergence and spread patterns of antibiotic resistance genes during cultivating aerobic granular sludge under different feeding modes

Aerobic granular sludge (AGS) technology, which is a promising approach for wastewater treatment, encounters challenges associated with antibiotic resistance genes (ARGs) in biological treatment systems. This study investigates the dynamics of ARGs during AGS cultivation under anaerobic plug-flow (APM), anaerobic mixed-flow (AMM), and aerobic feeding mode (AFM). Significant differences (P < 0.05) are observed in the shifts of ARGs among these modes. The abundance of ARGs notably increases under all modes, with AMM exhibiting a lower average relative abundance (0.063 ± 0.025) compared to that of APM (0.148 ± 0.061) and AFM (0.147 ± 0.047). The observed variations in bacterial communities and the co-selection of ARGs among the feeding modes are likely influential factors contributing to the documented variations of ARGs. Furthermore, the study identifies tnpA and intl1 as key contributors to horizontal gene transfer (HGT) during APM and AFM operations. Additionally, a heightened level of HGT is observed in both APM and AFM, with a larger potential host range (12 hosts each) harboring ARGs, in contrast to AMM (4 hosts). The study highlights the pivotal role played by distinct bacterial communities and the co-selection of ARGs in ARG variations across feeding modes. AMM emerges as an effective strategy for mitigating ARG proliferation, thereby enhancing the safety and sustainability of AGS technology in wastewater treatment.

Co-localization of clinically relevant antibiotic- and heavy metal resistance genes on plasmids in Klebsiella pneumoniae from marine bivalves.

Klebsiella pneumoniae is an opportunistic pathogen frequently associated with antibiotic resistance and present in a wide range of environments, including marine habitats. However, little is known about the development, persistence, and spread of antibiotic resistance in such environments. This study aimed to obtain the complete genome sequences of antibiotic-resistant K. pneumoniae isolated from marine bivalves in order to determine the genetic context of antibiotic- and heavy metal resistance genes in these isolates. Five antibiotic-resistant K. pneumoniae isolates, of which four also carried heavy metal resistance genes, were selected for complete genome sequencing using the Illumina MiSeq platform and the Oxford Nanopore Technologies GridION device. Conjugation experiments were conducted to examine the transfer potential of selected plasmids. The average length of the complete genomes was 5.48 Mbp with a mean chromosome size of 5.27 Mbp. Seven plasmids were detected in the antibiotic-resistant isolates. Three IncFIB, one IncFIB/IncFII, and one IncFIB/IncHIB plasmid, respectively, carried antibiotic resistance genes such as qnrS1, aph(6)-Id and aph(3')-Ia, aadA1, and aadA2. Four of these plasmids also carried genes encoding resistance to copper (pco), silver (sil), and arsenic (ars). One plasmid carrying tet(D) and blaSHV-1 as well as pco, sil, and ars genes was transferred to Escherichiacoli by conjugation. We show the co-occurrence of antibiotic- and heavy metal resistance genes on a conjugative IncFIB plasmid from K. pneumoniae from marine bivalves. Our study highlights the importance of the marine environment and seafood as a possible dissemination route for antimicrobial resistance and provides insights into the potential for co-selection of antibiotic resistance genes by heavy metals.

Limited impacts of high doses of dietary copper on the gut bacterial metal resistome explain negligible co-selection of antibiotic resistance

High dietary intake of Cu has previously been linked to the selection of Cu resistance and co-selection of antibiotic resistance in specific gut bacteria. Based on a novel HT-qPCR metal resistance gene chip as combined with 16S rRNA gene amplicon sequencing and phenotypic resistance typing of Escherichia coli isolates, we here report the impacts of two contrasting Cu-based feed additives on the swine gut bacterial metal resistome and community assembly. DNA was extracted from fecal samples (n = 80) collected at day 26 and 116 of the experiment from 200 pigs allotted to five dietary treatments: negative control (NC) diet with 20 μg CuSO4 g−1 and four diets added 125 or 250 μg CuSO4 g−1 feed or 125 or 250 μg Cu2O g−1 feed to the NC diet. Dietary Cu supplementation reduced the relative abundance of Lactobacillus, but it had negligible impacts on bacterial community composition relative to the gut microbiome maturation effect (time). The relative importance of different bacterial community assembly processes was not markedly affected by the dietary Cu treatments, and differences in swine gut metal resistome composition could be explained primarily by differences in bacterial community composition rather than by dietary Cu treatments. High dietary Cu intake (250 μg Cu g−1) selected for phenotypic Cu resistance in E. coli isolates, but surprisingly it did not result in increased prevalence of the Cu resistance genes targeted by the HT-qPCR chip. In conclusion, the lacking impacts of dietary Cu on the gut bacterial metal resistome explain results from a previous study showing that even high therapeutic doses of dietary Cu did not cause co-selection of antibiotic resistance genes and mobile genetic elements known to harbor these genes.

Deciphering the interaction impacts between antiseptic benzethonium chloride and biofilm nitrification system: Performance, resistance mechanisms and biodegradation

Benzethonium chloride (BEC) is one of emerging bacteriostatic agents. BEC-bearing wastewater generated during sanitary applications in food and medication is easily combined with other wastewater streams to flow into wastewater treatment plants. This study focused on the long-term (231 days) impacts of BEC on the sequencing moving bed biofilm nitrification system. Nitrification performance was tolerant to low concentration of BEC (≤ 0.2 mg/L), but the nitrite oxidation was severely inhibited when the concentration of BEC was 1.0–2.0 mg/L. Partial nitrification maintained about 140 days with nitrite accumulation ratio over 80%, mainly caused by the inhibition of Nitrospira, Nitrotoga and Comammox. Notably, BEC exposure in the system might cause the co-selection of antibiotic resistance genes (ARGs) and disinfectant resistance genes (DRGs), and the resistance of biofilm system to BEC was strengthened by efflux pumps mechanism (qacEdelta1 and qacH) and antibiotic deactivation mechanism (aadA, aac(6′)-Ib and blaTEM). Extracellular polymeric substances secretion and BEC biodegradation were also contributed to the system microorganisms resisting BEC exposure. In addition, Klebsiella, Enterobacter, Citrobacter and Pseudomonas were isolated and identified as BEC degrading bacteria. The metabolites of N,N-dimethylbenzylamine, N-benzylmethylamine and benzoic acid were identified, and the biodegradation pathway of BEC was proposed. This study brought new knowledge about the fate of BEC in biological treatment units and laid a foundation for its elimination from wastewater.

Exposure to benzalkonium chloride disinfectants promotes antibiotic resistance in sewage sludge microbiomes

Disinfectants are routinely used in human environments to control and prevent the transmission of microbial disease, and this is particularly true during the current COVID-19 crisis. However, it remains unclear whether the increased disinfectant loadings to wastewater treatment plants facilitate the dissemination of antibiotic resistance genes (ARGs) in sewage sludge microbiomes. Here, we investigated the impacts of benzalkonium chlorides (BACs), widely used disinfectants, on ARGs profiles and microbial community structures in sewage sludge by using high-throughput quantitative PCR and Illumina sequencing. A total of 147 unique ARGs and 39 mobile genetic elements (MGEs) were detected in all sewage sludge samples. Our results show that exposure to BACs disinfectants at environmentally relevant concentrations significantly promotes both the diversity and absolute abundance of ARGs in sludge microbiomes, indicating the co-selection of ARGs by BACs disinfectants. The enrichment of ARGs abundance varied from 2.15-fold to 3.63-fold compared to controls. In addition, BACs exposure significantly alters bacterial and protistan communities, resulting in dysbiosis of the sludge microbiota. The Mantel test and Procrustes analysis confirm that bacterial communities are significantly correlated with ARGs profiles under BACs treatments. The structural equation model explains 83.8 % of the total ARGs variation and further illustrates that the absolute abundance of MGEs exerts greater impacts on the variation of absolute abundance of ARGs than microbial communities under BACs exposure, suggesting BACs may promote antibiotic resistance by enhancing the horizontal gene transfer of ARGs across sludge microbiomes. Collectively, our results provide new insights into the proliferation of antibiotic resistance through disinfectant usage during the pandemic and highlight the necessity to minimize the environmental release of disinfectants into the non-target environment for combating antibiotic resistance.

Heavy metals potentially drive co-selection of antibiotic resistance genes by shifting soil bacterial communities in paddy soils along the middle and lower Yangtze River

Heavy metals (HMs) and antibiotic resistance have become serious environmental problems affecting soil and human health. Soil microorganisms play key roles in pollutant degradation and biogeochemical cycling processes; however, the interactions among HMs, soil microbial communities, and antibiotic resistance genes (ARGs) in agricultural soils remain unclear. Using quantitative real-time polymerase chain reaction and NovaSeq sequencing, we evaluated heavy metal contents, abundances of ARGs, soil bacterial community structure and functions, and their correlations in paddy soils at 43 sampling sites along the middle and lower reaches of the Yangtze River, central and eastern China. Our results showed the co-occurrence of HMs, ARGs, and HM resistance genes across all paddy soils. Additionally, significant positive associations were detected between HMs and resistance genes. Cadmium, czcA, and int1 were positively correlated with bacterial community diversity. The Mantel test showed that bacterial community composition and functions were significantly associated with HMs and resistance genes, such as Cd, Cr, Zn, copA, czcA, int1, and sul1. Moreover, HMs and ARGs were the major factors shaping soil bacterial communities; thus, HMs triggered proliferation of HM and antibiotic resistances by influencing the mobile genetic element (int1) and soil microbial communities. Our study revealed that HMs potentially drive the co-selection of ARGs by shifting soil bacterial community structure and functions, thereby increasing the potential risks to human health as well as ecological environment in the paddy soils along the middle and lower reaches of the Yangtze River.

Differential impacts of salinity on antibiotic resistance genes during cattle manure stockpiling are linked to mobility potentials revealed by metagenomic sequencing

Livestock manure is an important source of antibiotic resistance genes (ARGs), and its salinity level can change during stockpiling. To understand how the salinity changes affect the fate of ARGs, cattle manure was adjusted of salinity and stockpiled in laboratory microcosms at low (0.3% salt), moderate (3.0%) and high salinity levels (10.0%) for 44 days. Amongst the five ARGs (tetO, blaTEM, sul1, tetM, and ermB) and the first-class integrase (intI1) monitored by qPCR, the relative abundance of tetO and blaTEM exhibited no clear trend in response to salinity levels, while that of sul1, tetM, ermB and intI1 showed clear downward trends over time at the lower salinity levels (0.3% and 3%) but not at the high salinity level (10%). Metagenomic contig construction of cattle manure samples revealed that sul1, tetM and ermB genes were more likely to associate with mobile genetic elements (MGEs) than tetO and blaTEM, suggesting that their slower decay at higher salinity levels was either caused by horizontal gene transfer or co-selection of ARGs and osmotic stress resistant determinants. Further analysis of metagenomic contigs showed that osmotic stress resistance can also be located on MGEs or in conjunction with ARGs.

Co-occurrence of genes for antibiotic resistance and arsenic biotransformation in paddy soils

Paddy soils are potential hotspots of combined contamination with arsenic (As) and antibiotics, which may induce co-selection of antibiotic resistance genes (ARGs) and As biotransformation genes (ABGs), resulting in dissemination of antimicrobial resistance and modification in As biogeochemical cycling. So far, little information is available for these co-selection processes and specific patterns between ABGs and ARGs in paddy soils. Here, the 16S rRNA amplicon sequencing and high-throughput quantitative PCR and network analysis were employed to investigate the dynamic response of ABGs and ARGs to As stress and manure application. The results showed that As stress increased the abundance of ARGs and mobile genetic elements (MGEs), resulting in dissemination risk of antimicrobial resistance. Manure amendment increased the abundance of ABGs, enhanced As mobilization and methylation in paddy soil, posing risk to food safety. The frequency of the co-occurrence between ABGs and ARGs, the host bacteria carrying both ARGs and ABGs were increased by As or manure treatment, and remarkably boosted in soils amended with both As and manure. Multidrug resistance genes were found to have the preference to be co-selected with ABGs, which was one of the dominant co-occurring ARGs in all treatments, and manure amendment increased the frequency of Macrolide-Lincosamide-Streptogramin B resistance (MLSB) to co-occur with ABGs. Bacillus and Clostridium of Firmicutes are the dominant host bacteria carrying both ABGs and ARGs in paddy soils. This study would extend our understanding on the co-selection between genes for antibiotics and metals, also unveil the hidden environmental effects of combined pollution.

Heavy Metals Drive Co-Selection of Antibiotic Resistance Genes by Shifting Soil Bacterial Communities in Paddy Soils Along Middle and Lower Yangtze River

Heavy Metals Drive Co-Selection of Antibiotic Resistance Genes by Shifting Soil Bacterial Communities in Paddy Soils Along Middle and Lower Yangtze River

Cadmium enhances conjugative plasmid transfer to a fresh water microbial community.

Co-selection of antibiotic resistance genes (ARGs) by heavy metals might facilitate the spread of ARGs in the environments. Cadmium contamination is ubiquitous, while, it remains unknown the extent to which cadmium (Cd2+) impact plasmid-mediated transfer of ARGs in aquatic bacterial communities. In the present study, we found that Cd2+ amendment at sub-inhibitory concentration significantly increased conjugation frequency of RP4 plasmid from Pseudomonas putida KT2442 to a fresh water microbial community by liquid mating method. Cd2+ treatment (1-100mg/L) significantly increased the cell membrane permeability and antioxidant activities of conjugation mixtures. Amendments of 10 and 100mg/L Cd2+ significantly enhanced the mRNA expression levels of mating pair formation gene (trbBp) and the DNA transfer and replication gene (trfAp) due to the repression of regulatory genes (korA, korB and trbA). Phylogenetic analysis of transconjugants indicated that Proteobacteria was the dominant recipients and high concentration of Cd2+ treatment resulted in expanded recipient taxa. This study suggested that sub-inhibitory Cd2+ contamination would facilitate plasmid conjugation and contributed to the maintenance and spread of plasmid associated ARGs, and highlighted the urgent need for effective remediation of Cd2+ in aquatic environments.

Co-selection of antibiotic resistance genes, and mobile genetic elements in the presence of heavy metals in poultry farm environments

Environmental selection of antibiotic resistance genes (ARGs) is considered to be caused by antibiotic or metal residues, frequently used in livestock. In this study we examined three commercial poultry farms to correlate the co-occurrence patterns of antibiotic and metal residues to the presence of ARGs. We quantified 283 ARGs, 12 mobile genetic elements (MGEs), 49 targeted antibiotics, 7 heavy metals and sequenced 16S rRNA genes. The abundance and type of ARG were significantly enriched in manure while soil harbored the most diverse bacterial community. Procrustes analysis displayed significant correlations between ARGs/MGEs and the microbiome. Cadmium (Cd), arsenic (As), zinc (Zn), copper (Cu) and lead (Pb) were responsible for a majority of positive correlations to ARGs when compared to antibiotics. Integrons and transposons co-occurred with ARGs corresponding to 9 classes of antibiotics, especially Class1 integrase intI-1LC. Redundancy analysis (RDA) and Variance partitioning analysis (VPA) showed that antibiotics, metals, MGEs and bacteria explain solely 0.7%, 5.7%, 12.4%, and 21.9% of variances of ARGs in the microbial community, respectively. These results suggested that bacterial composition and horizontal gene transfer were the major factors shaping the composition of ARGs; Metals had a bigger effect on ARG profile than detected antibiotics in this study.

Coexistence of Antibiotic Resistance Genes and Virulence Factors Deciphered by Large-Scale Complete Genome Analysis.

ABSTRACTWidespread use of antibiotics has enhanced the evolution of highly resilient pathogens and poses a severe risk to human health via coselection of antibiotic resistance genes (ARGs) and virulence factors (VFs). In this study, we rigorously evaluate the abundance relationship and physical linkage between ARGs and VFs by performing a comprehensive analysis of 9,070 bacterial genomes isolated from multiple species and hosts. The coexistence of ARGs and VFs was observed in bacteria across distinct phyla, pathogenicities, and habitats, especially among human-associated pathogens. The coexistence patterns of gene elements in different habitats and pathogenicity groups were similar, presumably due to frequent gene transfer. A shorter intergenic distance between mobile genetic elements and ARGs/VFs was detected in human/animal-associated bacteria, indicating a higher transfer potential. Increased accumulation of exogenous ARGs/VFs in human pathogens highlights the importance of gene acquisition in the evolution of human commensal bacteria. Overall, the findings provide insights into the genic features of combinations of ARG-VF and expand our understanding of ARG-VF coexistence in bacteria.IMPORTANCE Antibiotic resistance has become a serious global health concern. Despite numerous case studies, a comprehensive analysis of ARG and VF coexistence in bacteria is lacking. In this study, we explore the coexistence profiles of ARGs and VFs in diverse categories of bacteria by using a high-resolution bioinformatics approach. We also provide compelling evidence of unique ARG-VF gene pairs coexisting in specific bacterial genomes and reveal the potential risk associated with the coexistence of ARGs and VFs in organisms in both clinical settings and environments.

Evolution and distribution of resistance genes and bacterial community in water and biofilm of a simulated fish-duck integrated pond with stress.

Integrated fish-duck pond is a common circular farming model in South China, besides, it is also hot-spot for the co-selection of antibiotic resistance genes (ARGs). The aim of this study was to investigate the effects of duck manure, As and cefotaxime on the bacterial community, and the evolution and distribution of ARGs and metal(loid) resistance genes (MRGs) in water and biofilm. Five groups of fish tanks included a control and four test groups. The experimental period lasted for 100 days. Six ARGs (CIT, DHA, EBC, FOX, MOX, TEM), two MRGs (arsB, arsC), and two integron genes (intI1, intI2) were tracked and detected in water and biofilm. The results showed that duck manure brought ARGs and MRGs into fish tanks. Stress factors (cefotaxime, As) increased the relative abundance of resistance genes, and this was positively correlated with stress concentrations. The biofilm was visible significantly at the end of stage 3, and the total relative abundance of resistance genes in biofilm was higher than water from stage 5 onwards. Evolution of AmpC β-lactamase resistance genes was more obvious than MRGs, especially for MOX, which increased by 3 orders of magnitude. The abundance of Flavobacterium was higher in biofilm than in water. Moreover, correlation analysis showed that both arsB and MOX were significantly correlated with intI1 (p<0.05), which suggested a potentially dissemination risk of resistance genes. This study provides a reference for health risk assessment in integrated aquaculture environment contaminated with duck manure, antibiotics and metalloids.

Heavy metal-induced co-selection of antibiotic resistance genes in the gut microbiota of collembolans

Heavy metal induced co-selection of antibiotic resistance genes (ARGs) has become an emerging environmental issue. The guts of soil fauna offer a unique habitat in the terrestrial ecosystem and harbor a variety of microorganisms. However, the effects of heavy metals on the gut-associated ARGs of soil fauna are poorly understood. In the present study, collembolans were cultivated with four types of heavy metals (Zn, Cu, Cd, and Cr) and one antibiotic (oxytetracycline), to investigate their impact on the gut-associated ARGs. High-throughput quantitative PCR and 16S rRNA gene amplicon sequencing were used to examine changes in the gut-associated ARGs and microbial composition caused by the metals and antibiotic. The results showed that heavy metals alone induced co-selection of ARGs in the collembolan gut, but the effects were weaker than selection by oxytetracycline. When Zn or Cu was present together with oxytetracycline, there was a strong synergistic effect between the compounds, which increased the selection of ARGs in the collembolan guts. Furthermore, redundancy analysis revealed that the gut microbiota and mobile genetic elements (MGEs) were significantly correlated with the ARG composition. These results extend our understanding on effects of heavy metals on the dispersal of ARGs in the soil food web.

Evidence for co-selection of antibiotic resistance genes and mobile genetic elements in metal polluted urban soils

Antibiotic resistance genes (ARGs) constitute emerging environmental pollutants and pose risks to public health. Toxic metals are known to select for metal-resistant bacteria in metal-contaminated soils, but there is growing concern that metal contaminants can also act as co-selective agents thereby causing environmental proliferation of antibiotic resistance. In this study, we quantified ARGs and selected mobile genetic elements (MGEs) known to constitute potential ARG hosts in 50 archived urban and suburban soils from the Belfast metropolitan area using a high-throughput qPCR ARG chip. ARG prevalence was linked to concentrations of individual metals and a soil metal toxicity index calculated based on the relative toxicity of different metals to soil microbial processes. A total of 164 ARGs were detected across the 50 soils analyzed with an average absolute abundance of 3.4 × 107 ARG gene copies per gram of soil. A significant correlation between abundance of ARGs and MGEs was observed, suggesting the importance of horizontal gene transfer for ARG dissemination. Network analysis revealed significant co-occurrence patterns between specific metals (As, Cd, Co, Cr, Cu. Hg, Ni and Zn) and associated ARGs. Path analysis further indicated that the soil metal toxicity index significantly affected the number of detected ARGs (λ = 0.32, P < 0.001) and the abundance of metal co-occurring ARGs (λ = 0.612, P < 0.001) via effects on MGEs. Collectively, our results indicate a role of soil metals in co-selection of ARGs and MGEs in urban and semi-urban soils and suggest a risk for environmental ARG dissemination via horizontal gene transfer.

Increase of zinc resistance in German human derived livestock-associated MRSA between 2000 and 2014

Problem addressedLivestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA), particularly of the clonal complex (CC) 398, emerged as zoonotic pathogens predominantly among humans with direct or indirect livestock contact, but also in healthcare settings. The factors contributing to the success of LA-MRSA are only poorly understood. ObjectiveDuring the past years, the use of heavy metal compounds as feed-supplements was found to influence the co-selection of LA-MRSA in pig herds. This study aimed to determine the prevalence of zinc resistance among MRSA CC398 isolated from patients of a German university hospital located in a pig farming-dense area. Methods and approachIn comparison to concurrent healthcare-associated MRSA (HA-MRSA), LA-MRSA CC398 comprising isolates from their first appearance in 2000 to recent isolates from 2014 were included. ResultsAmong MRSA CC398, the overall resistance rate towards zinc chloride was 57% compared to only 3% among concurrently isolated HA-MRSA. Zinc resistance correlated with the presence of the czrC gene in 100% of the MRSA CC398 and in 67% of the HA-MRSA. ConclusionsThe zinc resistance rate in MRSA CC398 significantly increased from 2009 to 2014 with a maximum in 2014. Alarmingly, zinc resistance has become a frequent phenotype of human LA-MRSA in Germany potentially facilitating co-selection of antibiotic resistance genes.

Co-occurrence of antibiotic and metal resistance genes revealed in complete genome collection.

The high frequency of antibiotic resistance is a global public health concern. More seriously, widespread metal pressure in the environment may facilitate the proliferation of antibiotic resistance via coselection of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). Given the lack of comprehensive understanding of the ARG and MRG coselection, in this study both abundance relationship and genetic linkage between ARGs and MRGs were rigorously investigated by performing a genomic analysis of a large complete genome collection. Many more ARGs were enriched in human-associated bacteria compared with those subjected to less anthropogenic interference. The signatures of ARG and MRG co-occurrence were much more frequent and the distance linkages between ARGs and MRGs were much more intimate in human pathogens than those less human-associated bacteria. Moreover, the co-occurrence structures in the habitat divisions were significantly different, which could be attributed to their distinct gene transfer potentials. More exogenous ARGs and MRGs on the genomes of human pathogens indicated the importance of recent resistance acquisition in resistome development of human commensal flora. Overall, the study emphasizes the potential risk associated with ARG and MRG coselection of both environmental and medical relevance.