Analysis of extracellular and intracellular antibiotic resistance genes in commercial organic fertilizers reveals a non-negligible risk posed by extracellular genes.

Livestock manure is known to be a significant reservoir of antibiotic resistance genes (ARGs), posing a major threat to human health and animal safety. ARGs are found in both intracellular and extracellular DNA fractions. However, there has been no comprehensive analysis of these fractions in commercial organic fertilizers (COFs). The present study conducted a systematic survey of the profiles of intracellular ARGs (iARGs) and extracellular ARGs (eARGs) and their contributing factor in COFs in Northern China. Results showed that the ARG diversity in COFs (i.e., 57 iARGs and 53 eARGs) was significantly lower than that in cow dung (i.e., 68 iARGs and 69 eARGs). The total abundance of iARGs and eARGs decreased by 85.7% and 75.8%, respectively, after compost processing, and there were no significant differences between iARGs and eARGs in COFs (P > 0.05). Notably, the relative abundance of Campilobacterota decreased significantly (99.1-100.0%) after composting, while that of Actinobacteriota and Firmicutes increased by 21.1% and 29.7%, respectively, becoming the dominant bacteria in COFs. Co-occurrence analysis showed that microorganisms and mobile genetic elements (MGEs) were more closely related to eARGs than iARGs in COFs. And structural equation models (SEMs) further verified that microbial community was an essential factor regulating iARGs and eARGs variation in COFs, with a direct influence (λ = 0.74 and 0.62, P < 0.01), following by similar effects of MGEs (λ = 0.59 and 0.43, P < 0.05). These findings indicate the need to separate eARGs and iARGs when assessing the risk of dissemination and during removal management in the environment.

Deciphering changes in the abundance of intracellular and extracellular antibiotic resistance genes and mobile genetic elements under anaerobic fermentation: Driven by bacterial community.

Antibiotic resistance genes (ARGs) are considered to be a new environmental pollutant and the removal of ARGs from swine manure by anaerobic fermentation was a crucial topic. This research discusses effects of initial pH values (3, 5, 7, 11) on intracellular and extracellular ARGs (iARGs and eARGs) as well as mobile genetic elements (MGEs) during anaerobic fermentation of swine manure had been examined. The initial pH during fermentation was found to be acidic (pH 3 and 5) in results, which was conducive to the removal of six eARGs and seven iARGs. Similarly, intracellular and extracellular MGEs were effectively eliminated with an initial pH of 3 and 5. The abundance of MGEs and four ARGs were enriched with an initial pH of 7 and 11. Acidic conditions can greatly deduce the diversity as well as abundance of the microbial community, ensuing removal of MEGs and ARGs. These findings are critical for risk assessment and management of ARGs.

Deciphering the extracellular and intracellular antibiotic resistance genes in multiple environments reveals the persistence of extracellular ones.

The extracellular and intracellular antibiotic resistance genes (eARGs and iARGs) together constitute the entire resistome in environments. However, the systematic analysis of eARGs and iARGs was still inadequate. Three kinds of environments, i.e., livestock manure, sewage sludge, and lake sediment, were analyzed to reveal the comprehensive characteristics of eARGs and iARGs. Based on the metagenomic data, the diversities, relative abundances, and compositions of eARGs and iARGs were similar. The extracellular and intracellular integrons and insertion sequences (ISs) also did not show any significant differences. However, the degree and significance of the correlation between total relative abundances of integrons/ISs and ARGs were lower outside than inside the cells. Gene cassettes carried by class 1 integron were amplified in manure and sludge samples, and sequencing results showed that the identified ARGs extracellularly and intracellularly were distinct. By analyzing the genetic contexts, most ARGs were found located on chromosomes. Nevertheless, the proportion of ARGs carried by plasmids increased extracellularly. qPCR was employed to quantify the absolute abundances of sul1, sul2, tetO, and tetW, and their extracellular proportions were found highest in sludge samples. These findings together raised the requirements of considering eARGs and iARGs separately in terms of risk evaluation and removal management.

Estuarine sediments are key hotspots of intracellular and extracellular antibiotic resistance genes: A high-throughput analysis in Haihe Estuary in China.

Estuaries lie between terrestrial/freshwater and marine ecosystems, receive considerable pollutant input from land-based sources, and are considerably influenced by human activities. However, little attention has been paid to combined research on extracellular antibiotic resistance genes (eARGs) and intracellular ARGs (iARGs) in the estuarine environment. In this study, we profiled eARGs and iARGs in sediments from Haihe Estuary, China by adopting high-throughput quantitative PCR and investigated their relationship with mobile genetic elements (MGEs), the bacterial community and environmental factors. The results showed that the abundance of eARGs ranged from 9.06 × 106 to 1.32 × 108 copies/g and that of iARGs ranged from 3.31 × 107 to 2.93 × 108 copies/g, indicating that estuarine sediments were key hotspots of eARGs and iARGs. Additionally, multidrug resistance genes were both highly diverse and abundant in Haihe Estuary, especially in coastal samples. The high abundance of vancomycin and carbapenemase resistance genes may pose a potential health risk to human. Salinity altered the composition and structure of the bacterial community. Partial redundancy analysis showed that the bacterial community and MGEs appeared to be the major drivers of ARG variance in estuarine sediment. This study provides an overview of the distribution of eARG and iARG along the Haihe Estuary and draws attention to the need to control pollutants in estuary ecosystems.

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

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

Assessment of extracellular antibiotic resistance genes (eARGs) in typical environmental samples and the transforming ability of eARG.

The emergence and spread of antibiotic resistance has pose a huge threat to both human health and environmental ecosystem. However, little is known regarding the pool of ARGs in extracellular DNA (eDNA). In this study ten ARGs (sul1, sul2, tetW, tetX, ermA, ermB, blaTEM, ampC, cat and cmr) and class I integron (intI1) in the sludge from hospital, pharmaceutical industry, wastewater treatment plant (WWTP), and swine manure, and sediment in urban lake in the form of both eDNA and intracellular DNA (iDNA) were evaluated by quantitative polymerase chain reaction (qPCR). The results showed that every gram of sludge dry weight contained from 7.31 × 103 to 1.16 × 1010 copies of extracellular ARGs (eARGs) and from 1.04 × 105 to 2.74 × 1012 copies of intracellular ARGs (iARGs). The sludge from hospital with the highest ratio of eARGs to total ARGs (11.02-89.63%), followed by the sediment from urban lake, implying that most of the ARGs in these regions were contributed by eARGs. The relative abundance of eARGs were higher than iARGs in sludge from WWTP and pharmaceutical industry, moreover, 1/3 and 5/9 detected eARGs were higher than the ARGs in the iDNA extracted from sludge of hospital and sediment from urban lake, respectively. Furthermore, the transforming ability of eARGs suggesting that adsorbed eARG is more preferentially coupled to the competent cells than free eARG. These findings highlight the need to focus attention on the contribution of eARGs to the dissemination of antibiotic resistance into environment, and also future needs in mitigating the spread of eARGs in the environment.

Profiling of intracellular and extracellular antibiotic resistance genes in tap water.

Antibiotic resistance genes (ARGs) have gained global attention due to their public health threat. Extracelluar ARGs (eARGs) can result in the dissemination of antibiotic resistance via free-living ARGs in natural environments, where they promote ARB transmission in drinking water distribution systems. However, eARG pollution in tap water has not been well researched. In this study, concentrations of eARGs and intracellular ARGs (iARGs) in tap water, sampled at Tianjin, China, were investigated for one year. Fourteen eARG types were found at the highest concentration of 1.3 × 105 gene copies (GC)/L. TetC was detected in 66.7% of samples, followed by sul1, sul2, and qnrA with the same detection frequency of 41.7%. Fifteen iARGs (including tetA, tetB, tetM, tetQ, tetX, sul1, sul2, sul3, ermB, blaTEM, and qnrA) were continuously detected in all collected tap water samples with sul1 and sul2 the most abundant. Additionally, both eARG and iARG concentrations in tap water presented a seasonal pattern with most abundant prevalence in summer. The concentration of observed intracellular sulfonamide resistance genes showed a significantly positive correlation with total nitrogen concentrations. This study suggested that eARG and iARG pollution of drinking water systems pose a potential risk to human public health.

Chlorine disinfection increases both intracellular and extracellular antibiotic resistance genes in a full-scale wastewater treatment plant.

The emergence and spread of antibiotic resistance has posed a major threat to both human health and environmental ecosystem. Although the disinfection has been proved to be efficient to control the occurrence of pathogens, little effort is dedicated to revealing potential impacts of disinfection on transmission of antibiotic resistance genes (ARGs), particularly for free-living ARGs in final disinfected effluent of urban wastewater treatment plants (UWWTP). Here, we investigated the effects of chlorine disinfection on the occurrence and concentration of both extracellular ARGs (eARGs) and intracellular ARGs (iARGs) in a full-scale UWWTP over a year. We reported that the concentrations of both eARGs and iARGs would be increased by the disinfection with chlorine dioxide (ClO2). Specifically, chlorination preferentially increased the abundances of eARGs against macrolide (ermB), tetracycline (tetA, tetB and tetC), sulfonamide (sul1, sul2 and sul3), ß-lactam (ampC), aminoglycosides (aph(2')-Id), rifampicin (katG) and vancomycin (vanA) up to 3.8 folds. Similarly, the abundances of iARGs were also increased up to 7.8 folds after chlorination. In terms of correlation analyses, the abundance of Escherichia coli before chlorination showed a strong positive correlation with the total eARG concentration, while lower temperature and higher ammonium concentration were assumed to be associated with the concentration of iARGs. This study suggests the chlorine disinfection could increase the abundances of both iARGs and eARGs, thereby posing risk of the dissemination of antibiotic resistance in environments.