Comprehensive insights into profiles and bacterial sources of intracellular and extracellular antibiotic resistance genes in groundwater.

Antibiotic resistance genes (ARGs), especially last-resort ARGs (LARGs), are receiving extensive attention as emerging environmental contaminants in groundwater. However, their prevalent intracellular and extracellular patterns and bacterial sources in groundwater remain unclear. Herein, groundwater samples were collected in Tianjin, and characterized based on the profiles of intracellular ARGs (iARGs) and extracellular ARGs (eARGs), as well as the resident bacterial communities and extracellular DNA (eDNA)-releasing bacterial communities. The quantitative real-time PCR assays showed that eARGs presented fewer subtypes than iARGs and generally displayed lower detection frequencies than the corresponding iARGs. Similarly, LARGs exhibited lower detection frequencies than common ARGs, but the total abundance showed no significant differences between them. Genes vanA and blaVIM were the observed dominant LARGs, and aadA was the observed common ARG independent of location inside or outside the bacteria. Furthermore, the top 10 phyla showed much difference between the main eDNA-releasing bacteria and the dominant resident bacteria. Proteobacteria was the predominant resident bacterial phyla while dominating the source of eDNA in groundwater. Despite representing a minor portion of the abundance in the resident bacteria, Actinobacteriota, Acidobacteriota, and Chloroflex surprisingly accounted for a large majority of eDNA release. Co-occurrence patterns among persistent ARGs, the resident bacteria, and eDNA-releasing bacteria revealed that the dominant common iARG aadA and intracellular LARGs blaVIM and vanA had significant positive correlations with Methylobacterium_Methylorubrum and Shewanella. Meanwhile, the dominant extracellular LARG blaVIM may be released by bacteria belonging to at least five genera, including Ellin6067, Bifidobacterium, Blautia, Veillonella, and Dechloromonas. Collectively, the findings of this study extend our understanding regarding the distribution of ARGs and their bacterial sources in groundwater, and indicate the serious pollution of LARGs in groundwater, which poses potential risks to public health.

Antibiotic resistance genes in gut of breast-fed neonates born by caesarean section originate from breast milk and hospital ward air.

The human gut is a reservoir of antibiotic resistance genes (ARGs). Even in the absence of antibiotics, ARGs are present in large quantities in faeces of adults, children and even newborns. However, where and when ARGs are acquired remains unclear, as does the types of ARGs acquired. Herein, we recruited 82 pairs of women and their caesarean section newborns. Conventional culture methods and quantitative PCR were employed to detect nine species and six ARG types in meconia, faeces from 3-day-old newborns, amniotic fluid, colostrum, and hospital ward air samples. Furthermore, ARG transfer was explored by tracking Staphylococcus epidermidis isolated from faeces of 3-day-old newborns, colostrum and ward air samples using multi-locus sequence typing (MLST). No ARGs or microorganisms were detected in meconia or amniotic fluid. One or more ARGs were detected in 90.2% of faeces from 3-day-old newborns, and the mecA gene exhibited the highest detection rate (45.1%). ARGs were detected in 85.4% of colostra consistent with ARGs in faeces from 3-day-old newborns. Some ARGs were detected in ward air, and might also be a source of ARGs in neonatal faeces. Isolation of S. epidermidis from neonatal faeces was consistent with antibiotic resistance and gene profiles for colostrum samples. Traceability analysis of S. epidermidis showed that ARGs in neonatal faeces mainly originated from colostrum, and partly from ward air. After birth, neonates born by caesarean section obtain a variety of ARGs mainly from colostrum, and partly from ward air.

A reduced graphene oxide-Fe3O4 composite functionalized with cetyltrimethylammonium bromide for efficient adsorption of SARS-CoV-2 spike pseudovirus and human enteric viruses.

The latent dangers of waterborne viral transmission have become a major public health concern. In this study, reduced graphene oxide (rGO)-Fe3O4 nanoparticles were decorated with cetyltrimethylammonium bromide (CTAB) to adsorb severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike pseudovirus and three human enteric viruses (HuNoV, HRV, and HAdV). The successful combination of CTAB with rGO-Fe3O4 was confirmed by transmission electron microscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, zeta potential, Brunner-Emmet-Teller, and vibrating sample magnetometer measurements. The adsorption of HuNoV and HAdV followed pseudo-first-order kinetics, while that of HRV conformed to the pseudo-second-order model. CTAB-functionalized rGO-Fe3O4 exhibited exceptionally high adsorption of HuNoV, HRV, HAdV and SARS-CoV-2 spike pseudovirus, with maximum adsorption capacities of 3.55 × 107, 7.01 × 107, 2.21 × 107 and 6.92 × 106 genome copies mg-1, respectively. Moreover, the composite could effectively adsorb the four types of virus particles from coastal, tap, and river water. In addition, concentrating the virions using CTAB functionalized rGO-Fe3O4 composites before qPCR analysis significantly improved the detection limit. The results indicate that viruses are captured on the surface of CTAB functionalized rGO-Fe3O4 composites through electrostatic interactions and the intrinsic adsorption ability of rGO. Overall, CTAB-functionalized rGO-Fe3O4 composites are promising materials for the adsorption and detection of human enteric viruses as well as SARS-CoV-2 from complex aqueous environments.

Intestinal Microbiota Mediates High-Fructose and High-Fat Diets to Induce Chronic Intestinal Inflammation.

An unhealthy diet has been linked to increased incidence of chronic diseases. To investigate the relationship between diet and intestinal inflammation, mice in two experimental groups were fed on a high-fat diet or high-fructose diet, respectively. The result showed that the defecation volume of the experimental groups was significantly reduced compared with that of the control group, and the levels of pro-inflammatory cytokines (interleukin (IL)-1ß and IL-6) and IgG in serum were increased significantly. In addition, inflammatory cell infiltration was observed in intestinal tissue, indicating that a high-fructose or high-fat diet can lead to constipation and inflammation. Further analysis showed that the microbial composition of the experimental groups changed significantly, including a decrease of the Bacteroidetes/Firmicutes ratio and increased levels of Bacteroides, Akkermansia, Lactobacillus, and Ruminococcus, which might be associated with inflammation. The results of pro-inflammatory metabolites analysis showed that the levels of arachidonic acid, stearic acid, and indoxylsulfuric acid were significantly increased in the experimental groups, which were related significantly to Bacteroides, Enterococcus, and Akkermansia. Meanwhile, the content of 5-hydroxytryptamine (5-HT) was significantly decreased, which might cause constipation by reducing intestinal peristalsis. Moreover, transplantation of fecal bacteria from inflammatory mice caused constipation and inflammation in normal mice, which could be relieved by feeding a normal diet. The results of the present study indicated that changes in intestinal microbiota and microbial metabolites may underlie chronic intestinal inflammation and constipation caused by high-fructose and high-fat diets.

Decreased Antibiotic Susceptibility in Pseudomonas aeruginosa Surviving UV Irradition.

Given its excellent performance against the pathogens, UV disinfection has been applied broadly in different fields. However, only limited studies have comprehensively investigated the response of bacteria surviving UV irradiation to the environmental antibiotic stress. Here, we investigated the antibiotic susceptibility of Pseudomonas aeruginosa suffering from the UV irradiation. Our results revealed that UV exposure may decrease the susceptibility to tetracycline, ciprofloxacin, and polymyxin B in the survival P. aeruginosa. Mechanistically, UV exposure causes oxidative stress in P. aeruginosa and consequently induces dysregulation of genes contributed to the related antibiotic resistance genes. These results revealed that the insufficient ultraviolet radiation dose may result in the decreased antibiotic susceptibility in the pathogens, thus posing potential threats to the environment and human health.

Levels of human Rotaviruses and Noroviruses GII in urban rivers running through the city mirror their infection prevalence in populations.

Enteric viruses exposed to water pose a huge threat to global public health and can lead to waterborne disease outbreaks. A sudden increase in enteric viruses in some water matrices also underpins the prevalence of corresponding waterborne diseases in communities over the same time period. However, few efforts have been focused on water matrices whose viral pollution may best reflect the clinical prevalence in communities. Here, a one-year surveillance of human enteric viruses including Enteroviruses (EnVs), Rotaviruses (HRVs), Astroviruses (AstVs), Noroviruses GII (HuNoVsGII) and Mastadenoviruses (HAdVs) in four representative water matrices: an urban river (UR) running through city, effluent from Wastewater Treatment Plant (EW), raw water for Urban Water Treatment Plant (RW), and tap water (TW) were performed by qPCR. The relationship between the virus detection frequency at each site and their prevalence in clinical PCR assay was further analyzed. We found that the detection frequencies of HRVs, HuNoVsGII, and AstVs in stools peaked in winter, while EnVs peaked in autumn. No EnVs occurred in EW, RW, or TW, but HuNoVsGII and AstVs occurred intensively in winter. For UR, all types of enteric viruses could be detected and the levels of acute gastroenteritis viruses (HRVs, HuNoVsGII, AstVs, and HAdVs) were highest in autumn or winter, whereas EnVs peaked in summer. In terms of correlation analyses, only HRVs and HuNoVsGII levels in UR showed a strong positive correlation with their prevalence in clinical stool samples. This study indicated that HRVs and HuNoVsGII levels in URs may mirror the local virus prevalence, thereby implying the possibility of revealing their local epidemiology by monitoring them in the URs.

Contamination sources of the enteric virus in recreational marine water shift in a seasonal pattern.

Human enteric virus occurrence in bathing beaches poses a potential health risk to swimmers. They may come from several sources, but the understanding of the seasonal contribution of contamination sources to virus occurrence is still lacking. Here, the surveillance of human enteric viruses at the First Bathing Beach in Qingdao was performed January-December 2018. The occurrence of Enteric viruses, assayed with quantitative polymerase chain reaction (qPCR), was analyzed at temporal and spatial levels to determine the viral contamination sources. The results showed that only Astroviruses (AstVs) and Adenoviruses (HAdVs) were found in the swimming area. Their occurrence correlated significantly with the sewage-polluted area, but HAdVs were only found in autumn and AstVs in spring. Meanwhile, enteric viruses in the swimming area showed significantly higher levels than the surrounding area, particularly AstVs in summer with the swimmer crowd. All these data imply that sewage discharge and swimmers co-contribute to the viral occurrence in a seasonal pattern, with the former being more focused in warm seasons (spring and autumn) and the latter in hot seasons (summer). These results indicate that sewage discharge and crowd swimmers, as unsafe swimming conditions, should be avoided to improve public health at the bathing beaches.

Chlorine disinfection promotes the exchange of antibiotic resistance genes across bacterial genera by natural transformation.

Chlorine disinfection to drinking water plays an important role in preventing and controlling waterborne disease outbreaks globally. Nevertheless, little is known about why it enriches the antibiotic resistance genes (ARGs) in bacteria after chlorination. Here, ARGs released from killed antibiotic-resistant bacteria (ARB), and culturable chlorine-injured bacteria produced in the chlorination process as the recipient, were investigated to determine their contribution to the horizontal transfer of ARGs during disinfection treatment. We discovered Escherichia coli, Salmonella aberdeen, Pseudomonas aeruginosa and Enterococcus faecalis showed diverse resistance to sodium hypochlorite, and transferable RP4 could be released from killed sensitive donor consistently. Meanwhile, the survival of chlorine-tolerant injured bacteria with enhanced cell membrane permeabilisation and a strong oxidative stress-response demonstrated that a physiologically competent cell could be transferred by RP4 with an improved transformation frequency of up to 550 times compared with the corresponding untreated bacteria. Furthermore, the water quality factors involving chemical oxygen demand (CODMn), ammonium nitrogen and metal ions (Ca2+ and K+) could significantly promote above transformation frequency of released RP4 into injured E. faecalis. Our findings demonstrated that the chlorination process promoted the horizontal transfer of plasmids by natural transformation, which resulted in the exchange of ARGs across bacterial genera and the emergence of new ARB, as well as the transfer of chlorine-injured opportunistic pathogen from non-ARB to ARB. Considering that the transfer elements were quite resistant to degradation through disinfection, this situation poses a potential risk to public health.

Chlorine injury enhances antibiotic resistance in Pseudomonas aeruginosa through over expression of drug efflux pumps.

Adaption to adverse environments plays an important role in bacterial survival and is receiving increasing globe attention now. Here, cultivable chlorine-injured Pseudomonas aeruginosa, produced on the chlorination process, was investigated about their resistance to antibiotics. Then, global transcriptional analyses, quantitative PCR (qPCR) validation and antioxidant enzymes measurement were performed to explore the underlying mechanisms. The results showed that chlorine injury enhanced antibiotic resistance in P. aeruginosa and cultivable chlorine-injured P. aeruginosa exposed to 4 mg/L sodium hypochlorite (half of the lethal dose) improved antibiotic resistance against ceftazidime, chloramphenicol and ampicillin by 1.4-5.6 fold. This increase in antibiotic resistance was not hereditable and over expression of the MexEF-OprN efflux pump resulting from oxidative stress contributed to it. These results demonstrate temporal physiological persistence to antibiotics in cultivable chlorine-injured pathogens, suggesting their survival from adverse environments with antibiotic exposure and thereby posing lasting hazards to human health.

Presence of bacteroidales as a predicator of human enteric viruses in Haihe River of Tianjin City, China.

Traditional microbe indicators including total bacteria, total coliforms, fecal coliforms, Escherichia coli, enterococci, and F+ coliphage are all frequently used to characterize the microbial contamination state of water bodies for their correlation with pathogenic bacteria. However, these indicators have a poor relationship with viruses, which pose serious threat to economic and human health. Alternative indicators such as bacteroidales may be suitable complementary alternatives to traditional microbe indicators and are being increasingly reported. In the present study, water was analyzed for selected sites along Haihe River in Tianjin for traditional indicators, an alternative indicator (bacteroidales), pathogenic bacteria (Salmonella, Escherichia coli (E. coli) O157:H7, and Vibrio parahaemolyticus), viruses (enteric adenovirus, norovirus, enterovirus, poliovirus and rotavirus), and physicochemical parameters. Results indicated that traditional microbe indicators detected in this study showed good correlation with pathogenic bacteria, and the alternative indicator (bacteroidales) had a surprisingly good relationship with viral presence. We propose that bacteroidales might be a suitable complementary indicator for viral contamination in water bodies.

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.