Meta-analysis of wastewater microbiome for antibiotic resistance profiling.

The microbial composition and stress molecules are main drivers influencing the development and spread of antibiotic resistance bacteria (ARBs) and genes (ARGs) in the environment. A reliable and rapid method for identifying associations between microbiome composition and resistome remains challenging. In the present study, secondary metagenome data of sewage and hospital wastewaters were assessed for differential taxonomic and ARG profiling. Subsequently, Random Forest (RF)-based ML models were used to predict ARG profiles based on taxonomic composition and model validation on hospital wastewaters. Total ARG abundance was significantly higher in hospital wastewaters (15 ppm) than sewage (5 ppm), while the resistance towards methicillin, carbapenem, and fluoroquinolone were predominant. Although, Pseudomonas constituted major fraction, Streptomyces, Enterobacter, and Klebsiella were characteristic of hospital wastewaters. Prediction modeling showed that the relative abundance of pathogenic genera Escherichia, Vibrio, and Pseudomonas contributed most towards variations in total ARG count. Moreover, the model was able to identify host-specific patterns for contributing taxa and related ARGs with >90% accuracy in predicting the ARG subtype abundance. More than >80% accuracy was obtained for hospital wastewaters, demonstrating that the model can be validly extrapolated to different types of wastewater systems. Findings from the study showed that the ML approach could identify ARG profile based on bacterial composition including 16S rDNA amplicon data, and can serve as a viable alternative to metagenomic binning for identification of potential hosts of ARGs. Overall, this study demonstrates the promising application of ML techniques for predicting the spread of ARGs and provides guidance for early warning of ARBs emergence.

Oxygen mediated mobilization and co-occurrence of antibiotic resistance in lab-scale bioreactor using metagenomic binning.

Sub-lethal levels of antibiotic stimulate bacteria to generate reactive oxygen species (ROS) that promotes emergence and spread of antibiotic resistance mediated by mobile genetic elements (MGEs). Nevertheless, the influence of dissolved oxygen (DO) levels on mobility of antibiotic resistance genes (ARGs) in response to ROS-induced stress remains elusive. Thus, the study employs metagenomic assembly and binning approaches to decipher mobility potential and co-occurrence frequency of ARGs and MGEs under hyperoxic (5.5-7 mgL- 1), normoxic (2.5-4 mgL- 1), and hypoxic (0.5-1 mgL- 1) conditions in lab-scale bioreactor for 6 months. Among 163 high-quality metagenome-assembled genomes (MAGs) recovered from 13 metagenomes, 42 MAGs harboured multiple ARGs and were assigned to priority pathogen group. Total ARG count increased by 4.3 and 2.5% in hyperoxic and normoxic, but decreased by 0.53% in hypoxic conditions after 150 days. On contrary, MGE count increased by 7.3-1.3% in all the DO levels, with only two ARGs showed positive correlation with MGEs in hypoxic compared to 20 ARGs under hyperoxic conditions. Opportunistic pathogens (Escherichia, Klebsiella, Clostridium, and Proteus) were detected as potential hosts of ARGs wherein co-localisation of critical ARG gene cassette (sul1, dfr1,adeF, and qacC) were identified in class 1 integron/Tn1 family transposons. Thus, enhanced co-occurrence frequency of ARGs with MGEs in pathogens suggested promotion of ARGs mobility under oxidative stress. The study offers valuable insights into ARG dissemination and hosts dynamics that is essential for controlling oxygen-related stress for mitigating MGEs and ARGs in the environment.

Deterministic effect of oxygen level variation on shaping antibiotic resistome.

An increase in acquisition of antibiotic resistance genes (ARGs) by pathogens under antibiotic selective pressure poses public health threats. Sub-inhibitory antibiotics induce bacteria to generate reactive oxygen species (ROS) dependent on dissolved oxygen (DO) levels, while molecular connection between ROS-mediated ARG emergence through DNA damage and metabolic changes remains elusive. Thus, the study investigates antibiotic resistome dynamics, microbiome shift, and pathogen distribution in hyperoxic (5-7 mg L-1), normoxic (2-4 mg L-1), and hypoxic (0.5-1 mg L-1) conditions using lab-scale bioreactor. Composite inoculums in the reactor were designed to represent comprehensive microbial community and AR profile from selected activated sludge. RT-qPCR and metagenomic analysis showed an increase in ARG count (100.98 ppm) with enrichment of multidrug efflux pumps (acrAB, mexAB) in hyperoxic condition. Conversely, total ARGs decreased (0.11 ppm) under hypoxic condition marked by a major decline in int1 abundance. Prevalence of global priority pathogens increased in hyperoxic (22.5%), compared to hypoxic (0.9%) wherein major decrease were observed in Pseudomonas, Shigella, and Borrelia. The study observed an increase in superoxide dismutase (sodA, sodB), DNA repair genes (nfo, polA, recA, recB), and ROS (10.4 µmol L-1) in adapted biomass with spiked antibiotics. This suggests oxidative damage that facilitates stress-induced mutagenesis providing evidence for observed hyperoxic enrichment of ARGs. Moreover, predominance of catalase (katE, katG) likely limit oxidative damage that deplete ARG breeding in hypoxic condition. The study proposes a link between oxygen levels and AR development that offers insights into mitigation and intervention of AR by controlling oxygen-related stress and strategic selection of bacterial communities.

Mapping the spread and mobility of antibiotic resistance in wastewater due to COVID-19 surge.

Large amounts of antibiotics have been discharged into wastewater during the COVID-19 pandemic due to overuse and misuse of antibiotics to treat patients. Wastewater-based surveillance can be used as an early warning for antibiotic resistance (AR) emergence. The present study analyzed municipal wastewater corresponding to the major pandemic waves (WW1, WW2, and WW3) in India along with hospital wastewater (Ho) taken as a benchmark for AR. Commonly prescribed antibiotics during a pandemic, azithromycin and cefixime residues, were found in the range of 2.1-2.6 µg/L in Ho and WW2. Total residual antibiotic concentration was less in WW2; however, the total antibiotic resistance gene (ARG) count was 1065.6 ppm compared to 85.2 ppm in Ho. Metagenome and RT-qPCR analysis indicated a positive correlation between antibiotics and non-corresponding ARGs (blaOXA, aadA, cat, aph3, and ere), where 7.2-7.5% was carried by plasmid in the bacterial community of WW1 and WW2. Moreover, as the abundance of the dfrA and int1 genes varied most among municipal wastewater, they can be suggested as AR markers for the pandemic. The common pathogens Streptococcus, Escherichia, Shigella, and Aeromonas were putative ARG hosts in metagenome-assembled genomes. The ARG profile and antibiotic levels varied between municipal wastewaters but were fairly similar for WW2 and Ho, suggesting the impact of the pandemic in shaping the resistome pattern. The study provides insights into the resistome dynamic, AR markers, and host-ARG association in wastewater during the COVID-19 surge. Continued surveillance and identification of intervention points for AR beyond the pandemic are essential to curbing the environmental spread of ARGs in the near future.

Potential of camel rumen derived Bacillus subtilis and Bacillus velezensis strains for application in plant biomass hydrolysis.

Rumen inhabiting Bacillus species possesses a high genetic potential for plant biomass hydrolysis and conversion to value-added products. In view of the same, five camel rumen-derived Bacillus strains, namely B. subtilis CRN 1, B. velezensis CRN 2, B. subtilis CRN 7, B. subtilis CRN 11, and B. velezensis CRN 23 were initially assayed for diverse hydrolytic activities, followed by genome mining to unravel the potential applications. CRN 1 and CRN 7 showed the highest endoglucanase activity with 0.4 U/ml, while CRN 23 showed high ß-xylosidase activity of 0.36 U/ml. The comprehensive genomic insights of strains resolve taxonomic identity, clusters of an orthologous gene, pan-genome dynamics, and metabolic features. Annotation of Carbohydrate active enzymes (CAZymes) reveals the presence of diverse glycoside hydrolases (GH) GH1, GH5, GH43, and GH30, which are solely responsible for the effective breakdown of complex bonds in plant polysaccharides. Further, protein modeling and ligand docking of annotated endoglucanases showed an affinity for cellotrioside, cellobioside, and ß-glucoside. The finding indicates the flexibility of Bacillus-derived endoglucanase activity on diverse cellulosic substrates. The presence of the butyrate synthesis gene in the CRN 1 strain depicts its key role in the production of important short-chain fatty acids essential for healthy rumen development. Similarly, antimicrobial peptides such as bacilysin and non-ribosomal peptides (NRPS) synthesized by the Bacillus strains were also annotated in the genome. The findings clearly define the role of Bacillus sp. inside the camel rumen and its potential application in various plant biomass utilizing industry and animal health research sectors.

Differential colonization and functioning of microbial community in response to phosphate levels.

Microbes play a major role in phosphate cycling and regulate its availability in various environments. The metagenomic study highlights the microbial community divergence and interplay of phosphate metabolism functional genes in response to phosphate rich (100 mgL-1), limiting (25 mgL-1), and stressed (5 mgL-1) conditions at lab-scale bioreactor. Total five core phyla were found responsive toward different phosphate (Pi) levels. However, major variations were observed in Proteobacteria and Actinobacteria with 33-81% and 5-56% relative abundance, respectively. Canonical correspondence analysis reflects the colonization of Sinorhizobium (0.8-4%), Mesorhizobium (1-4%), Rhizobium (0.5-3%) in rich condition whereas, Pseudomonas (1-2%), Rhodococcus (0.2-2%), Flavobacterium (0.2-1%) and Streptomyces (0.3-4%) colonized in limiting and stress condition. The functional profiling demonstrates that Pi limiting and stress condition subjected biomass were characterized by abundant PQQ-Glucose dehydrogenase, alkaline phosphatase, 5'-nucleotidase, and phospholipases C genes. The finding implies that the major abundant genera belonging to phosphate solubilization enriched in limiting/stressed conditions decide the functional turnover by modulating the metabolic flexibility for Pi cycling. The study gives a better insight into intrinsic ecological responsiveness mediated by microbial communities in different Pi conditions that would help to design the microbiome according to the soil phosphate condition. Furthermore, this information assists in sustainably maintaining the ecological balance by omitting excessive chemical fertilizers and eutrophication.

Genome plasticity as a paradigm of antibiotic resistance spread in ESKAPE pathogens.

The major reason behind the spread of antibiotic resistance genes (ARGs) is persistent selective pressure in the environment encountered by bacteria. Genome plasticity plays a crucial role in dissemination of antibiotic resistance among bacterial pathogens. Mobile genetic elements harboring ARGs are reported to dodge bacterial immune system and mediate horizontal gene transfer (HGT) under selective pressure. Residual antibiotic pollutants develop selective pressures that force the bacteria to lose their defense mechanisms (CRISPR-cas) and acquire resistance. The present study targets the ESKAPE organisms (namely, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) causing various nosocomial infections and emerging multidrug-resistant species. The role of CRISPR-cas systems in inhibition of HGT in prokaryotes and its loss due to presence of various stressors in the environment is also focused in the study. IncF and IncH plasmids were identified in all strains of E. faecalis and K. pneumoniae, carrying Beta-lactam and fluoroquinolone resistance genes, whereas sal3, phiCTX, and SEN34 prophages harbored aminoglycoside resistance genes (aadA, aac). Various MGEs present in selected environmental niches that aid the bacterial genome plasticity and transfer of ARGs contributing to its spread are also identified.

Environmental Distribution, Metabolic Fate, and Degradation Mechanism of Chlorpyrifos: Recent and Future Perspectives.

Pesticides play a significant role in crop production and have become an inevitable part of the modern environment due to their extensive distribution throughout the soil ecosystem. Prophylactic applications of chlorpyrifos (CP) affect soil fertility, modify soil microbial community structure, and pose potential health risks to the nontarget organisms. Bioremediation through microbial metabolism is found to be an ecofriendly and cheaper process for CP removal from the environment. So far, various bacterial and fungal communities have been reported for CP and its metabolites degradation. Organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) are crucial bacterial enzymes for CP degradation as they efficiently hydrolyze the unbreakable P-O and P = S bond. This review discusses the prospects of toxicity level, persistency, and harmful effects of CP on the environment. CP degradation mechanisms, metabolic pathways, and key enzymes, along with their structural details, are also featured. The highlights on molecular docking with OPH and MPH enzyme for CP show the best binding affinity with OPH; hence, it is an essential part of CP degradation. Simultaneously, metagenomic analysis of soil from contaminated agricultural lands and wastewater was analyzed with the goal to identify the dominant CP degraders and enzymes. The identification of potent degraders, key enzymes, and evaluation of microbial community dynamics upon pesticide exposure can be used as a warning for its dissemination and biomagnification into the food chain.

Polybrominated diphenyl ethers in the environment: a wake-up call for concerted action in India.

Polybrominated diphenyl ethers (PBDEs) are a class of persistent organic pollutants (POPs) used as flame retardants in the products utilized in day-to-day life. Their bioaccumulation, low volatility, and high persistence in the environment have led to their global spread even to remote and distant regions. The present study identifies gaps in the investigation of the neurotoxic potential of PBDEs, their effects on brain development, toxicokinetic, and their potential as a carcinogen. In India, to date, only human breast milk was assessed for levels of PBDEs, and it is suggested that other human tissues can also be explored. No data on the reproductive toxicity of PBDEs are reported from Indian cohorts. Long-range transport and deposition of PBDEs in colder regions necessitates monitoring of Himalayan regions in India. An inventory of PBDEs is required to be made for addressing the worrisome situation of the unregulated import of E-waste from the developed countries in India. The study also emphasizes providing guidelines for the articulation of policies regarding sound surveillance and management of PBDE production, consumption, and release in the Indian context. It is recommended that a separate cell for monitoring and follow-up of PBDEs should be established in India. Also, the development of better alternatives and environment-friendly remediation technologies for PBDEs is the need of the hour.

Mobility of antibiotic resistance and its co-occurrence with metal resistance in pathogens under oxidative stress.

The bacterial communities are challenged with oxidative stress during their exposure to bactericidal antibiotics, metals, and different levels of dissolved oxygen (DO) encountered in diverse environmental habitats. The frequency of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) co-selection is increased by selective pressure posed by oxidative stress. Hence, study of resistance acquisition is important from an evolutionary perspective. To understand the dependence of oxidative stress on the dissemination of ARGs and MRGs through a pathogenic bacterial population, 12 metagenomes belonging to gut, water and soil habitats were evaluated. The metagenome-wide analysis showed the chicken gut to pose the most diverse pool of ARGs (30.4 ppm) and pathogenic bacteria (Simpson diversity = 0.98). The most common types of resistances found in all the environmental samples were efflux pumps (13.22 ppm) and genes conferring resistance to vancomycin (12.4 ppm), tetracycline (12.1 ppm), or beta-lactam (9.4 ppm) antibiotics. Additionally, limiting DO level in soil was observed to increase the abundance of excision nucleases (uvrA and uvrB), DNA polymerase (polA), catalases (katG), and other oxidative stress response genes (OSGs). This was further evident from major variations occurred in antibiotic efflux genes due to the effect of DO concentration on two human pathogens, namely Salmonella enterica and Shigella sonnei found in all the selected habitats. In conclusion, the microbial community, when challenged with oxidative stress caused by environmental variations in oxygen level, tends to accumulate higher amounts of ARGs with increased dissemination potential through triggering non-lethal mutagenesis. Furthermore, the genetic linkage or co-occurrence of ARGs and MRGs provides evidence for selecting ARGs under high concentrations of heavy metals.

Antibiotic resistance in the environment: a critical insight on its occurrence, fate, and eco-toxicity.

The overuse, misuse, and underuse of antibiotics tend to increase the antibiotic burden in the environment resulting into the evolution in microbial community to possess resistance that renders antibiotics ineffective against them. The current review recapitulates the present state of knowledge about the occurrence and fate of antibiotics in various environmental matrices. Also, the prevalence of antibiotic-resistant bacteria/antibiotic-resistant genes (ARB/ARGs) in various biological and non-biological systems, eco-toxicity of antibiotics on non-target organisms, and remediation methods for antibiotics and ARB/ARGs removal were critically reviewed. Furthermore, a comparison of various technologies for their efficiency to eliminate antibiotic residues and ARB/ARGs is made. The study identified gaps in the investigation of toxic effects of low concentration of antibiotics and the mixture of multiple antibiotics on non-target organisms. The study of antibiotics' phytotoxicity and toxicity towards sediment and soil-dwelling organisms are also recognized as a knowledge gap. The review also details policies implemented across the globe to fight against antibiotic resistance, and the scarcity of data on lab to land transferred remediation technology was identified. The present study entails a critical review of literature providing guidelines for the articulation of policies for prudent use of antibiotics, limits on the amount of antibiotics in pharmaceutical formulations, and regular surveillance in the Indian context.

Study of indiscriminate distribution of restrained antimicrobial resistome of different environmental niches.

Prophylactic usage and high persistent nature of several antibiotics have put selective pressure on the native microbial population that led to the emergence, propagation, and persistence of antibiotic resistance in nature. The surveillance of antibiotic resistome pattern and identification of points of intervention throughout the different environmental habitats will help to break the flow of antibiotic resistance from environmental bacteria to human pathogens. The present study compares the occurrence, diversity, and abundance of ARGs in industrial sludge, wetland sludge, and sediment sample contaminated with pharmaceutical discharge. Metagenomes were mined for the presence of ARGs against the ResFinder 3.2 database using BLASTn program. Pharmaceutical sample (2.52%) showed high degree of ARG abundance and richness as compared with ETP sludge (2.28%) and wetland sludge samples (1.29%). The modern resistome pattern represented by critically important resistance genes against tetracycline (tetA, tetC, tetW, tetT, and tetS/M) and quinolone (qnrS, qnrVC, and qnrD) was identified in pharmaceutical sediment sample. However, effluent treatment plant (ETP) sludge sample showed abundance of multidrug efflux pumps indicating the presence of primitive resistome profile. In conclusion, the indiscriminate distribution pattern of antibiotic resistance genes in three selected environmental sites suggests enrichment and distribution of environmental niche-driven resistance. The study also suggests effluent discharge site from pharmaceutical industries and ETPs as pivotal points of intervention for the mitigation of antibiotic resistance.