Metagenomics insights into microbiome and antibiotic resistance genes from free living amoeba in chlorinated wastewater effluents.

Free living amoeba (FLA) are among the organisms commonly found in wastewater and are well-established hosts for diverse microbial communities. Despite its clinical significance, there is little knowledge on the FLA microbiome and resistome, with previous studies relying mostly on conventional approaches. In this study we comprehensively analyzed the microbiome, antibiotic resistome and virulence factors (VFs) within FLA isolated from final treated effluents of two wastewater treatment plants (WWTPs) using shotgun metagenomics. Acanthamoeba has been identified as the most common FLA, followed by Entamoeba. The bacterial diversity showed no significant difference (p > 0.05) in FLA microbiomes obtained from the two WWTPs. At phylum level, the most dominant taxa were Proteobacteria, followed by Firmicutes and Actinobacteria. The most abundant genera identified were Enterobacter followed by Citrobacter, Paenibacillus, and Cupriavidus. The latter three genera are reported here for the first time in Acanthamoeba. In total, we identified 43 types of ARG conferring resistance to cephalosporins, phenicol, streptomycin, trimethoprim, quinolones, cephalosporins, tigecycline, rifamycin, and kanamycin. Similarly, a variety of VFs in FLA metagenomes were detected which included flagellar proteins, Type IV pili twitching motility proteins (pilH and rpoN), alginate biosynthesis genes AlgI, AlgG, AlgD and AlgW and Type VI secretion system proteins and general secretion pathway proteins (tssM, tssA, tssL, tssK, tssJ, fha, tssG, tssF, tssC and tssB, gspC, gspE, gspD, gspF, gspG, gspH, gspI, gspJ, gspK, and gspM). To the best of our knowledge, this is the first study of its kind to examine both the microbiomes and resistome in FLA, as well as their potential pathogenicity in treated effluents. Additionally, this study showed that FLA can host a variety of potentially pathogenic bacteria including Paenibacillus, and Cupriavidus that had not previously been reported, indicating that their relationship may play a role in the spread and persistence of antibiotic resistant bacteria (ARBs) and antibiotic resistance genes (ARGs) as well as the evolution of novel pathogens.

Insights into the role of extracellular polymeric substances (EPS) in the spread of antibiotic resistance genes.

Antibiotic resistance genes (ARG) are prevalent in aquatic environments. Discharge from wastewater treatment plants is an important point source of ARG release into the environment. It has been reported that biological treatment processes may enhance rather than remove ARG because of their presence in sludge. Attenuation of ARG in biotechnological processes has been studied in depth, showing that many microorganisms can secrete complex extracellular polymeric substances (EPS). These EPS can serve as multifunctional elements of microbial communities, involving aspects, such as protection, structure, recognition, adhesion, and physiology. These aspects can influence the interaction between microbial cells and extracellular ARG, as well as the uptake of extracellular ARG by microbial cells, thus changing the transformative capability of extracellular ARG. However, it remains unclear whether EPS can affect horizontal ARG transfer, which is one of the main processes of ARG dissemination. In light of this knowledge gap, this review provides insight into the role of EPS in the transmission of ARGs; furthermore, the mechanism of ARG spread is analyzed, and the molecular compositions and functional properties of EPS are summarized; also, how EPS influence ARG mitigation is addressed, and factors impacting how EPS facilitate ARG during wastewater treatment are summarized. This review provides comprehensive insights into the role of EPS in controlling the transport and fate of ARG during biodegradation processes at the mechanistic level.

Effects of different laying periods on airborne bacterial diversity and antibiotic resistance genes in layer hen houses.

Poultry farms are a complex environment for close contact between humans and animals. Accumulating evidence has indicated that pathogens and drug resistance genes in chicken houses may pose a serious threat to public health and economic concerns. However, insufficient knowledge of the indoor aerosol microbiome and resistome profiles of layer hen houses hampers the understanding of their health effects. Environmental surveillance of antibiotic resistance may contribute to a better understanding and management of the human exposure risk of bioaerosols under the environmental conditions of chicken houses. In addition, the chicken house has a long operation cycle, and the bacterial diversity and antibiotic resistance genes of aerosols in different periods may be different. In this study, air samples were collected from 18 chicken houses on three farms, including the early laying period (EL), peak laying period (PL), and late laying period (LL). 16S rRNA gene sequencing and metagenomics were used to study the composition of the bacteria and resistome in aerosols of layer hen houses and the results showed that they varied with laying period. The highest alpha diversity of bacteria was observed in PL bioaerosols. The dominant bacterial phyla included Firmicutes, Bacteroidetes and Proteobacteria. Three potential pathogenic bacterial genera (Bacteroides, Corynebacterium and Fusobacterium) were found. The most abundant ARG type was aminoglycosides in all laying periods. In total, 22 possible ARG host genera were detected. ARG subtypes and abundance were both higher in LL. Network analysis also showed higher co-occurrence patterns between the bacteria and resistome in bioaerosols. The laying period plays an important role in the bacterial community and resistome in layer house aerosols.

A Novel Phytogenic Formulation, EUBIO-BPSG, as a Promising One Health Approach to Replace Antibiotics and Promote Reproduction Performance in Laying Hens.

Gut microbiota play a key role in health maintenance and disease pathogenesis in animals. Dietary phytochemicals are crucial factors shaping gut bacteria. Here, we investigated the function and mechanism of a phytogenic formulation, EUBIO-BPSG (BP), in laying hens. We found that BP dose-dependently improved health and egg production in 54-week-old hens. Furthermore, BP was correlated with increased fecal Lactobacillus, decreased Escherichia coli and Salmonella enterica, and reduced antibiotic resistance (AR) and antibiotic resistance genes (ARG) in chicken stools. The 16S rDNA data showed that BP increased seven genera of probiotics and reduced 13 genera of pathogens in chicken feces. In vitro co-culture experiments showed that BP at 4 µg/mL and above promoted growth of L. reuteri while large 100- and 200-fold higher doses suppressed growth of E. coli and S. enterica, respectively. Mechanistic studies indicated that L. reuteri and its supernatants antagonized growth of E. coli and S. enterica but not vice-versa. Five short-chain fatty acids and derivatives (SCFA) produced from L. reuteri directly killed both pathogens via membrane destruction. Furthermore, BP inhibited conjugation and recombination of ARG via interference with conjugation machinery and integrase activity in E. coli. Collectively, this work suggests that BP promotes host health and reproductive performance in laying hens through regulation of gut microbiota through increasing probiotics and decreasing pathogens and spreading ARG.

Study of antibiotic resistance in freshwater ecosystems with low anthropogenic impact.

This study aimed to investigate the bacterial diversity and the background level of antibiotic resistance in two freshwater ecosystems with low anthropogenic impact in order to evaluate the presence of natural antimicrobial resistance in these areas and its potential to spread downstream. Water samples from a pre-Alpine and an Apennine river (Variola and Tiber, respectively) were collected in three different sampling campaigns and bacterial diversity was assessed by 16S sequencing, while the presence of bacteria resistant to five antibiotics was screened using a culturable approach. Overall bacterial load was higher in the Tiber River compared with the Variola River. Furthermore, the study revealed the presence of resistant bacteria, especially the Tiber River showed, for each sampling, the presence of resistance to all antibiotics tested, while for the Variola River, the detected resistance was variable, comprising two or more antibiotics. Screening of two resistance genes on a total of one hundred eighteen bacterial isolates from the two rivers showed that blaTEM, conferring resistance to ß-lactam antibiotics, was dominant and present in ~58 % of isolates compared to only ~9 % for mefA/E conferring resistance to macrolides. Moreover, ß-lactam resistance was detected in various isolates showing also resistance to additional antibiotics such as macrolides, aminoglycosides and tetracyclines. These observations would suggest the presence of co-resistant bacteria even in non-anthropogenic environments and this resistance may spread from the environment to humans and/or animals.

Good microbes, bad genes? The dissemination of antimicrobial resistance in the human microbiome.

A global rise in antimicrobial resistance among pathogenic bacteria has proved to be a major public health threat, with the rate of multidrug-resistant bacterial infections increasing over time. The gut microbiome has been studied as a reservoir of antibiotic resistance genes (ARGs) that can be transferred to bacterial pathogens via horizontal gene transfer (HGT) of conjugative plasmids and mobile genetic elements (the gut resistome). Advances in metagenomic sequencing have facilitated the identification of resistome modulators, including live microbial therapeutics such as probiotics and fecal microbiome transplantation that can either expand or reduce the abundances of ARG-carrying bacteria in the gut. While many different gut microbes encode for ARGs, they are not uniformly distributed across, or transmitted by, various members of the microbiome, and not all are of equal clinical relevance. Both experimental and theoretical approaches in microbial ecology have been applied to understand differing frequencies of ARG horizontal transfer between commensal microbes as well as between commensals and pathogens. In this commentary, we assess the evidence for the role of commensal gut microbes in encoding antimicrobial resistance genes, the degree to which they are shared both with other commensals and with pathogens, and the host and environmental factors that can impact resistome dynamics. We further discuss novel sequencing-based approaches for identifying ARGs and predicting future transfer events of clinically relevant ARGs from commensals to pathogens.

Knowledge gaps in the assessment of antimicrobial resistance in surface waters.

The spread of antibiotic resistance in the water environment has been widely described. However, still many knowledge gaps exist regarding the selection pressure from antibiotics, heavy metals and other substances present in surface waters as a result of anthropogenic activities, as well as the extent and impact of this phenomenon on aquatic organisms and humans. In particular, the relationship between environmental concentrations of antibiotics and the acquisition of ARGs by antibiotic-sensitive bacteria as well as the impact of heavy metals and other selective agents on antimicrobial resistance (AMR) need to be defined. Currently, established safety values are based on the effects of antibiotic toxicity neglecting the question of AMR spread. In turn, risk assessment of antibiotics in waterbodies remains a complex question implicating multiple variables and unknowns reinforced by the lack of harmonized protocols and official guidelines. In the present review, we discussed current state-of-the-art and the knowledge gaps related to pressure exerted by antibiotics and heavy metals on aquatic environments and their relationship to the spread of AMR. Along with this latter, we reflected on (i) the risk assessment in surface waters, (ii) selective pressures contributing to its transfer and propagation and (iii) the advantages of metagenomics in investigating AMR. Furthermore, the role of microplastics in co-selection for metal and antibiotic resistance, together with the need for more studies in freshwater are highlighted.

[Mutual Influence Between Microbial Community, Wastewater Characteristics, and Antibiotic Resistance Genes During Spiramycin Production Wastewater Treatment].

Wastewater from antibiotic production usually contains a huge amount of antibiotic resistance genes (ARG). Therefore, it is essential to study the dissemination and control of antibiotic resistance during the treatment of antibiotic production wastewater. The mutual influence between microbial community evolution, wastewater characteristics, and ARG was investigated using high-throughput sequencing and a variety of statistical analysis methods. Results showed that the influent characteristics had only a marginal influence on the microbial community of each treatment section. Methanogenic bacteria and sulfate-reducing bacteria were the dominant microbes in the anaerobic and anoxic tank. Chemical oxygen demand (COD), NO2--N, and PO43--P exhibited an intimate relationship with the microbial community, whereas biomass, NH4+-N, and COD showed a strong correlation with ARG and mobile genetic elements (MGE). In the sludge, more genera (including pathogenic bacteria) were significantly correlated with ARG and MGE than that in the wastewater, indicating that bacteria in the sludge had a greater chance of acquiring pathogenicity and resistance. Therefore, more attnetion should be given to waste sludge from the treatment plants of antibiotic production wastewater. This research could provide further understanding of antibiotic resistance dissemination and control during wastewater treatment, especially for antibiotic production wastewater.

Fecal indicators, pathogens, antibiotic resistance genes, and ecotoxicity in Galveston Bay after Hurricane Harvey.

Unprecedented rainfall after Hurricane Harvey caused a catastrophic flood in the southern coast of Texas, and flushed significant floodwater and sediments into Galveston Bay, the largest estuary along the Texas Gulf Coast. This study investigated the immediate and long-term (6 months post-Harvey) fecal indicators, pathogenic bacteria, antibiotic resistance genes (ARGs), and ecotoxicity in the Galveston Bay. Dramatic decrease of salinity profile to zero, increased levels of fecal indicator bacteria and pathogenic bacteria, and detection of various ARGs were observed in the water and sediment samples collected 2 weeks post-Harvey. High levels of BlaTEM and cytotoxicity measured by yeast bioluminescent assay (BLYR) were also observed especially near the river mouths. While Vibrio spp. was dominant in water, much higher abundance of fecal indicator bacteria and pathogen were detected in the sediments. A decreasing trend of BlaTEM and cytotoxicity was observed in March 2018 samples, suggesting the Bay has returned to its pre-hurricane conditions 6 months post-Harvey. Interestingly, the abundance of fecal indicator bacteria and pathogens were shifted dramatically according to high-streamflow and low-streamflow seasons in the Bay. The data are useful to construct the model of risk assessment in coastal estuaries system and predict the effects of extreme flooding events in the future.

[Occurrence of Antibiotic Resistance Genes and Bacterial Community Structure of Different Sludge Samples During Microwave Pretreatment-Anaerobic Digestion].

The waste sludge of municipal wastewater treatment plants is an important reservoir for antibiotic resistance genes (ARG). It is necessary to explore the fate of ARG, microbial community succession, and the correlations between them. Therefore, the distribution of ARG and the microbial community structure of waste sludge from wastewater treatment plants with A2O and A2O-MBR processes during microwave pretreatment and anaerobic digestion were studied in this research. The results showed that the occurrence of ARG and the microbial community structure were quite different in the waste sludge of A2O and A2O-MBR processes. The microwave pretreatment did not change the microbial community much, whereas the community structure of the digested sludge with pretreatment showed significant differences. Anaerobic digestion had a conformity effect on the distribution of ARG and MGE in the digested sludge with or without pretreatment. Among genes, ermF, qnrS, and blaNDM-1 were the most difficult to be reduced ARG and were prone to propagation during anaerobic digestion. The influence of biomass, ammonia nitrogen, and phosphorus on the distribution of ARG and MGE was higher than that of other environmental factors. The sludge characteristics also showed important impacts on the microbial community, especially on some genera with specific functions. These results could help people to better understand the spread and control of ARG during sludge anaerobic digestion.

Evaluation of Potential ARG Packaging by Two Environmental T7-Like Phage during Phage-Host Interaction.

The increase in antimicrobial resistance is a threat to both human and animal health. The transfer of antibiotic resistance genes (ARG) via plasmids has been studied in detail whereas the contribution of bacteriophage-mediated ARG transmission is relatively little explored. We isolated and characterized two T7-like lytic bacteriophages that infected multidrug-resistant Escherichia coli hosts. The morphology and genomic analysis indicated that both phage HZP2 and HZ2R8 were evolutionarily related and their genomes did not encode ARGs. However, ARG-like raw reads were detected in offspring sequencing data with a different abundance level implying that potential ARG packaging had occurred. PCR results demonstrated that six fragments of genes (qnrS, cmlA, tetM, blaTEM, sul3, mcr-1) were potentially packaged by phage HZP2 and four (qnrS, cmlA, blaTEM, mcr-1) by phage HZ2R8. Further quantitative results showed that ARG abundance hierarchies were similar. The gene blaTEM was the most abundant (up to 1.38 × 107 copies/mL) whereas cmlA and qnrS were the least. Moreover, the clinically important mcr-1 gene was the second most abundant ARG indicating a possibility for spread through generalized transduction. Together, our results indicated that these structurally similar phage possessed similar characteristics and potential packaging during phage-host interaction displayed an ARG preference rather than occurring randomly.

River Ganges water as reservoir of microbes with antibiotic and metal ion resistance genes: High throughput metagenomic approach.

The large scale usage of antibiotics and trace elements leads to their progressive release in the environment, and ultimately the spread of antibiotic resistance genes (ARGs) and metal ion resistance genes (MRGs) in bacteria. A high-throughput metagenomic sequencing of the microbial community in water and sediments in the river Ganges harboring resistance genes was performed. The results revealed that the river harbors a broad spectrum of resistance genes with high abundance in sediments. The highly dominant ARGs type was beta-lactam, multidrug/efflux and elfamycin. The ARGs such as (tuf, parY, ileS, mfd) were highly abundant in water and sediments. The MRGs subtype acn was the most abundant metal resistance gene in water and sediments. Majority of ARGs types showed significant (p ≤ 0.05) positive correlation with the MRGs types in the river environment suggesting their distribution and transfer to be possibly linked. Taxonomic classification revealed that Proteobacteria and Actinobacteria were the two most abundant phyla in water and sediments. Arcobacter, Terrimicrobium, Acidibacter and Pseudomonas were the most abundant genera. This study suggests that antibiotics and metals are the driving force for the emergence of resistance genes, and their subsequent propagation and accumulation in the environmental bacteria. The present metagenomic investigation highlights significance of such study, and attracts attention for the mitigation of pollutants associated with the propagation of ARGs and MRGs in the river environment.

Characterization of Bacterial Communities and Their Antibiotic Resistance Profiles in Wastewaters Obtained from Pharmaceutical Facilities in Lagos and Ogun States, Nigeria.

In Nigeria, pharmaceutical wastewaters are routinely disseminated in river waters; this could be associated with public health risk to humans and animals. In this study, we characterized antibiotic resistant bacteria (ARB) and their antibiotic resistance profile as well as screening for sul1 and sul2 genes in pharmaceutical wastewater effluents. Bacterial composition of the wastewater sources was isolated on non-selective media and characterized by the polymerase chain reaction (PCR) amplification of the 16S rRNA genes, with subsequent grouping using restriction fragment length polymorphism (RFLP) and sequencing. The antibiotics sensitivity profiles were investigated using the standard disk diffusion plate method and the minimum inhibitory concentrations (MICs) of selected antibiotics on the bacterial isolates. A total of 254 bacterial strains were isolated, and majority of the isolates were identified as Acinetobacter sp., Klebsiella pneumonia, Proteus mirabilis, Enterobacter sp. and Bacillus sp. A total of 218 (85.8%) of the bacterial isolates were multidrug resistant. High MICs values were observed for all antibiotics used in the study. The result showed that 31.7%, 21.7% and 43.3% of the bacterial isolates harbored sul1, sul2, and Intl1 genes, respectively. Pharmaceuticals wastewaters are potential reservoirs of ARBs which may harbor resistance genes with possible risk to public health.

The role of class I integrons in the dissemination of sulfonamide resistance genes in the Pearl River and Pearl River Estuary, South China.

Antibiotic resistance genes (ARGs), as a newly emerging contaminant, are unique because they are disseminated through horizontal gene transfer in the environment. In the present study, a class 1 integron gene (int1) and various ARGs (sul1, sul2, sul3, qnrS, and ermB) were measured in water and sediment samples from the Pearl River (PR) to the Pearl River Estuary (PRE), where there is a distinct gradient in anthropogenic impact. The int1, sul1, and sul2 genes were detected in all samples, and their concentrations exhibited a clear trend of decline consistent with anthropogenic impact. Both the int1 and sul genes had dynamically migrated between water and sediments. The relative abundance of the int1 gene normalized to the 16S rRNA gene correlated significantly with the total concentrations of antibiotics in water and sediments. Good correlations were also observed between the abundance of int1 and each type of sul gene in the samples. However, the sul1 gene showed a much stronger relationship with int1 in different seasons, probably due to the presence of sul1 in the conserved region of class 1 integron. Our results strongly support that integrons play an important role in the dissemination of ARGs in human-impacted aquatic environments.