In developing countries, where high levels of antimicrobial resistance are observed in hospitals, the surveillance of this phenomenon in wastewater treatment plants (WWTPs) and the environment is very limited, especially using cutting-edge culture-independent methods. In this study, the composition of bacterial communities, the resistome and mobilome (the pool of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs), respectively) at a WWTP were determined using shotgun metagenomics and culture-based approaches. Wastewater samples were collected at four sampling points of a WWTP in Antioquia, Colombia. A total of 24 metagenomes were analyzed. Specifically, there were marked differences in bacterial community composition, resistome, and mobilome, according to the WWTP sampling points. Bacterial families of clinical importance such as Moraxellaceae, Aeromonadaceae, and Enterobacteriaceae were mainly detected in the WWTP influent and effluent samples. Genes encoding resistance to macrolide-lincosamide-streptogramin, β-lactams, and those conferring multidrug resistance (e.g., acrB, adeG, and mexD) were the most abundant. Moreover, some clinically important ARGs such as blaKPC₋₂ and blaCTX₋M, and others not reported locally, such as blaTEM₋₁₉₆, blaGES₋₂₃, blaOXA₋₁₀, mcr-3, and mcr-5 were frequently detected. Co-occurrence network analyses indicated a significant association of ARGs such as blaOXA₋₅₈ and blaKPC genes with Aeromonadaceae and Enterobacteriaceae. Among the markers of MGEs, intI1 and ISCR8 were the most frequently detected. Altogether, this work reveals the importance of shotgun metagenomics and culture-based approaches in antimicrobial resistance studies. The findings also support that WWTPs are hotspots for antimicrobial resistance, whose analysis constitutes a powerful tool to predict the impact of antimicrobial resistance in a population.

A comparative study was conducted to (1) assess the potential of raw sewage used for urban agriculture to disseminate bacterial resistance in two cities of different size in Cameroon (Central Africa) and (2) compare the outcome with data obtained in Burkina Faso (West Africa). In each city, raw sewage samples were sampled from open-air canals in three neighbourhoods. After DNA extraction, the microbial population structure and function, presence of pathogens, antibiotic resistance genes and Enterobacteriaceae plasmids replicons were analysed using whole genome shotgun sequencing and bioinformatics. Forty-three pathogen-specific virulenc e factor genes were detected in the sewage. Eighteen different incompatibility groups of Enterobacteriaceae plasmid replicon types (ColE, A/C, B/O/K/Z, FIA, FIB, FIC, FII, H, I, N, P, Q, R, T, U, W, X, and Y) implicated in the spread of drug-resistance genes were present in the sewage samples. One hundred thirty-six antibiotic resistance genes commonly associated with MDR plasmid carriage were identified in both cities. Enterobacteriaceae plasmid replicons and ARGs found in Burkina Faso wastewaters were also present in Cameroon waters. The abundance of Enterobacteriaceae, plasmid replicons and antibiotic resistance genes was greater in Yaounde, the city with the greater population.In conclusion, the clinically relevant environmental resistome found in raw sewage used for urban agriculture is common in West and Central Africa. The size of the city impacts on the abundance of drug-resistant genes in the raw sewage while ESBL gene abundance is related to the prevalence of Enterobacteriaceae along with plasmid Enterobacteriaceae abundance associated to faecal pollution.

The study of aerosol dispersion characteristics in wastewater treatment plants (WWTPs) has attracted extensive attention. Oxidation ditch (OD) is a commonly implemented process during biological wastewater treatment. This study assessed the component characteristics, source apportionment, and exposure risks of aerosols generated from a WWTP using the OD process (AWO). The results indicated that the aeration part of oxidation ditch (ODA) exhibited the highest concentrations and proportions of the respiratory fractions (RF) of bacteria, Enterobacteriaceae, Staphylococcus aureus, and Pseudomonas aeruginosa. Some pathogenic or opportunistic-pathogenic bacteria and carcinogenic metal(loid)s were detected in the AWO. The source apportionment results indicated that the outdoor wastewater treatment processes and ambient air contributed to the constitution of the AWO. The indoor aerosols were mainly constituted by composition of the wastewater treatment process such as the sludge dewatering room (SDR). The pathogenic or opportunistic-pathogenic bacteria with eight genera (Colinsella, Dermatophilus, Enterobactor, Erycherichia-Shigella, Ledionella, Selenomonas, Xanthobacter, and Veillonella) were largely attributed to wastewater or sludge. The risk assessment suggested that inhalation was the main exposure pathway for aerosols (including bacteria and metal(loid)s). Additionally, As indicated the highest non-carcinogenic risks. Furthermore, As, Cd, and Co were associated with high carcinogenic risks. The ODA and sludge dewatering room (SDR) indicated the highest carcinogenic and non-carcinogenic risks of metal(loid)s, respectively. Thus, the AWO should be sufficiently researched and monitored to mitigate their harmful effects on human health, particularly with regard to the health of the site workers.

Discharge of antimicrobial residues and resistant bacteria in hospital effluents is supposed to have strong impacts on the spread of antibiotic resistant bacteria in the environment. This study aimed to characterize the effluents of the Gabriel Montpied teaching hospital, Clermont-Ferrand, France, by simultaneously measuring the concentration of ciprofloxacin and of biological indicators resistant to this molecule in biofilms formed in the hospital effluent and by comparing these data to ciprofloxacin consumption and resistant bacterial isolates of the hospital. Determination of the measured environmental concentration of ciprofloxacin by spot sampling and polar organic chemical integrative (POCIS) sampling over 2 weeks, and comparison with predicted environmental concentrations produced a hazard quotient >1, indicating a potential ecotoxicological risk. A negative impact was also observed with whole hospital effluent samples using the Tetrahymena pyriformis biological model.During the same period, biofilms were formed within the hospital effluent, and analysis of ciprofloxacin-resistant isolates indicated that Gamma-Proteobacteria were numerous, predominantly Aeromonadaceae (69.56%) and Enterobacteriaceae (22.61%). Among the 115 isolates collected, plasmid-mediated fluoroquinolone-resistant genes were detected, with mostly aac(6′)-lb-cr and qnrS. In addition, 60% of the isolates were resistant to up to six antibiotics, including molecules mostly used in the hospital (aminosides and third-generation cephalosporins).In parallel, 1247 bacteria isolated from hospitalized patients and resistant to at least one of the fluoroquinolones were collected. Only 5 of the 14 species identified in the effluent biofilm were also found in the clinical isolates, but PFGE typing of the Gram-negative isolates found in both compartments showed there was no clonality among the strains.Altogether, these data confirm the role of hospital loads as sources of pollution for wastewater and question the role of environmental biofilms communities as efficient shelters for hospital-released resistance genes.

Carbapenems are used as last-resort drugs to treat infections caused by multidrug-resistant bacteria. Despite the increasing number of reports of carbapenem-resistant Enterobacteriaceae (CRE), there is still limited information on their distribution or prevalence in the environment. Our aim was to assess the occurrence of CRE in the Lis river (Portugal) and to characterize the genetic platforms linked to carbapenemase genes. We collected six water samples from sites near a wastewater treatment plant (n = 4 samples) and livestock farms (n = 2). Twenty-four CRE were characterized by BOX element-polymerase chain reaction (BOX-PCR), and thirteen representative isolates were analysed by Pulsed-Field Gel Electrophoresis (PFGE) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility testing, PCR screening for carbapenemase-encoding genes, conjugation experiments and plasmid analysis were performed. Four isolates were chosen for whole-genome sequencing. All water samples contained CRE (4.0 CFU/mL on average). Representative isolates were multidrug-resistant (resistant to ciprofloxacin, trimethoprim-sulfamethoxazole and to all β-lactams tested) and were identified as K. pneumoniae, Enterobacter and Citrobacter. Isolates carried plasmids and harboured carbapenemase-encoding genes: blaKPC₋₃ in K. pneumoniae (n = 9), blaNDM₋₁ in Enterobacter (n = 3) and blaGES₋₅ in Citrobacter (n = 1). Conjugation experiments were successful in two Klebsiella isolates. Enterobacter PFGE profiles grouped in one cluster while Klebsiella were divided in three clusters and a singleton. Whole-genome sequencing analysis revealed blaGES₋₅ within a novel class 3 integron (In3-16) located on an IncQ/pQ7-like plasmid in Citrobacter freundii CR16. blaKPC₋₃ was present on IncFIA-FII pBK30683-like plasmids, which were subsequently confirmed in all K. pneumoniae (n = 9). Furthermore, blaKPC₋₃ was part of a genomic island in K. pneumoniae CR12. In E. roggenkampii CR11, blaNDM₋₁ was on an IncA/C₂ plasmid. The carbapenemase-encoding plasmids harboured other resistance determinants and mobile genetic elements. Our results demonstrate that Lis river is contaminated with CRE, highlighting the need for monitoring antibiotic resistance in aquatic environments, especially to last-resort drugs.

Carbapenems are β-lactam antibiotics with a broad spectrum of activity and are usually considered the last resort for the treatment of severe infections caused by multidrug-resistant pathogens. The clinically most significant carbapenemases are KPC, NDM, and OXA-48-like enzymes, whose genes have been increasingly reported worldwide in members of the family Enterobacteriaceae. In this study, we quantified the abundance of these genes in wastewater effluents from different Tunisian hospitals. The blaNDM and blaOXA-48-like genes were detected at similar concentrations in all hospital wastewater effluents. In contrast, the blaKPC gene was detected at lower concentration than other genes and it was only detected in three of the seven effluents analyzed. To the best of our knowledge, this study quantified for the first time the abundance of blaKPC, blaNDM, and blaOXA-48-like genes in wastewater effluents from Tunisian hospitals, highlighting the widespread distribution of these carbapenemase genes.

The spread of carbapenemase-producing bacteria is a worldwide concern as it challenges healthcare, especially considering the insufficient development of antimicrobials. These microorganisms have been described not only in hospitals, but also in several environmental settings including recreational waters. Community exposure to antimicrobial-resistant bacteria through recreation might be relevant for human health, but risk assessment studies are lacking. Absence of effective and feasible monitoring in recreational aquatic matrices contributes to such a knowledge gap. Here, we aimed at assessing predictors of occurrence of medically relevant carbapenemase-producing bacteria in coastal waters. We quantitatively assessed recovery of carbapenemase-producing Enterobacteriaceae, Pseudomonas spp., Acinetobacter spp. and Aeromonas spp. in superficial coastal waters showing distinct pollution history across one year, and registered data regarding tide regimen, 7-days pluviosity, salinity, pH, water temperature. We analyzed data using General Estimating Equation (GEE) to assess predictors of such occurrence. Our results suggest that the sampling site had the strongest effect over concentration of these antimicrobial-resistant microorganisms, followed by pollution indexes and tide regimen. Increased salinity, advanced sampling time, water temperature, rainfall and decrease of pH were related to decrease concentrations. We provide a list of factors that could be easily monitored and further included in models aiming at predicting occurrence of carbapenemase producers in coastal waters. Our study may encourage researchers to further improve this list and validate the model proposed, so that monitoring and future public policies can be developed to control the spread of antimicrobial resistance in the environment.

The severe and pervasive effects of multispecies foodborne microbial biofilms highlight the importance of rapid detection and diagnosis of contamination risk in the field using epifluorescence-based techniques (EBT) combined with automatic image-counting software. This study screened the hygiene quality of the environment, the carcass and the slaughtering equipment in the El-Kharga abattoir, New Valley Province, Egypt, to assess possible contamination during slaughter process. In addition, biofilm was assessed, and bacteria was enumerated by epifluorescence microscopy. Using both conventional and EBT, the highest bacterial counts were observed for the slaughtering equipment (6.6 and 5.2 cfu/cm2, respectively), followed by different parts of the carcass (4.1 and 4.4 cfu/cm2, respectively) and environmental samples (3.9 and 4.1 cfu/cm2, respectively). A high prevalence of E. coli O157:H7 was observed on the slaughtering equipment (25%), which also led to carcass (1%) contamination. Moreover, Enterobacteriaceae members were detected during examination, such as Klebsiella pneumoniae, Enterobacter aerogenes, and Raoultella ornithinolytica. Despite the relatively good hygiene quality of the abattoir environment, there is also a high risk associated with biofilm formation by pathogenic microorganisms on the slaughtering equipment. Moreover, EBT showed different structures of the biofilm, including those formed at different maturation stages, such as voids, microbubbles, channels and mushroom shapes. (EBT) microscopy combined with image-counting software could be a candidate substitute to estimate efficiently, precisely and rapidly the microbial aggregation and exposure risk in field than the conventional counting techniques.

Freshwater environments are susceptible to possible contamination by residual antibiotics that are released through different sources, such as agricultural runoffs, sewage discharges and leaching from nearby farms. Freshwater environment can thus become reservoirs where an antibiotic impact microorganisms, and is an important public health concern. Degradation and dilution processes are fundamental for predicting the actual risk of antibiotic resistance dissemination from freshwater reservoirs. This study reviews major approaches for detecting and quantifying antibiotic resistance bacteria (ARBs) and genes (ARGs) in freshwater and their prevalence in these environments. Finally, the role of dilution, degradation, transmission and the persistence and fate of ARB/ARG in these environments are also reviewed. Culture-based single strain approaches and molecular techniques that include polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR) and metagenomics are techniques for quantifying ARB and ARGs in freshwater environments. The level of ARBs is extremely high in most of the river systems (up to 98% of the total detected bacteria), followed by lakes (up to 77% of the total detected bacteria), compared to dam, pond, and spring (<1%). Of most concern is the occurrence of extended-spectrum β-lactamase producing Enterobacteriaceae, methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE), which cause highly epidemic infections. Dilution and natural degradation do not completely eradicate ARBs and ARGs in the freshwater environment. Even if the ARBs in freshwater are effectively inactivated by sunlight, their ARG-containing DNA can still be intact and capable of transferring resistance to non-resistant strains. Antibiotic resistance persists and is preserved in freshwater bodies polluted with high concentrations of antibiotics. Direct transmission of indigenous freshwater ARBs to humans as well as their transitory insertion in the microbiota can occur. These findings are disturbing especially for people that rely on freshwater resources for drinking, crop irrigation, and food in form of fish.

The toxic sensitivity in different physiological levels of chromium (Cr) contaminated soils with environmentally equivalent concentrations (EEC) was fully unknown. The earthworm Eisenia fetida was exposed to a Cr-contaminated soil at the EEC level (referred to as Cr-CS) to characterize the induced toxicity at the whole body, organ, tissue, subcellular structure and metabolic levels. The results showed that the survival rate, weight and biodiversity of the gut microorganisms (organ) had no significant difference (p > 0.05) between control and Cr-CS groups. Qualitative histopathological and subcellular evaluations from morphology showed earthworms obvious injuries. The organelle injuries combined with the metabolic changes provided additional evidence that the Cr-CS damaged the nucleus and probably disturbed the nucleic acid metabolism of earthworms. 2-hexyl-5-ethyl-3-furansulfonate, dimethylglycine, betaine and scyllo-inositol were sensitive and relatively quantitative metabolites that were recommended as potential biomarkers for Cr-CS based on their significant weights in the multivariate analysis model. In addition, the relative abundance of Burkholderiaceae, Enterobacteriaceae and Microscillaceae of the earthworm guts in the Cr-CS group significantly increased, particularly for Burkholderiaceae (increased by 13.1%), while that of Aeromonadaceae significantly decreased by 5.6% in contrast with the control group. These results provided new insights into our understanding of the toxic effects of the EEC level of Cr contaminated soil from different physiological levels of earthworms and extend our knowledge on the composition and sensitivity of the earthworm gut microbiota in Cr contaminated soil ecosystems. Furthermore, these toxic responses from gut microorganisms to metabolites of earthworms provided important data to improve the adverse outcome pathway and toxic mechanism of the Cr-CS if the earthworm genomics and proteomics would be also gained in the future.