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- Comparison of culture- and quantitative PCR-Based indicators of antibiotic resistance in wastewater, recycled water, and tap waterPublication . Rocha, Jaqueline; Fernandes, Telma; Riquelme, Maria V.; Zhu, Ni; Pruden, Amy; Manaia, Célia M.Standardized methods are needed to support monitoring of antibiotic resistance in environmental samples. Culture-based methods target species of human-health relevance, while the direct quantification of antibiotic resistance genes (ARGs) measures the antibiotic resistance potential in the microbial community. This study compared measurements of tetracycline-, sulphonamide-, and cefotaxime-resistant presumptive total and fecal coliforms and presumptive enterococci versus a suite of ARGs quantified by quantitative polymerase chain reaction (qPCR) across waste-, recycled-, tap-, and freshwater. Cross-laboratory comparison of results involved measurements on samples collected and analysed in the US and Portugal. The same DNA extracts analysed in the US and Portugal produced comparable qPCR results (variation <28%), except for blaOXA-1 gene (0%–57%). Presumptive total and fecal coliforms and cefotaxime-resistant total coliforms strongly correlated with blaCTX-M and intI1 (0.725 ≤ R2 ≤ 0.762; p < 0.0001). Further, presumptive total and fecal coliforms correlated with the Escherichia coli-specific biomarkers, gadAB, and uidA, suggesting that both methods captured fecal-sourced bacteria. The genes encoding resistance to sulphonamides (sul1 and sul2) were the most abundant, followed by genes encoding resistance to tetracyclines (tet(A) and tet(O)) and β-lactams (blaOXA-1 and, blaCTX-M), which was in agreement with the culture-based enumerations. The findings can help inform future application of methods being considered for international antibiotic resistance surveillance in the environment.
- Investigating the impact of UV-C/H2O2 and sunlight/H2O2 on the removal of antibiotics, antibiotic resistance determinants and toxicity present in urban wastewaterPublication . G. Michael, Stella; Michael-Kordatou, Irene; Nahim-Granados, Samira; Polo-López, Maria Inmaculada; Rocha, Jaqueline; Martínez-Piernas, Ana B.; Fatta-Kassinos, Pilar; Manaia, Célia M.This work aimed at exploring the impact of UV-C/H2O2 and sunlight/H2O2 processes, applied at pilot scale, on removing: (i) ciprofloxacin and sulfamethoxazole, (ii) cultivable Escherichia coli and Pseudomonas aeruginosa grown in the presence and absence of sub-minimal inhibitory concentrations of ciprofloxacin and sulfamethoxazole and (iii) the genes 16S rRNA and selected antibiotic resistance genes (ARGs) (i.e., sul1, blaCTX-M, qnrS, tetM, etc.) from urban wastewater. The major antibiotic transformation products (TPs) formed, were elucidated and the chronic toxicity of the whole effluent mixture against Vibrio fischeri was evaluated. The capability of the processes, in terms of the elimination of the antibiotics present in urban wastewater, varied among the two light sources used: both antibiotics were fully removed during UV-C/Η2Ο2, whereas only ciprofloxacin was removed during the sunlight/H2O2. The photo-transformation of the antibiotics led to the identification of 21 and 18 TPs of ciprofloxacin and sulfamethoxazole, respectively, while all of them retained their core moiety, responsible for the antibacterial activity. All the UV-C/H2O2-treated samples were found to be toxic, whereas the luminescence of V. fischeri was not inhibited when tested in the sunlight/H2O2-treated samples. During both processes, E. coli, P. aeruginosa and the colonies of these species still viable in the presence of antibiotics, were successfully inactivated to values below the detection limit. However, sunlight/H2O2 has not achieved complete disinfection, as regrowth of E. coli and P. aeruginosa colonies was observed after 48 h of storage of the treated effluent. Finally, none of the technologies tested was able to completely remove the target ARGs, confirming their inability to prevent the spread of resistance determinants to the environment.
- Inter-laboratory calibration of quantitative analyses of antibiotic resistance genesPublication . Rocha, Jaqueline; Cacace, Damiano; Kampouris, Ioannis; Guilloteau, Hélène; Jäger, Thomas; Marano, Roberto B.M.; Karaolia, Popi; Manaia, Célia M.; Merlin, Christophe; Fatta-Kassinos, Despo; Cytryn, Eddie; Berendonk, Thomas U.; Schwartz, ThomasAntibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are widely distributed in the environment where they represent potential public health threats. Quantitative PCR (qPCR) is a suitable approach to detect and quantify ARGs in environmental samples. However, the comparison of gene quantification data between different laboratories is challenging since the data are predominantly obtained under non-harmonized conditions, using different qPCR protocols. This study aimed at carrying out an inter-laboratory calibration in order to assess the variability inherent to the qPCR procedures for quantification of ARGs. With this aim, samples of treated wastewater collected in three different countries were analysed based on common DNA extract pools and identical protocols as well as distinct equipment, reagents batches, and operators. The genes analysed were the 16S rRNA, vanA, blaTEM, qnrS, sul1, blaCTXM-32 and intI1 and the artificial pNORM1 plasmid containing fragments from the seven targeted genes was used as a reference. The 16S rRNA gene was the most abundant, in all the analysed samples, followed by intI1, sul1, qnrS, and blaTEM, while blaCTXM-32 and vanA were below the limit of quantification in most or all the samples. For the genes 16S rRNA, sul1, intI1, blaTEM and qnrS the inter-laboratory variation was below 28% (3–8%, 6–18%, 8–21%, 10–24%, 15–28%, respectively). While it may be difficult to fully harmonize qPCR protocols due to equipment, reagents and operator variations, the inter-laboratory calibration is an adequate and necessary step to increase the reliability of comparative data on ARGs abundance in different environmental compartments and/or geographic regions.
- Assessment of full-scale tertiary wastewater treatment by UV-C based-AOPs: removal or persistence of antibiotics and antibiotic resistance genes?Publication . Rodríguez-Chueca, Jorge; Giustina, Saulo Varella della; Rocha, Jaqueline; Fernandes, Telma; Pablos, Cristina; Encinas, Ángel; Barceló, Damià; Rodríguez-Mozaz, Sara; Manaia, Célia M.; Marugána, JavierAOPs based on the photolytic decomposition of H2O2 and peroxymonosulfate tested at low dosages (0.05-0.5 mM) and with very low UV-C contact time (4-18 s) demonstrated to be more efficient than UV-C radiation alone on the removal of the analyzed ABs. PMS (0.5 mM) combined with UV-C (7 s contact time) was the most efficient treatment in terms of AB removal: 7 out of 10 ABs detected in the waste water were removed more efficiently than using the other oxidants. In terms of ARGs removal efficiency, UV-C alone seemed the most efficient treatment, although H2O2/UV-C, PMS/UV-C and PMS/Fe(II)/UV-C were supposed to generate higher concentrations of free radicals. The results show that treatments with the highest removal of ABs and ARGs did not coincide, which could be attributed to the competition between DNA and oxidants in the absorption of UV photons, reducing the direct photolysis of the DNA. Whereas the photolytic ABs removal is improved by the generation of hydroxyl and sulfate radicals, the opposite behavior occurs in the case of ARGs. These results suggest that a compromise between ABs and ARGs removal must be achieved in order to optimize wastewater treatment processes.
- Bacterial lineages putatively associated with the dissemination of antibiotic resistance genes in a full-scale urban wastewater treatment plantPublication . Narciso-da-Rocha, Carlos; Rocha, Jaqueline; Vaz-Moreira, Ivone; Lira, Felipe; Tamames, Javier; Henriques, Isabel; Martinez, Jose Luis; Manaia, Célia M.Urban wastewater treatment plants (UWTPs) are reservoirs of antibiotic resistance. Wastewater treatment changes the bacterial community and inevitably impacts the fate of antibiotic resistant bacteria and antibiotic resistance genes (ARGs). Some bacterial groups are major carriers of ARGs and hence, their elimination during wastewater treatment may contribute to increasing resistance removal efficiency. This study, conducted at a full-scale UWTP, evaluated variations in the bacterial community and ARGs loads and explored possible associations among them. With that aim, the bacterial community composition (16S rRNA gene Illumina sequencing) and ARGs abundance (real-time PCR) were characterized in samples of raw wastewater (RWW), secondary effluent (sTWW), after UV disinfection (tTWW), and after a period of 3 days storage to monitoring possible bacterial regrowth (tTWW-RE). Culturable enterobacteria were also enumerated. Secondary treatment was associated with the most dramatic bacterial community variations and coincided with reductions of ~2 log-units in the ARGs abundance. In contrast, no significant changes in the bacterial community composition and ARGs abundance were observed after UV disinfection of sTWW. Nevertheless, after UV treatment, viability losses were indicated ~2 log-units reductions of culturable enterobacteria. The analysed ARGs (qnrS, blaCTX-M, blaOXA-A, blaTEM, blaSHV, sul1, sul2, and intI1) were strongly correlated with taxa more abundant in RWW than in the other types of water, and which associated with humans and animals, such as members of the families Campylobacteraceae, Comamonadaceae, Aeromonadaceae, Moraxellaceae and Bacteroidaceae. Further knowledge of the dynamics of the bacterial community during wastewater treatment and its relationship with ARGs variations may contribute with information useful for wastewater treatment optimization, aiming at a more effective resistance control.
- Do Klebsiella pneumoniae environmental strains maintain clinically relevant genomic and phenotypic traits?Publication . Rocha, Jaqueline; Ferreira, Catarina; Mil-Homens, Dalila; Brito, Margarida; Lameiras, Catarina; Fialho, Arsénio; Henriques, Isabel; Gomila, Margarita; Manaia, Célia M.
- Differential regulation of transition zone and centriole proteins contributes to ciliary base diversityPublication . Jana, Swadhin Chandra; Mendonça, Susana; Machado, Pedro; Werner, Sascha; Rocha, Jaqueline; Pereira, António; Maiato, Helder; Bettencourt-Dias, MónicaCilia are evolutionarily conserved structures with many sensory and motility-related functions. The ciliary base, composed of the basal body and the transition zone, is critical for cilia assembly and function, but its contribution to cilia diversity remains unknown. Hence, we generated a high-resolution structural and biochemical atlas of the ciliary base of four functionally distinct neuronal and sperm cilia types within an organism, Drosophila melanogaster. We uncovered a common scaffold and diverse structures associated with different localization of 15 evolutionarily conserved components. Furthermore, CEP290 (also known as NPHP6) is involved in the formation of highly diverse transition zone links. In addition, the cartwheel components SAS6 and ANA2 (also known as STIL) have an underappreciated role in basal body elongation, which depends on BLD10 (also known as CEP135). The differential expression of these cartwheel components contributes to diversity in basal body length. Our results offer a plausible explanation to how mutations in conserved ciliary base components lead to tissue-specific diseases
- Novel approaches on the characterization of the wastewater resistome : possible implications on human health and water quality managementPublication . Rocha, Jaqueline Maria Matias da; Rodrigues, Célia Maria Manaia; Henriques, Isabel da Silva; Ribas, Margarita GomilaAntibiotic resistant bacteria and antibiotic resistance genes are considered contaminants of emerging concern, nowadays widely disseminated in the environment. Urban wastewater treatment plants are major recipients and reservoirs for these contaminants. In urban these plants, wastewater is subjected to different types of treatment that reduce the levels of antibiotic resistant bacteria and antibiotic resistance genes, although not completely. Considering that most of the bacteria are not culturable and that this fraction might harbor antibiotic resistance genes, culture-independent methods are currently used to assess antibiotic resistance in the environment. Among these methods, quantitative PCR is considered the gold standard used to quantify antibiotic resistance genes in environmental samples, although the lack of harmonized methods seriously limits the reliable comparison of results obtained in different laboratories. Among the antibiotic resistant bacteria emitted by wastewater treatment plants, Enterobacteriaceae represent an important fraction and among these Klebsiella pneumoniae deserve special attention. Indeed, K. pneumoniae is a bacterial species that besides the clinical importance when associated to humans, can also be found in the environment, in soils, plants, water, and wastewater. The capacity of this bacterial species to thrive in different environments and in humans and animals increases its significance in terms of human health threat might constitute a human health threat. However, the traits that might be maintained or lost during the transit of K. pneumoniae through clinical and environmental contexts are still unknown. This thesis aimed to 1) advance the knowledge regarding the use of harmonized analytical quantitative PCR methods that might enable reliable comparisons of genes quantification ; 2) design a cell-based internal standard that could be used in different laboratories to assess losses during water samples filtration, DNA extraction and quantitative PCR quantification; and 3) contribute to a better understanding of the ecology of K. pneumoniae and infer about possible dynamics between clinic and environmental niches, with special focus on genetic diversity and antibiotic resistance stability. To tackle the first aim, genetic determinants encoding resistance to sulfonamides (sul1 and sul2), quinolones (qnrS), and β-lactams (blaTEM) and the 16S rRNA gene were monitored in DNA extracts supplied by partners who were investigating wastewater treatment processes, at full- or pilot-scale. In parallel, the influence on genes quantification of DNA shipment, quantitative PCR protocols, standards and equipment was studied in an interlaboratory comparison. These results and the literature available justified the efforts to meet the second aim. An internal standard, consisting in a cloned gene fragment not found in wastewater samples was designed and tested. This internal standard is to be used to spike wastewater or water samples aiming to control DNA losses during the processing of the sample and DNA extraction process. The emission of antibiotic resistant bacteria by wastewater treatment plants is an issue of concern, however it is not clear if these bacteria will survive and maintain their features once in the environment. To investigate this topic, K. pneumoniae was used as a model species and two distinct research approaches were used. A group of 3 rd generation cephalosporin-resistant K. pneumoniae isolates (25 wastewater; 34 clinical) was compared based on phenotypic, genotypic and genomic analyses (n=22) and a broader group of genomes collected from a public database (21 countries, 61 environmental; 78 clinical) was compared based on a core and pangenome approach and profiles of antibiotic and metal resistance, virulence, efflux systems, oxidative stress and quorum sensing traits. According to the results obtained and their analysis, it was concluded that wastewater treatment efficiency and wastewater quality regarding antibiotic resistance emissions should always be measured based on absolute abundance (per volume), rather than in relative abundance (per total bacteria). The interlaboratory comparison seemed to be reliable, although DNA extract quality and stability during shipment, as well as consumables and equipment specificities, may be critical for the monitoring findings. The use of a cell-based internal standard may contribute to overcome those limitations. This internal standard permitted to estimate the water matrix effect which was associated with an underestimation that ranged 0.1–0.9 log gene copy number mL−1 of sample, irrespective of the water type. Clinical and wastewater isolates were indistinguishable based on phenotypic and genotypic characterization, although distinct lineages may prevail in clinical or environmental settings. Genetic determinants related to efflux, oxidative stress or quorum sensing functions were common to clinical and wastewater isolates, while antibiotic and metal resistance or virulence genes, were variable across the genomes and associated with mobile genetic elements, mostly transposons, insertion sequences or integrative and conjugative elements. The analysis of a larger and geographically more diverse group of genomes, suggested that antibiotic and metal resistance and virulence gene alleles were more prevalent and diverse in clinical isolates, while some quorum sensing, efflux systems and oxidative stress genetic determinants alleles were more prevalent and diverse in environmental isolates. The studies performed unveil a promising opportunity to implement comparable and reliable antibiotic resistance monitoring schemes. The harmonization of some procedures and the use of internal standards will enable worldwide comparisons of antibiotic resistance genes in wastewaters, and therefore improve and promote surveillance studies worldwide. The double evidence that antibiotic resistance features observed in clinical K. pneumoniae are maintained in the environment and that it is among the environmental isolates that stress dwelling features seem to be more diverse, supports the high capacity of K. pneumoniae for spreading through wastewater or in the environment, enhancing the risks of transmission back to humans.
- Antibiotic resistance in wastewater treatment plants: tackling the black boxPublication . Manaia, Célia M.; Rocha, Jaqueline; Scaccia, Nazareno; Marano, Roberto; Radu, Elena; Biancullo, Francesco; Cerqueira, Francisco; Fortunato, Gianuário; Iakovides, Iakovos C.; Zammit, Ian; Kampouris, Ioannis; Vaz-Moreira, Ivone; Nunes, Olga C.Wastewater is among the most important reservoirs of antibiotic resistance in urban environments. The abundance of carbon sources and other nutrients, a variety of possible electron acceptors such as oxygen or nitrate, the presence of particles onto which bacteria can adsorb, or a fairly stable pH and temperature are examples of conditions favouring the remarkable diversity of microorganisms in this peculiar habitat. The wastewater microbiome brings together bacteria of environmental, human and animal origins, many harbouring antibiotic resistance genes (ARGs). Although numerous factors contribute, mostly in a complex interplay, for shaping this microbiome, the effect of specific potential selective pressures such as antimicrobial residues or metals, is supposedly determinant to dictate the fate of antibiotic resistant bacteria (ARB) and ARGs during wastewater treatment. This paper aims to enrich the discussion on the ecology of ARB&ARGs in urban wastewater treatment plants (UWTPs), intending to serve as a guide for wastewater engineers or other professionals, who may be interested in studying or optimizing the wastewater treatment for the removal of ARB&ARGs. Fitting this aim, the paper overviews and discusses: i) aspects of the complexity of the wastewater system and/or treatment that may affect the fate of ARB&ARGs; ii) methods that can be used to explore the resistome, meaning the whole ARB&ARGs, in wastewater habitats; and iii) some frequently asked questions for which are proposed addressing modes. The paper aims at contributing to explore how ARB&ARGs behave in UWTPs having in mind that each plant is a unique system that will probably need a specific procedure to maximize ARB&ARGs removal.
- One health approach reveals the absence of methicillin-resistant staphylococcus aureus in autochthonous cattle and their environmentsPublication . Correia, Susana; Silva, Vanessa; García-Díez, Juan; Teixeira, Paula; Pimenta, Kevin; Pereira, José E.; Oliveira, Soraia; Rocha, Jaqueline; Manaia, Célia M.; Igrejas, Gilberto; Poeta, PatríciaAntimicrobial resistance represents one of the greatest challenges of the twenty-first century, and it is globally recognized that addressing this problem requires a concerted One Health approach involving humans, animals, and the environment. Methicillin-resistant Staphylococcus aureus (MRSA) currently represents a global burden; it is resistant to almost all beta-lactams and some MRSA strains are highly multiresistant. S. aureus infection in cattle results in major economic losses in the food industry. Moreover, cases of livestock-associated MRSA strains responsible for invasive life-threatening infections have been reported among human patients in contact with infected or colonized animals. The autochthonous Maronesa cattle breed is a threatened rustic traditional Portuguese breed of mountain cattle of high importance for the Vila Real region. It has been used for centuries as motive power in all kinds of agricultural work and also for meat production, which is its current dominant use and the main source of economic value, being the Maronesa meat commercialized with PDO - Protected Designation of Origin. This study aimed to determine the prevalence and transmission of MRSA in cattle of the Maronesa breed, through a concerted One Health approach comprising human, water, and soil samples of the animals’ handlers and environments. In a total of 195, 63, 40, and 43 cattle, human, water, and soil samples screened in selective ORSAB media supplemented with 2 mg/L oxacillin; only one human sample harbored a MRSA isolate which was ascribed to spa-type t9413 and to ST30, one of the most common genetic lineages associated with community-acquired MRSA. Considering the increasing reports of MRSA isolation from cattle and handlers in Europe, the absence of this major human and animal pathogen in Maronesa cattle and their production systems represents a serendipitous result, valuing this important autochthonous breed. To our knowledge, this is the first study to determine MRSA prevalence and transmission in Maronesa cattle. Through a concerted One Health approach, this study revealed that the Maronesa cattle and their surrounding environments do not represent reservoirs for Methicillin-resistant Staphylococcus aureus.