Molecular epidemiology and mechanisms of carbapenem and colistin resistance in Klebsiella and other Enterobacterales from treated wastewater in Croatia.

Among the most problematic bacteria with clinical relevance are the carbapenem-resistant Enterobacterales (CRE), as there are very limited options for their treatment. Treated wastewater can be a route for the release of these bacteria into the environment and the population. The aim of this study was to isolate CRE from treated wastewater from the Zagreb wastewater treatment plant and to determine their phenotypic and genomic characteristics. A total of 200 suspected CRE were isolated, 148 of which were confirmed as Enterobacterales by MALDI-TOF MS. The predominant species was Klebsiella spp. (n = 47), followed by Citrobacter spp. (n = 40) and Enterobacter cloacae complex (cplx.) (n = 35). All 148 isolates were carbapenemase producers with a multidrug-resistant phenotype. Using multi-locus sequence typing and whole-genome sequencing (WGS), 18 different sequence types were identified among these isolates, 14 of which were associated with human-associated clones. The virulence gene analysis of the sequenced Klebsiella isolates (n = 7) revealed their potential pathogenicity. PCR and WGS showed that the most frequent carbapenemase genes in K. pneumoniae were blaOXA-48 and blaNDM-1, which frequently occurred together, while blaKPC-2 together with blaNDM-1 was mainly detected in K. oxytoca, E. cloacae cplx. and Citrobacter spp. Colistin resistance was observed in 40% of Klebsiella and 57% of Enterobacter isolates. Underlying mechanisms identified by WGS include known and potentially novel intrinsic mechanisms (point mutations in the pmrA/B, phoP/Q, mgrB and crrB genes) and acquired mechanisms (mcr-4.3 gene). The mcr-4.3 gene was identified for the first time in K. pneumoniae and is probably located on the conjugative IncHI1B plasmid. In addition, WGS analysis of 13 isolates revealed various virulence genes and resistance genes to other clinically relevant antibiotics as well as different plasmids possibly associated with carbapenemase genes. Our study demonstrates the important role that treated municipal wastewater plays in harboring and spreading enterobacterial pathogens that are resistant to last-resort antibiotics.

Comparison of MALDI-TOF mass spectrometry and 16S rDNA sequencing for identification of environmental bacteria: a case study of cave mussel-associated culturable microorganisms.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment - Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches.

Large-Scale Biogeographical Shifts of Abundance of Antibiotic Resistance Genes and Marine Bacterial Communities as Their Carriers along a Trophic Gradient.

The role of marine environments in the global spread of antibiotic resistance still remains poorly understood, leaving gaps in the One Health-based research framework. Antibiotic resistance genes (ARGs) encoding resistance to five major antibiotic classes, including sulfonamides (sul1, sul2), tetracyclines (tetA, tetB), ß-lactams (blaCTX-M, blaTEMblaVIM), macrolides (ermB, mphA), aminoglycosides (aac3-2), and integrase gene (intl1) were quantified by RT-qPCR, and their distribution was investigated in relation to environmental parameters and the total bacterial community in bottom layer and surface waters of the central Adriatic (Mediterranean), over a 68 km line from the wastewater-impacted estuary to coastal and pristine open sea. Seasonal changes (higher in winter) were observed for antibiotic resistance frequency and the relative abundances of ARGs, which were generally higher in eutrophic coastal areas. In particular, intl1, followed by blaTEM and blaVIM, were strongly associated with anthropogenic influence and Gammaproteobacteria as their predominant carriers. Water column stratification and geographic location had a significant influence on ARGs distribution in the oligotrophic zone, where the bacterial community exhibited a seasonal shift from Gammaproteobacteria in winter to Marine group II in summer.

Treated municipal wastewater as a source of high-risk and emerging multidrug-resistant clones of E. coli and other Enterobacterales producing extended-spectrum ß-lactamases.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales are a major public health problem, and wastewater from municipal wastewater treatment plants (WWTPs) is a potential means of spreading them into the environment and community. Our objective was to isolate ESBL-producing E. coli and other Enterobacterales from wastewater after treatment at Croatia's largest WWTP and to characterize these isolates by phenotypic and genotypic testing. Of the 200 bacterial isolates, 140 were confirmed as Enterobacterales by MALDI-TOF MS, with Escherichia coli and Klebsiella spp. predominating (69% and 7%, respectively). All 140 enterobacterial isolates were multidrug-resistant (MDR) and produced ESBLs. The most prevalent ESBL genes among the isolates tested were blaCTX-M-15 (60%), blaTEM-116 (44%), and blaCTX-M-3 (13%). Most isolates (94%) carried more than one ESBL gene in addition to blaCTX-M. Genes encoding plasmid-mediated AmpC, most notably blaEBC, were detected in 22% of isolates, whereas genes encoding carbapenemases (blaOXA-48, blaNDM-1, blaVIM-1) were less represented (10%). In E. coli, 9 different sequence types (ST) were found, with the emerging high-risk clones ST361 (serotype A-O9:H30) and pandemic ST131 (serotype B2-O25:H4) predominating (32% and 15%, respectively). Other high-risk E. coli clones included ST405 (3%), ST410 (3%), CC10 (3%), ST10 (3%), and ST38 (2%), and emerging clones included ST1193 (2%) and ST635 (2%). Whole-genome sequencing of three representative E. coli from two dominant clone groups (ST361 and ST131) and one extensively drug-resistant K. oxytoca revealed the presence of multiple plasmids and resistance genes to several other antibiotic classes, as well as association of the blaCTX-M-15 gene with transposons and insertion sequences. Our findings indicate that treated municipal wastewater contributes to the spread of emerging and pandemic MDR E. coli clones and other enterobacterial strains of clinical importance into the aquatic environment, with the risk of reintroduction into humans.

Integrons, transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies.

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.

Characterization of Third Generation Cephalosporin- and Carbapenem-Resistant Aeromonas Isolates from Municipal and Hospital Wastewater.

Antibiotic resistance (AR) remains one of the greatest threats to global health, and Aeromonas species have the potential to spread AR in the aquatic environment. The spread of resistance to antibiotics important to human health, such as third-generation cephalosporins (3GCs) and carbapenems, is of great concern. We isolated and identified 15 cefotaxime (3GC)- and 51 carbapenem-resistant Aeromonas spp. from untreated hospital and treated municipal wastewater in January 2020. The most common species were Aeromonas caviae (58%), A. hydrophila (17%), A. media (11%), and A. veronii (11%). Almost all isolates exhibited a multidrug-resistant phenotype and harboured a diverse plasmidome, with the plasmid replicons ColE, IncU, and IncR being the most frequently detected. The most prevalent carbapenemase gene was the plasmid-associated blaKPC-2 and, for the first time, the blaVIM-2, blaOXA-48, and blaIMP-13 genes were identified in Aeromonas spp. Among the 3GC-resistant isolates, the blaGES-5 and blaMOX genes were the most prevalent. Of the 10 isolates examined, three were capable of transferring carbapenem resistance to susceptible recipient E. coli. Our results suggest that conventionally treated municipal and untreated hospital wastewater is a reservoir for 3GC- and carbapenem-resistant, potentially harmful Aeromonas spp. that can be introduced into aquatic systems and pose a threat to both the environment and public health.

Resistance to critically important antibiotics in hospital wastewater from the largest Croatian city.

The emergence of extended-spectrum ß-lactamase (ESBL)- and especially carbapenemases in Enterobacterales has led to limited therapeutic options. Therefore, it is critical to fully understand all potential routes of transmission, especially in high-risk sources such as hospital wastewater. This study aimed to quantify four enteric opportunistic pathogens (EOPs), total, ESBL- and carbapenem-resistant coliforms and their corresponding resistance genes (two ESBL and five carbapenemase genes) and to characterize enterobacterial isolates from hospital wastewater from two large hospitals in Zagreb over two seasons. Culturing revealed similar average levels of total and carbapenem-resistant coliforms (3.4 × 104 CFU/mL), and 10-fold lower levels of presumptive ESBL coliforms (3 × 103 CFU/mL). Real-time PCR revealed the highest E. coli levels among EOPs (105 cell equivalents/mL) and the highest levels of the blaKPC gene (up to 10-1 gene copies/16S copies) among all resistance genes examined. Of the 69 ESBL- and 90 carbapenemase-producing Enterobacterales (CPE) isolates from hospital wastewater, all were multidrug-resistant and most were identified as Escherichia coli, Citrobacter, Enterobacter, and Klebsiella. Among ESBL isolates, blaCTX-M-15 was the most prevalent ESBL gene, whereas in CPE isolates, blaKPC-2 and blaNDM-1 were the most frequently detected CP genes, followed by blaOXA-48. Molecular epidemiology using PFGE, MLST and whole-genome sequencing (WGS) revealed that clinically relevant variants such as E. coli ST131 (blaCTX-M-15/blaTEM-116) and ST541 (blaKPC-2), K. pneumoniae ST101 (blaOXA-48/blaNDM-1), and Enterobacter cloacae complex ST277 (blaKPC-2/blaNDM-1) were among the most frequently detected clone types. WGS also revealed a diverse range of resistance genes and plasmids in these and other isolates, as well as transposons and insertion sequences in the flanking regions of the blaCTX-M, blaOXA-48, and blaKPC-2 genes, suggesting the potential for mobilization. We conclude that hospital wastewater is a potential secondary reservoir of clinically important pathogens and resistance genes and therefore requires effective pretreatment before discharge to the municipal sewer system.

Yesterday's contamination-A problem of today? The case study of discontinued historical contamination of the Mreznica River (Croatia).

The remnants of historical industrial contamination can be detected in many aquatic ecosystems worldwide even at present time. Mreznica is a river in Croatia that has been, for more than a hundred years, continually exposed to effluents of various industries, which have, in modern time, mostly ceased to operate. Our aim was to establish the level of current contamination and pollution of the Mreznica river-water and sediments. The study of river contamination at three sites (reference site; site nearby former cotton industry facility in Duga Resa - DRF; industrial zone of Karlovac town - KIZ) in three sampling campaigns (May 2020, April and September 2021) encompassed analyses of physico-chemical water parameters, screening of 369 pesticides, measurement of metal (loid) concentrations in the sediments, and in the dissolved and particulate phases of the river-water. The sediment pollution was assessed through the analyses of total bacteria abundance (by targeting 16S rRNA genes), and their associated metal resistance genes (cnrA, pbrT and czcD) and class 1 integrons (intl1). At the DRF site, industrial organic contaminants that can be traced to textile production were detected (dye and nylon components), as well as increased levels of some metals bound to suspended particulate matter and sediments. At the most downstream KIZ site, occasional high level of industrial herbicide neburon was measured in the river-water, and metal contamination of suspended particulate matter and sediments was evident. Although, based on the comparison with legislation and literature data, the level of contamination was rather mild, the effects on microbial communities were unquestionable, confirmed by increased abundance of the czcD gene at DRF site and the intI1 gene at both industrially impacted sites. Obtained results indicated long-term sediment retention of some industrial contaminants at the places of historical freshwater contamination, and, thus, the necessity for their monitoring even after the termination of contamination sources.

Pharmaceutical pollution of the world's rivers.

Environmental exposure to active pharmaceutical ingredients (APIs) can have negative effects on the health of ecosystems and humans. While numerous studies have monitored APIs in rivers, these employ different analytical methods, measure different APIs, and have ignored many of the countries of the world. This makes it difficult to quantify the scale of the problem from a global perspective. Furthermore, comparison of the existing data, generated for different studies/regions/continents, is challenging due to the vast differences between the analytical methodologies employed. Here, we present a global-scale study of API pollution in 258 of the world's rivers, representing the environmental influence of 471.4 million people across 137 geographic regions. Samples were obtained from 1,052 locations in 104 countries (representing all continents and 36 countries not previously studied for API contamination) and analyzed for 61 APIs. Highest cumulative API concentrations were observed in sub-Saharan Africa, south Asia, and South America. The most contaminated sites were in low- to middle-income countries and were associated with areas with poor wastewater and waste management infrastructure and pharmaceutical manufacturing. The most frequently detected APIs were carbamazepine, metformin, and caffeine (a compound also arising from lifestyle use), which were detected at over half of the sites monitored. Concentrations of at least one API at 25.7% of the sampling sites were greater than concentrations considered safe for aquatic organisms, or which are of concern in terms of selection for antimicrobial resistance. Therefore, pharmaceutical pollution poses a global threat to environmental and human health, as well as to delivery of the United Nations Sustainable Development Goals.

Prevalence of enteric opportunistic pathogens and extended-spectrum cephalosporin- and carbapenem-resistant coliforms and genes in wastewater from municipal wastewater treatment plants in Croatia.

Extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing Enterobacterales are a critical global health problem and wastewater treatment plants (WWTPs) can promote their spread into the environment; yet their efficacy is not well characterized. Here, we have used conventional culturing to monitor coliform bacteria and quantitative PCR to monitor 2 ESBL and 5 carbapenemase (CP) genes and 4 enteric opportunistic pathogens (EOPs) in the influent and effluent of 7 Croatian WWTPs in two seasons. In general, levels of total, cefotaxime- and carbapenem-resistant coliforms were significantly reduced but not eliminated by conventional treatment in most WWTPs. Most WWTPs efficiently removed EOPs such as K. pneumoniae and A. baumannii, while E. coli and Enterococcus spp. were reduced but still present in relatively high concentrations in the effluent. ESBL genes (blaTEM and blaCTX-M-32) were only slightly reduced or enriched after treatment. CP genes, blaKPC-3, blaNDM and blaOXA-48-like, were sporadically detected, while blaIMP and blaVIM were frequently enriched during treatment and correlated with plant size, number or size of hospitals in the catchment area, and COD effluent concentration. Our results suggest that improvements in wastewater treatment technologies are needed to minimize the risk of environmental contamination with top priority EOPs and ARGs and the resulting public health.

Characterization of macrolide resistance in bacteria isolated from macrolide-polluted and unpolluted river sediments and clinical sources in Croatia.

Environments polluted with excessively high levels of antibiotics released from manufacturing sites can act as a source of transferable antibiotic resistance (AR) genes to human commensal and pathogenic bacteria. The aim of this study was to evaluate AR of bacteria isolated from the Sava river sediments (Croatia) at the discharge site of effluents from azithromycin production compared to those from the upstream site and isolates collected in Croatian hospitals. A total of 228 environmental strains of azithromycin-resistant bacteria were isolated and identified, with 124 from the discharge site and 104 from the upstream site. In addition, a total of 90 clinical, azithromycin-resistant streptococcal and staphylococcal isolates obtained from the Croatian Reference Center for Antibiotic Resistance Surveillance were analyzed. PCR screening of isolates on 11 relevant macrolide-resistance genes (MRGs) showed that discharge isolates had greater detection frequencies for 4 gene targets (ermB, msrE, mphE and ermF) compared to upstream isolates. Among clinical isolates, the most frequently detected gene was ermB, followed by msrD, mefE and mefC. The discharge site demonstrated a greater abundance of isolates with co-occurrence of two different MRGs (predominantly msrE-mphE) than the upstream site, but a lower abundance than the clinical sources (most commonly msrD-mefE). The simultaneous presence of three or even four MRGs was specific for the discharge and clinical isolates, but not for the upstream isolates. When MRG results were sorted by gene mechanism, the ribosomal methylation (erm) and protection genes (msr) were the most frequently detected among both the discharge and the clinical isolates. Following sequencing, high nucleotide sequence similarity was observed between ermB in the discharge isolates and the clinical streptococcal isolates, suggesting a possible transfer of the ermB gene between bacteria of clinical and environmental origin. Our study highlights the importance of environmental bacterial populations as reservoirs for clinically relevant macrolide-resistance genes.

Every fifth published metagenome is not available to science.

Have you ever sought to use metagenomic DNA sequences reported in scientific publications? Were you successful? Here, we reveal that metagenomes from no fewer than 20% of the papers found in our literature search, published between 2016 and 2019, were not deposited in a repository or were simply inaccessible. The proportion of inaccessible data within the literature has been increasing year-on-year. Noncompliance with Open Data is best predicted by the scientific discipline of the journal. The number of citations, journal type (e.g., Open Access or subscription journals), and publisher are not good predictors of data accessibility. However, many publications in high-impact factor journals do display a higher likelihood of accessible metagenomic data sets. Twenty-first century science demands compliance with the ethical standard of data sharing of metagenomes and DNA sequence data more broadly. Data accessibility must become one of the routine and mandatory components of manuscript submissions-a requirement that should be applicable across the increasing number of disciplines using metagenomics. Compliance must be ensured and reinforced by funders, publishers, editors, reviewers, and, ultimately, the authors.

Effects of industrial effluents containing moderate levels of antibiotic mixtures on the abundance of antibiotic resistance genes and bacterial community composition in exposed creek sediments.

Environmental discharges of very high (mg/L) antibiotic levels from pharmaceutical production contributed to the selection, spread and persistence of antibiotic resistance. However, the effects of less antibiotic-polluted effluents (µg/L) from drug-formulation on exposed aquatic microbial communities are still scarce. Here we analyzed formulation effluents and sediments from the receiving creek collected at the discharge site (DW0), upstream (UP) and 3000 m downstream of discharge (DW3000) during winter and summer season. Chemical analyses indicated the largest amounts of trimethoprim (up to 5.08 mg/kg) and azithromycin (up to 0.39 mg/kg) at DW0, but sulfonamides accumulated at DW3000 (total up to 1.17 mg/kg). Quantitative PCR revealed significantly increased relative abundance of various antibiotic resistance genes (ARGs) against ß-lactams, macrolides, sulfonamides, trimethoprim and tetracyclines in sediments from DW0, despite relatively high background levels of some ARGs already at UP site. However, only sulfonamide (sul2) and macrolide ARG subtypes (mphG and msrE) were still elevated at DW3000 compared to UP. Sequencing of 16S rRNA genes revealed pronounced changes in the sediment bacterial community composition from both DW sites compared to UP site, regardless of the season. Numerous taxa with increased relative abundance at DW0 decreased to background levels at DW3000, suggesting die-off or lack of transport of effluent-originating bacteria. In contrast, various taxa that were more abundant in sediments than in effluents increased in relative abundance at DW3000 but not at DW0, possibly due to selection imposed by high sulfonamide levels. Network analysis revealed strong correlation between some clinically relevant ARGs (e.g. blaGES, blaOXA, ermB, tet39, sul2) and taxa with elevated abundance at DW sites, and known to harbour opportunistic pathogens, such as Acinetobacter, Arcobacter, Aeromonas and Shewanella. Our results demonstrate the necessity for improved management of pharmaceutical and rural waste disposal for mitigating the increasing problems with antibiotic resistance.

Industrial wastewater treatment plant enriches antibiotic resistance genes and alters the structure of microbial communities.

Antibiotic resistance is an emerging global health crisis, driven largely by overuse and misuse of antibiotics. However, there are examples in which the production of these antimicrobial agents has polluted the environment with active antibiotic residues, selecting for antibiotic resistant bacteria and the genes they carry. In this work, we have used shotgun metagenomics to investigate the taxonomic structure and resistance gene composition of sludge communities in a treatment plant in Croatia receiving wastewater from production of the macrolide antibiotic azithromycin. We found that the total abundance of antibiotic resistance genes was three times higher in sludge from the treatment plant receiving wastewater from pharmaceutical production than in municipal sludge from a sewage treatment plant in Zagreb. Surprisingly, macrolide resistance genes did not have higher abundances in the industrial sludge, but genes associated with mobile genetic elements such as integrons had. We conclude that at high concentrations of antibiotics, selection may favor taxonomic shifts towards intrinsically resistant species or strains harboring chromosomal resistance mutations rather than acquisition of mobile resistance determinants. Our results underscore the need for regulatory action also within Europe to avoid release of antibiotics into the environment.

Antibiotic-manufacturing sites are hot-spots for the release and spread of antibiotic resistance genes and mobile genetic elements in receiving aquatic environments.

High antibiotic releases from manufacturing facilities have been identified as a risk factor for antibiotic resistance development in bacterial pathogens. However, the role of antibiotic pollution in selection and transferability of antibiotic resistance genes (ARGs) is still limited. In this study, we analyzed effluents from azithromycin-synthesis and veterinary-drug formulation facilities as well as sediments from receiving river and creek taken at the effluent discharge sites, upstream and downstream of discharge. Culturing showed that the effluent discharge significantly increased the proportion of antibiotic resistant bacteria in exposed sediments compared to the upstream ones. Quantitative real-time PCR revealed that effluents from both industries contained high and similar relative abundances of resistance genes [sul1, sul2, qacE/qacEΔ1, tet(A)], class 1 integrons (intI1) and IncP-1 plasmids (korB). Consequently, these genes significantly increased in relative abundances in receiving sediments, with more pronounced effects being observed for river than for creek sediments due to lower background levels of the investigated genes in the river. In addition, effluent discharge considerably increased transfer frequencies of captured ARGs from exposed sediments into Escherichia coli CV601 recipient as shown by biparental mating experiments. Most plasmids exogenously captured from effluent and polluted sediments belonged to the broad host range IncP-1ε plasmid group, conferred multiple antibiotic resistance and harbored class 1 integrons. Discharge of pharmaceutical waste from antibiotic manufacturing sites thus poses a risk for development and dissemination of multi-resistant bacteria, including pathogens.

Pollution from azithromycin-manufacturing promotes macrolide-resistance gene propagation and induces spatial and seasonal bacterial community shifts in receiving river sediments.

Effluents from antibiotic manufacturing may contain high concentrations of antibiotics, which are the main driving force behind the selection and spread of antibiotic resistance genes in the environment. However, our knowledge about the impact of such effluent discharges on the antibiotic resistome and bacterial communities is still limited. To gain insight into this impact, we collected effluents from an azithromycin-manufacturing industry discharge site as well as upstream and downstream sediments from the receiving Sava river during both winter and summer season. Chemical analyses of sediment and effluent samples indicated that the effluent discharge significantly increased the amount of macrolide antibiotics, heavy metals and nutrients in the receiving river sediments. Quantitative PCR revealed a significant increase of relative abundances of macrolide-resistance genes and class 1 integrons in effluent-impacted sediments. Amplicon sequencing of 16S rRNA genes showed spatial and seasonal bacterial community shifts in the receiving sediments. Redundancy analysis and Mantel test indicated that macrolides and copper together with nutrients significantly correlated with community shift close to the effluent discharge site. The number of taxa that were significantly increased in relative abundance at the discharge site decreased rapidly at the downstream sites, showing the resilience of the indigenous sediment bacterial community. Seasonal changes in the chemical properties of the sediment along with changes in effluent community composition could be responsible for sediment community shifts between winter and summer. Altogether, this study showed that the discharge of pharmaceutical effluents altered physicochemical characteristics and bacterial community of receiving river sediments, which contributed to the enrichment of macrolide-resistance genes and integrons.

Corrigendum: Functional Repertoire of Antibiotic Resistance Genes in Antibiotic Manufacturing Effluents and Receiving Freshwater Sediments.

[This corrects the article DOI: 10.3389/fmicb.2017.02675.].

Biotransformation of macrolide antibiotics using enriched activated sludge culture: Kinetics, transformation routes and ecotoxicological evaluation.

The biotransformation of three prominent macrolide antibiotics (azithromycin, clarithromycin and erythromycin) by an activated sludge culture, which was adapted to high concentrations of azithromycin (10 mg/L) was investigated. The study included determination of removal kinetics of the parent compounds, identification of their major biotransformation products (TPs) and assessment of ecotoxicological effects of biotransformation. The chemical analyses were performed by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry, which enabled a tentative identification of TPs formed during the experiments. The ecotoxicological evaluation included two end-points, residual antibiotic activity and toxicity to freshwater algae. The enriched activated sludge culture was capable of degrading all studied macrolide compounds with high removal efficiencies (>99%) of the parent compounds at elevated concentrations (10 mg/L). The elimination of all three macrolide antibiotics was associated with the formation of different TPs, including several novel compounds previously unreported in the literature. Some of the TPs were rather abundant and contributed significantly to the overall mass balance at the end of the biodegradation experiments. Biodegradation of all investigated macrolides was associated with a pronounced reduction of the residual antibiotic activity and algal toxicity, indicating a rather positive ecotoxicological outcome of the biotransformation processes achieved by the enriched sludge culture.

Negative environmental impacts of antibiotic-contaminated effluents from pharmaceutical industries.

Effluents from pharmaceutical industries are recognized as significant contributors to aquatic pollution with antibiotics. Although such pollution has been mostly reported in Asia, knowledge on industrial discharges in other regions of the world, including Europe, and on the effects associated with such exposures is still limited. Thus, we performed chemical, microbiological and ecotoxicological analyses of effluents from two Croatian pharmaceutical industries during four seasons. In treated effluents of the company synthesizing macrolide antibiotic azithromycin (AZI), the total concentration of AZI and two macrolide by-products from its synthesis was 1-3 orders of magnitude higher in winter and springtime (up to 10.5 mg/L) than during the other two seasons (up to 638 µg/L). Accordingly, the highest total concentrations (up to 30 µg/L) in the recipient river were measured in winter and spring. Effluents from second company formulating veterinary antibiotics contained fluoroquinolones, trimethoprim, sulfonamides and tetracyclines ranging from low µg/L to approx. 200 µg/L. Low concentrations of these antibiotics, from below the limit of quantification to approx. few µg/L, have also been measured in the recipient stream. High frequency of culturable bacteria resistant to AZI (up to 83%) or sulfamethazine (up to 90%) and oxytetracycline (up to 50%) were also found in studied effluents. Finally, we demonstrated that toxicity to algae and water fleas often exceeded the permitted values. Most highly contaminated effluents induced multiple abnormalities in zebrafish embryos. In conclusion, using a wide array of analyses we have demonstrated that discharges from pharmaceutical industries can pose a significant ecological and public health concern due to their toxicity to aquatic organisms and risks for promoting development and spread of antibiotic resistance.