污水处理厂中四环素和磺胺类抗生素抗性基因的分布、传播及去除

近年来,抗生素滥用造成的抗性基因(ARGs)污染问题引起了人们的关注.四环素及磺胺类抗生素由于价格低廉被广泛使用,大量残留的四环素和磺胺通过各种途径进入污水处理厂,并进一步导致ARGs的污染.为深入了解四环素和磺胺类ARGs的污染及治理现状,本研究对污水处理厂中四环素和磺胺类ARGs的分布情况及传播机制进行了综述,并重点讨论了不同污水处理工艺对ARGs的去除效果.在此基础上,从加大污水处理厂ARGs污染调查、改进污水处理工艺以及探讨ARGs传播机制等方面进行了展望.     

Abundances of Tetracycline,Sulphonamide and Beta-Lactam Antibiotic Resistance Genes in Conventional Wastewater Treatment Plants (WWTPs) with Different Waste Load

Antibiotics and antibiotic resistant bacteria enter wastewater treatment plants (WWTPs), an environment where resistance genes can potentially spread and exchange between microbes. Several antibiotic resistance genes (ARGs) were quantified using qPCR in three WWTPs of decreasing capacity located in Helsinki, Tallinn, and Tartu, respectively: sulphonamide resistance genes (sul1 and sul2), tetracycline resistance genes (tetM and tetC), and resistance genes for extended spectrum beta-lactams (bla_oxa-58, bla_shv-34, and bla_ctx-m-32). To avoid inconsistencies among qPCR assays we normalised the ARG abundances with 16S rRNA gene abundances while assessing if the respective genes increased or decreased during treatment. ARGs were detected in most samples; sul1, sul2, and tetM were detected in all samples. Statistically significant differences (adjusted p<0.01) between the inflow and effluent were detected in only four cases. Effluent values for bla_oxa-58 and tetC decreased in the two larger plants while tetM decreased in the medium-sized plant. Only bla_shv-34 increased in the effluent from the medium-sized plant. In all other cases the purification process caused no significant change in the relative abundance of resistance genes, while the raw abundances fell by several orders of magnitude. Standard water quality variables (biological oxygen demand, total phosphorus and nitrogen, etc.) were weakly related or unrelated to the relative abundance of resistance genes. Based on our results we conclude that there is neither considerable enrichment nor purification of antibiotic resistance genes in studied conventional WWTPs.     

A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria

The arenas and detailed mechanisms for transfer of antibiotic resistance genes between environmental bacteria and pathogens are largely unclear. Selection pressures from antibiotics in situations where environmental bacteria and human pathogens meet are expected to increase the risks for such gene transfer events. We hypothesize that waste-water treatment plants (WWTPs) serving antibiotic manufacturing industries may provide such spawning grounds, given the high bacterial densities present there together with exceptionally strong and persistent selection pressures from the antibiotic-contaminated waste. Previous analyses of effluent from an Indian industrial WWTP that processes waste from bulk drug production revealed the presence of a range of drugs, including broad spectrum antibiotics at extremely high concentrations (mg/L range). In this study, we have characterized the antibiotic resistance profiles of 93 bacterial strains sampled at different stages of the treatment process from the WWTP against 39 antibiotics belonging to 12 different classes. A large majority (86%) of the strains were resistant to 20 or more antibiotics. Although there were no classically-recognized human pathogens among the 93 isolated strains, opportunistic pathogens such as Ochrobactrum intermedium, Providencia rettgeri, vancomycin resistant Enterococci (VRE), Aerococcus sp. and Citrobacter freundii were found to be highly resistant. One of the O. intermedium strains (ER1) was resistant to 36 antibiotics, while P. rettgeri (OSR3) was resistant to 35 antibiotics. Class 1 and 2 integrons were detected in 74/93 (80%) strains each, and 88/93 (95%) strains harbored at least one type of integron. The qPCR analysis of community DNA also showed an unprecedented high prevalence of integrons, suggesting that the bacteria living under such high selective pressure have an appreciable potential for genetic exchange of resistance genes via mobile gene cassettes. The present study provides insight into the mechanisms behind and the extent of multi-drug resistance among bacteria living under an extreme antibiotic selection pressure.     

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267) kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.     

污水处理厂生物气溶胶抗生素抗性污染特征、来源及潜在风险

污水处理厂是抗生素抗性基因（antibiotic resistance genes,ARGs）和抗生素抗性细菌（antibiotic resistant bacteria,ARB）重要的源和汇,生物气溶胶是ARGs和ARB自污水处理厂向周边环境释放的关键载体。目前缺乏对污水处理厂生物气溶胶抗生素抗性污染特征、来源及潜在风险的系统性总结。本文从采样方法、检测方法、逸散特征、来源、潜在危害和风险评估等方面对污水处理厂抗生素抗性污染研究现状进行综述。惯性采样法和过滤法是常用的污水处理厂抗生素抗性生物气溶胶主要采集方法,而宏基因组测序、组装和分箱为其ARGs组成、可移动性和宿主提供了有效的检测方法,抗多药类、抗杆菌肽类、抗氨基糖苷类、抗四环素类、抗β-内酰胺类、抗磺胺类、抗大环内酯类和抗糖肽类等抗性基因在污水处理厂PM₁₀、PM_2.5和PM_1.0颗粒物中广泛检出。格栅间、生化反应池和污泥处理单元是污水处理厂PM₁₀、PM_2.5和PM_1.0负载ARGs和ARB的主要释放单元。污水处理厂不同粒径生物气溶胶中致病性ARB的存在增加了抗生素治疗的难度,而污水和污泥对ARGs和ARB的释放起到了重要的源的贡献。本文在研究内容、研究技术和控制策略等方面也提出了相关展望,以期为污水厂生物气溶胶抗生素抗性污染的监测和防护提供参考和借鉴。     

Metagenomic analysis reveals the prevalence and persistence of antibiotic- and heavy metal-resistance genes in wastewater treatment plant

The increased antibiotic resistance among microorganisms has resulted into growing interest for investigating the wastewater treatment plants (WWTPs) as they are reported to be the major source in the dissemination of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) in the environment. In this study, we investigated the prevalence and persistence of ARGs and HMRGs as well as bacterial diversity and mobile genetic elements (MGEs) in influent and effluent at the WWTP in Gwangju, South Korea, using high-throughput sequencing based metagenomic approach. A good number of broad-spectrum of resistance genes (both ARG and HMRG) were prevalent and likely persistent, although large portion of them were successfully removed at the wastewater treatment process. The relative abundance of ARGs and MGEs was higher in effluent as compared to that of influent. Our results suggest that the resistance genes with high abundance and bacteria harbouring ARGs and MGEs are likely to persist more through the treatment process. On analyzing the microbial community, the phylum Proteobacteria, especially potentially pathogenic species belonging to the genus Acinetobacter, dominated in WWTP. Overall, our study demonstrates that many ARGs and HMRGs may persist the treatment processes in WWTPs and their association to MGEs may contribute to the dissemination of resistance genes among microorganisms in the environment.     

Horizontal transfer of antibiotic resistance genes in a membrane bioreactor

Growing attention has been paid to the dissemination of antibiotic resistance genes (ARGs) in wastewater microbial communities. The application of membrane bioreactors (MBRs) in wastewater treatment is becoming increasingly widespread. We hypothesized that the transfer of ARGs among bacteria could occur in MBRs, which combine a high density of bacterial cells, biofilms, and antibiotic resistance bacteria or ARGs. In this study, the transfer discipline and dissemination of the RP4 plasmid in MBRs were investigated by the counting plate method, the MIDI microorganism identification system, and quantitative polymerase chain reaction (qPCR) techniques. The results showed that the average transfer frequency of the RP4 plasmid from the donor strain to cultivable bacteria in activated sludge was 2.76 × 10⁻⁵ per recipient, which was greater than the transfer frequency in wastewater and bacterial sludge reported previously. In addition, many bacterial species in the activated sludge had received RP4 by horizontal transfer, while the genera of Shewanella spp., Photobacterium spp., Pseudomonas spp., Proteus spp., and Vibrio spp. were more likely to acquire this plasmid. Interestingly, the abundance of the RP4 plasmid in total DNA remained at high levels and relatively stable at 10⁴ copies/mg of biosolids, suggesting that ARGs were transferred from donor strains to activated sludge bacteria in our study. Thus, the presence of ARGs in sewage sludge poses a potential health threat.     

Transfer of ampicillin resistance from Salmonella Typhimurium DT104 to Escherichia coli K12 in food

Aims: To investigate the transfer of antibiotic resistance from a donor Salmonella Typhimurium DT104 strain to a recipient Escherichia coli K12 strain. Methods and Results: Mating experiments were conducted in broth, milk and ground meat (beef) at incubation temperatures of 4, 15, 25 and 37°C for 18 and 36 h. Ampicillin‐resistance transfer was observed at similar frequencies in all transfer media at 25 and 37°C (10^?4 to 10^?5 log₁₀ CFU ml g^?1, transconjugants per recipient) for 18 h. At 15°C, transfer was observed in ground meat in the recipient strain (10^?6, log₁₀ CFU g^?1, transconjugants per recipient), but not in broth or milk. At 4°C, transfer did not occur in any of the examined mediums. Further analysis of the E. coli K12 nal^R transconjugant strain revealed the presence of a newly acquired plasmid (21 kbp) bearing the β‐lactamase gene bla_TEM. Transconjugants isolated on the basis of resistance to ampicillin did not acquire any other resistant markers. Conclusion: This study demonstrates the transfer of antibiotic resistance in food matrices at mid‐range temperatures. Significance and Impact of the Study: It highlights the involvement of food matrices in the dissemination of antibiotic‐resistant genes and the evolution of antibiotic‐resistant bacteria.     

Comparison of microbial communities in sequencing batch reactors (SBRs) exposed to trace erythromycin and erythromycin-H2O

Wastewater treatment plants (WWTPs) are major collection pools of antibiotics of which low concentrations may induce antibiotic resistance in their microbial communities and pose threat to human health. However, information is still limited on the microbial community alteration in WWTPs upon exposure to low-dose antibiotics due to absence of negative control systems without input of resistant bacteria and resistance genes. Here we report the impact of trace erythromycin (ERY) and dehydrated erythromycin (ERY-H₂O) on microbial community dynamics in three long-term (1 year) running sequencing batch reactors (SBRs), R1 (ERY-H₂O), R2 (ERY), and negative control R3. The PhyloChip microarray analysis showed that ERY-H₂O and ERY significantly altered their microbial communities based on bacterial richness (e.g., 825 operational taxonomic units (OTUs) in R1, 699 OTUs in R2, and 920 OTUs in R3) and population abundance (15 and 48 subfamilies with >80 % abundance decrease in R1 and R2, respectively). ERY-H₂O and ERY have broad but distinct antimicrobial spectrums. For example, bacteria of all the major phyla (i.e., Proteobacteria, Actinobacteria, Bacteroidetes, and Chloroflexi) present in SBRs were severely inhibited by ERY-H₂O and ERY, but bacteria of Acidobacteria, Chlorobi, Firmicutes, Nitrospira and OP10 phyla were only inhibited by ERY. Very limited bacterial groups showed antibiotic resistance to ERY-H₂O or ERY through forming biofilms (e.g., Zoogloea) or synthesizing resistant proteins (e.g., Thauera, Candidatus Accumulibacter, Candidatus Competibacter, and Dechloromonas) in the SBRs. Inhibition was observed to be the main effect of ERY-H₂O and ERY on microbial communities in the reactors. The results would broaden our knowledge of effects of low-dose antibiotics on microbial communities in WWTPs.     

Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq

Background

Gram-negative multidrug-resistant (MDR) bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq.

Methodology/Principal Findings

In this study, all MDR Enterobacteriaceae (n = 38) and randomly selected non-MDR counterparts (n = 41) isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥3) plasmids compared to their non-MDR counterparts, which carried ≤2 plasmids (p<0.01). Various large plasmids (∼52 to 100 kb) from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA), β-lactam (bla _TEM1, bla _AMPC, bla _CTX-M-15, bla _OXA-1, bla _VIM-2 and bla _SHV), sulfamethoxazole/trimethoprim (sul/dfr), tetracycline (tet) and chloramphenicol (cat) resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N) were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates.

Conclusions/Significance

This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary relationship with other parts of world. The large plasmids, carrying resistance genes and transfer-associated genes, may be potential factors for regional dissemination of antibiotic resistance.     

Abundance and Dynamics of Antibiotic Resistance Genes and Integrons in Lake Sediment Microcosms

Antibiotic resistance in bacteria causing disease is an ever growing threat to the world. Recently, environmental bacteria have become established as important both as sources of antibiotic resistance genes and in disseminating resistance genes. Low levels of antibiotics and other pharmaceuticals are regularly released into water environments via wastewater, and the concern is that such environmental contamination may serve to create hotspots for antibiotic resistance gene selection and dissemination. In this study, microcosms were created from water and sediments gathered from a lake in Sweden only lightly affected by human activities. The microcosms were exposed to a mixture of antibiotics of varying environmentally relevant concentrations (i.e., concentrations commonly encountered in wastewaters) in order to investigate the effect of low levels of antibiotics on antibiotic resistance gene abundances and dynamics in a previously uncontaminated environment. Antibiotic concentrations were measured using liquid chromatography-tandem mass spectrometry. Abundances of seven antibiotic resistance genes and the class 1 integron integrase gene, intI1, were quantified using real-time PCR. Resistance genes sulI and ermB were quantified in the microcosm sediments with mean abundances 5 and 15 gene copies/10⁶ 16S rRNA gene copies, respectively. Class 1 integrons were determined in the sediments with a mean concentration of 3.8×10⁴ copies/10⁶ 16S rRNA gene copies. The antibiotic treatment had no observable effect on antibiotic resistance gene or integron abundances.     

Amplicon-based profiling of bacteria in raw and secondary treated wastewater from treatment plants across Australia

In this study, we investigated the use of Illumina high-throughput sequencing of 16S ribosomal RNA (rRNA) amplicons to explore microbial diversity and community structure in raw and secondary treated wastewater (WW) samples from four municipal wastewater treatment plants (WWTPs A–D) across Australia. Sequence reads were analyzed to determine the abundance and diversity of bacterial communities in raw and secondary treated WW samples across the four WWTPs. In addition, sequence reads were also characterized to phenotypic features and to estimate the abundance of potential pathogenic bacterial genera and antibiotic-resistant genes in total bacterial communities. The mean coverage, Shannon diversity index, observed richness (S _obs), and abundance-based coverage estimate (ACE) of richness for raw and secondary treated WW samples did not differ significantly (P > 0.05) among the four WWTPs examined. Generally, raw and secondary treated WW samples were dominated by members of the genera Pseudomonas, Arcobacter, and Bacteroides. Evaluation of source contributions to secondary treated WW, done using SourceTracker, revealed that 8.80–61.4% of the bacterial communities in secondary treated WW samples were attributed to raw WW. Twenty-five bacterial genera were classified as containing potential bacterial pathogens. The abundance of potentially pathogenic genera in raw WW samples was higher than that found in secondary treated WW samples. Among the pathogenic genera identified, Pseudomonas and Arcobacter had the greatest percentage of the sequence reads. The abundances of antibiotic resistance genes were generally low (<0.5%), except for genes encoding ABC transporters, which accounted for approximately 3% of inferred genes. These findings provided a comprehensive profile of bacterial communities, including potential bacterial pathogens and antibiotic-resistant genes, in raw and secondary treated WW samples from four WWTPs across Australia and demonstrated that Illumina high-throughput sequencing can be an alternative approach for monitoring WW quality in order to protect environmental and human health.

     

Metagenomic Profiling of Antibiotic Resistance Genes and Mobile Genetic Elements in a Tannery Wastewater Treatment Plant

Antibiotics are often used to prevent sickness and improve production in animal agriculture, and the residues in animal bodies may enter tannery wastewater during leather production. This study aimed to use Illumina high-throughput sequencing to investigate the occurrence, diversity and abundance of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in aerobic and anaerobic sludge of a full-scale tannery wastewater treatment plant (WWTP). Metagenomic analysis showed that Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria dominated in the WWTP, but the relative abundance of archaea in anaerobic sludge was higher than in aerobic sludge. Sequencing reads from aerobic and anaerobic sludge revealed differences in the abundance of functional genes between both microbial communities. Genes coding for antibiotic resistance were identified in both communities. BLAST analysis against Antibiotic Resistance Genes Database (ARDB) further revealed that aerobic and anaerobic sludge contained various ARGs with high abundance, among which sulfonamide resistance gene sul1 had the highest abundance, occupying over 20% of the total ARGs reads. Tetracycline resistance genes (tet) were highly rich in the anaerobic sludge, among which tet33 had the highest abundance, but was absent in aerobic sludge. Over 70 types of insertion sequences were detected in each sludge sample, and class 1 integrase genes were prevalent in the WWTP. The results highlighted prevalence of ARGs and MGEs in tannery WWTPs, which may deserve more public health concerns.     

Magnetic isotope effect of magnesium <Superscript>25</Superscript>Mg on <Emphasis Type="Italic">E. coli</Emphasis> resistance to antibiotics

Effects of synergism and antagonism of antibacterial drugs and magnetic isotope of magnesium ²⁵Mg on antibiotic resistance of bacteria E. coli were discovered. Fourteen antibiotics from seven different groups were tested. The increase in antibiotic resistance in the presence of the ion ²⁵Mg²⁺ was discovered in E. coli cells incubated with quinolones/fluoroquinolones, indicating the inhibiting effect of the magnetic moments of nuclei ²⁵Mg on DNA synthesis. The change in antibiotic resistance was also detected in bacteria affected by magnesium ²⁵Mg and certain antibiotics from aminoglycoside and lincosamide groups.     

Quantifying Nonspecific TEM β-Lactamase (blaTEM) Genes in a Wastewater Stream

To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are β-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by β-lactamases. Of the β-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM β-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla_TEM genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla_TEM are more likely to survive the treatment process. Thus, β-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.     

Fate of Antibiotic Resistant Bacteria and Genes during Wastewater Chlorination: Implication for Antibiotic Resistance Control

This study investigated fates of nine antibiotic-resistant bacteria as well as two series of antibiotic resistance genes in wastewater treated by various doses of chlorine (0, 15, 30, 60, 150 and 300 mg Cl₂ min/L). The results indicated that chlorination was effective in inactivating antibiotic-resistant bacteria. Most bacteria were inactivated completely at the lowest dose (15 mg Cl₂ min/L). By comparison, sulfadiazine- and erythromycin-resistant bacteria exhibited tolerance to low chlorine dose (up to 60 mg Cl₂ min/L). However, quantitative real-time PCRs revealed that chlorination decreased limited erythromycin or tetracycline resistance genes, with the removal levels of overall erythromycin and tetracycline resistance genes at 0.42 ± 0.12 log and 0.10 ± 0.02 log, respectively. About 40% of erythromycin-resistance genes and 80% of tetracycline resistance genes could not be removed by chlorination. Chlorination was considered not effective in controlling antimicrobial resistance. More concern needs to be paid to the potential risk of antibiotic resistance genes in the wastewater after chlorination.     

Abundance of amoA genes of ammonia-oxidizing archaea and bacteria in activated sludge of full-scale wastewater treatment plants

In this study, the abundance and sequences of amoA genes of ammonia-oxidizing archaea (AOA) and bacteria (AOB) were determined in seven wastewater treatment plants (WWTPs) whose ammonium concentrations in influent and effluent wastewaters varied considerably (5.6-422.3 mgN l⁻¹ and 0.2-29.2 mgN l⁻¹, respectively). Quantitative real-time PCR showed that the comparative abundance of AOA and AOB amoA genes differed among the WWTPs. In all three industrial WWTPs, where the influent and effluent contained the higher levels of ammonium (36.1-422.3 mgN l⁻¹ and 5.3-29.2 mgN l⁻¹, respectively), more than four orders of magnitude higher numbers of AOB amoA genes than AOA amoA genes arose (with less than the limit of detection of AOA amoA genes). In contrast, significant numbers of AOA amoA genes occurred in all municipal WWTPs (with ammonium levels in the influent and effluent of 5.6-11.0 mgN l⁻¹ and 0.2-3.0 mgN l⁻¹, respectively). Statistical analysis suggested that compared to other plants’ parameters, the ammonium levels in the plants’ effluent showed correlation with the highest p value to the abundance of AOA amoA genes.     

Antibiotic Resistance Characteristics of Environmental Bacteria from an Oxytetracycline Production Wastewater Treatment Plant and the Receiving River

We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as Gammaproteobacteria. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (tet) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with tet(A) being the most common (67.0%), followed by tet(W), tet(C), tet(J), tet(L), tet(D), tet(Y), and tet(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the tet genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.The widespread emergence of antibiotic resistance, particularly multidrug resistance (MDR), among bacterial pathogens has become one of the most serious challenges in clinical therapy (22, 46). Some pathogens, such as MDR Klebsiella pneumoniae and Acinetobacter baumannii, are now virtually untreatable with current antibiotics (14, 30). Acquisition of resistance genes through horizontal transfer has been found to be ubiquitous in clinical pathogens (22). Environmental bacteria have been shown to be a reservoir of antibiotic resistance genes and a potential source of novel resistance genes in clinical pathogens (10, 12). Horizontal transfer of genes between bacterial strains could be facilitated by mobile genetic elements, such as plasmids, transposons, bacteriophages, integrons, insertion elements (IS), and genomic islands (13). Some elements, including class I integrons, conjugative plasmids, and transposons, are frequently linked to antibiotic resistance as they harbor rather diverse resistance genes and possibly promote the distribution of these genes in phylogenetically diverse bacteria (29). In light of the potential health risk, many studies have focused on antibiotic-resistant bacteria recovered from various ecosystems (1, 18, 45). Environments that contain antibiotic residues are particularly worrisome because antibiotics could exert selective pressure and might contribute to the appearance of resistant bacteria. Hospital sewage was once considered the major source of antibiotics in aquatic environments, followed by municipal, agricultural, and aquacultural wastewater (WW), which have also been shown to be important sources of these compounds and resistant bacteria (40). It has also been reported that treated antibiotic production wastewater contains much higher concentrations of antibiotic residues than other aquatic environments (20, 26, 27) and can serve as an important reservoir of resistant bacteria and genes (25).In the current study, we investigated resistance profiles of bacterial isolates from a unique wastewater treatment plant (WWTP) that is used solely for treating oxytetracycline (OTC), avermectin, and ivermectin production wastewater from the facility of the North China Pharmaceutical Group Corporation in Hebei Province, China. Avermectin and ivermectin are both broad-spectrum antiparasitic agents without any antibacterial or antifungal activities. The effluent of the WWTP is discharged directly into the receiving river without disinfection, and residual levels of OTC in wastewater and downstream sampling points nearly approach those in human blood after drug administration (15, 26). Thus, the characteristics of bacterial strains in heavily OTC-contaminated wastewater and surface water could be unique. In a 1-year (2002-2003) survey of antibiotic resistance in 140 clinical enterococcal isolates from a hospital in this region, the prevalence of tetracycline resistance ranged from 14.9% to 25.0% (47). This rate increased to 47.9 to 75.9% for 302 clinical enterococcal isolates from the same hospital over the following 3 years (2004-2006) (28), indicating that tetracycline resistance among human pathogens in this region is becoming more prevalent. Elucidation of the resistance characteristics of bacterial isolates from OTC wastewater and surface water might help explain the prevalence of increased resistance among human pathogens. To gain a comprehensive understanding of this relationship, we isolated bacterial strains from wastewater (WW) and river water downstream (RWD) and river water upstream (RWU) samples using nonselective culture media. Previously, it was shown that long-term administration of a single antibiotic can lead to MDR, a phenomenon that has been observed in bacteria obtained from the urinary tract, intestine, and other niches in both clinical therapy and livestock raising (11, 23, 34). Thus, the combination of long-term treatment and discharge of antibiotic production wastewater might contribute to MDR in environmental bacteria in this specific ecosystem. To test this hypothesis, we determined the resistance profiles of bacterial isolates for 10 antibiotics representing seven classes.OTC belongs to the tetracycline class of antibiotics, which includes tetracycline, chlortetracycline, doxycycline, minocycline, and glycylcyclines (6). This class of antibiotics represents broad-spectrum agents that act against a range of Gram-positive and Gram-negative bacteria by inhibiting protein synthesis. The tetracyclines have played an important role in human and veterinary medicine, and some have been used as growth promoters for livestock and aquaculture. However, resistance to tetracyclines in many commensal and pathogenic bacteria emerged soon after the widespread application of this class of antibiotic. More than 40 types of tetracycline resistance determinants have been described to date and grouped into three main classes: energy-dependent membrane-associated efflux proteins, which export tetracycline out of the cell; ribosomal protection proteins, which interact with the ribosome and disrupt the tetracycline binding site; and tetracycline inactivation enzymes (6, 37). Many studies of tetracycline resistance (tet) genes in various environmental niches have been reported. Resistance genes encoding efflux proteins and ribosomal protection proteins have been identified in phylogenetically diverse bacterial genera. Extensive reviews with detailed information about tet genes and their distribution are available (4, 6, 24, 37). Notably lacking, however, is information about tet genes in aquatic environments with high levels of antibiotic residue. Studies have shown that several tet genes are associated with mobile genetic elements (6, 37) and that the SOS response induced by antibiotics and other factors can promote horizontal dissemination of mobile genetic elements like genomic islands, as well as integron recombination, among bacterial populations (5, 17, 41). In the current study, the presence and distribution of 23 tet genes in bacterial isolates from wastewater and river water were determined by PCR. Class I integrons were also examined in the bacterial isolates to evaluate the possibility of horizontal transfer of these resistance genes and/or the promoted dissemination of mobile genetic elements in such heavily antibiotic-polluted environments. We also compared the characteristics of the recovered bacteria with those of isolates from samples from water upstream of the discharge site in the river. Our results reveal the potential environmental influence of wastewater discharge on acquired antibiotic resistance.     

Wastewater pollution differently affects the antibiotic resistance gene pool and biofilm bacterial communities across streambed compartments

Wastewater discharges introduce antibiotic residues and antibiotic‐resistant bacteria (ARB) into surface waters. Both inputs directly affect the streambed resistome, either by exerting a selective pressure that favour the proliferation of resistant phenotypes or by enriching the resident communities with wastewater‐associated ARB. Here, we investigated the impact of raw and treated urban wastewater discharges on epilithic (growing on rocks) and epipsammic (growing on sandy substrata) streambed biofilms. The effects were assessed by comparing control and impact sites (i) on the composition of bacterial communities; (ii) on the abundance of twelve antibiotic resistance genes (ARGs) encoding resistance to β‐lactams, fluoroquinolones, sulphonamides, tetracyclines, macrolides and vancomycin, as well as the class 1 integron‐integrase gene (intI1); (iii) on the occurrence of wastewater‐associated bacteria, including putative pathogens, and their potential linkage to target ARGs. We measured more pronounced effects of raw sewage than treated wastewater at the three studied levels. This effect was especially noticeable in epilithic biofilms, which showed a higher contribution of wastewater‐associated bacteria and ARB than in epipsammic biofilms. Comparison of correlation coefficients obtained between the relative abundance of both target ARGs and operational taxonomic units classified as either potential pathogens or nonpathogens yielded significant higher correlations between the former category and genes intI1, sul1, sul2 and ermB. Altogether, these results indicate that wastewater‐associated micro‐organisms, including potential pathogens, contribute to maintain the streambed resistome and that epilithic biofilms appear as sensitive biosensors of the effect of wastewater pollution in surface waters.