Propagation of New Delhi Metallo-β-lactamase Genes (blaNDM‑1) froma Wastewater Treatment Plant to Its Receiving RiverFengxia Yang,†,‡ Daqing Mao,*,‡ Hao Zhou,† Xiaolong Wang,† and Yi Luo*,†

‡School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China†College of Environmental Science and Engineering, Ministry of Education Key Laboratory of Pollution Processes and EnvironmentalCriteria, Nankai University, Tianjin 300071, China

*S Supporting Information

ABSTRACT: The emergence and spread of NDM-1 (NewDelhi metallo-β-lactamase-1) are of great concern to publichealth. Our previous study reported the occurrence andpersistence of NDM-1 genes in wastewater treatment plants(WWTPs). In this study, the occurrence and fate of NDM-1genes and host bacteria were investigated in a WWTPdischarge-receiving river. A considerable level of NDM-1genes occurred in the receiving river, whereas no NDM-1genes were detected upstream of the WWTP. This findingtogether with the DNA sequencing of NDM-1 genesdemonstrated that the river NDM-1 is derived from theWWTP. Opportunistic pathogens, like Shigella sonnei, Enter-ococcus faecium, andWautersiella falsenii, were isolated from both the receiving water and the WWTP. This study underscores theneed to mitigate the release of NDM-1 from WWTPs and indicates that more attention should to be paid to the propagation ofthese genes to the receiving environment to alleviate their worldwide dissemination.

■ INTRODUCTION

The rapid emergence and dissemination of carbapenem-resistant bacteria pose a growing threat to human health.1

Carbapenemase-producing Enterobacteriaceae have caused anunprecedented public health crisis of global dimensions2

because of their ability to efficiently defend against a broadspectrum of β-lactams, including almost all carbapenems.3

NDM-1 (New Delhi metallo-β-lactamase-1), a newly emergentcarbapenemase, was first identified in Klebsiella pneumoniaeisolated from a Swedish patient who had been hospitalized inNew Delhi, India.4 To date, NDM-1 has drawn worldwidepublic concern because of its resistance to all antimicrobialagents except polymyxins.1,5,6 Initially, most reported NDM-1-positive isolates were found in India, Bangladesh, and Pakistan.7

However, numerous cases of clinical infection caused by NDM-1 producers have been reported in many other countries,including Australia,8 New Zealand,9 Canada,10 France,11 Iraq,12

Japan,13,14 Switzerland,15 China,16−20 Egypt,21 Germany,22 andthe United States,23,24 which heralds a global distribution of theblaNDM‑1 genes.Multidrug-resistant bacteria harboring the blaNDM‑1 gene have

their primary reservoir in the clinic, with an increasing numberof reports of their detection and occurrence.25 Worryingly,NDM-1 genes have been found in some environmentalcompartments, including hospital sewage,26 municipal waste-water,27 seepage, and tap water,28,29 indicating the risk thatNDM-1 producers could widely disseminate the NDM-1 genefrom medical origins. The occurrence of NDM-1 genes in the

environment has serious implications for human health. Forinstance, in New Delhi, NDM-1-producing bacteria in anenvironmental setting (seepage and tap water) would mostlikely influence the people who are reliant on public water andsanitation facilities.28 Our recent study revealed that blaNDM‑1genes occurred in and even survived the sanitizing processes oftwo wastewater treatment plants (WWTPs).27 The release ofNDM-1 genes increases the opportunity for blaNDM‑1 horizontaltransfer. Our previous study provided evidence of NDM-1 genetransfer to the indigenous bacteria from the river.27 Obviously,this increases the extent of dissemination of blaNDM‑1 genes intothe natural environment. Furthermore, WWTPs link humanactivities with receiving water and may also facilitate thetransfer of blaNDM‑1 genes from clinical pathogens to environ-mental microorganisms.30−33 Notably, the treated wastewaterfrom WWTPs is often used as a recycled water resource inmany countries where freshwater resources are limited. Tianjinis one of these cities with very limited fresh water resources,and the WWTP effluent is drained directly into a river primarilyused for farm irrigation and aquaculture, which represents apotential pathway for spreading blaNDM‑1 genes elsewhere.In this study, the WWTP and its receiving river water were

investigated to study how the WWTP effluent containing

Received: February 6, 2016Revised: March 10, 2016Accepted: March 10, 2016

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© XXXX American Chemical Society A DOI: 10.1021/acs.estlett.6b00036Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

blaNDM‑1 genes impacts its receiving water. Here we quantifiedblaNDM‑1 abundance, isolated NDM-1-producing bacteria, andtested the transferability of blaNDM‑1-positive plasmids todemonstrate the occurrence and fate of the blaNDM‑1 geneand its host bacteria in the receiving water and to verify thatblaNDM‑1 genes occurring in the river are derived fromwastewater. To the best of our knowledge, this is the firststudy to provide direct evidence that the emergence of NDM-1genes in the river is derived from the discharges of the upstreamWWTP.

■ MATERIALS AND METHODSStudy Site and Water Sampling. Nine sampling sites

were selected in January 2015 in the effluent of a WWTP andits receiving river as shown in Figure 1. The WWTP with

conventional activated sludge serves a large population of 1million in Tianjin. The WWTP effluent and river water sampleswere collected. The effluent samples were collected afterchlorinated disinfection. The river water samples were collectedfrom 0, 100, 200, 300, 400, 500, and 1000 m along the WWTPdischarges into the receiving water. The control, which isassumed not to be influenced by the WWTP effluent, wassampled approximately 300 m upstream of the WWTP. Watersamples were collected in triplicate for each sample site. Allwater samples (2.5 L) from the top 0.5 m of the surface werecollected in sterile polypropylene bottles and were transportedto the laboratory in a portable icebox before further analysis.Sample Preparation and DNA Extraction. All water

samples were filtered through 0.22 μm filters using a vacuumfiltration apparatus, and the filters were stored at 4 °C untilDNA was extracted. DNA extraction was performed using anultraclean water DNA kit (MoBio Laboratories Inc., Carlsbad,CA) according to the manufacturer’s instructions. Meanwhile,Escherichia coli DH5α cloned with the CESA9 gene as aninternal standard was used to determine DNA extractionefficiency, as described previously.34 The extracted DNA wasfurther purified using the DNA pure-spin kit (Vigorousbio,Beijing, China) to minimize polymerase chain reaction (PCR)inhibition. DNA extraction recoveries at different sites are listedin Table S2 of the Supporting Information. All the DNAsamples were stored at −20 °C until further analysis.

PCR Assays and Quantification of blaNDM‑1 Genes.Qualitative PCR (qPCR) amplifications were performed usinga Biometra T100 gradient (Biometra). Primers for blaNDM‑1,which amplified a 984 bp product, were used for qPCR toensure accurate target genes, and it is good for indicating DNAdamage. The quantification of blaNDM‑1 was accomplished withreal-time PCR on a Bio-Rad iQ5 instrument (Bio-Rad,Hercules, CA), and the 16S rRNA gene was quantified toevaluate the abundance of the total bacterial community.Primers that amplified 214 and 126 bp products were used forqPCR assays of the blaNDM‑1 and 16S rRNA genes, respectively.The primer sequences are listed in Table S1. Calibrationstandard curves for positive controls were generated asdescribed previously.34 Negative controls contained all thecomponents of the PCR mixture without DNA template. Thedetails of PCR and qPCR assays are described in SI-1 and SI-2of the Supporting Information.

Isolation of blaNDM‑1-Positive Bacteria. Selective Luria-Bertani (LB) agar plates containing ampicillin (100 mg/L),kanamycin (25 mg/L), sulfamethoxazole (50 mg/L), eryth-romycin (50 mg/L), chloromycetin (50 mg/L), streptomycin(50 mg/L), neomycin (50 mg/L), and tetracycline (50 mg/L)were used to isolate NDM-1-positive strains from watersamples based on the criteria of the Clinical and LaboratoryStandards Institute (CLSI). The blaNDM‑1 gene in isolates wasassessed by PCR with specific primers listed in Table S1. ThenNDM-1-producing isolates were identified by 16S rRNAsequencing combined with the Biolog Microbial Identificationsystem (BIOLOG, Hayward, CA). The minimum inhibitoryconcentrations (MICs) of various antibiotics in these blaNDM‑1-positive isolates were determined by the agar dilution methodas set by the CLSI.35 The E. coli ATCC25922 strain was used asa control strain in this study. More specialized details areavailable in SI-3 of the Supporting Information.

Conjugative Transfer. To assess the transferability ofblaNDM‑1 genes, two isolates, Wautersiella falsenii andAcinetobacter seohaensis, isolated from the effluent and receivingriver, respectively, were used as the donor of conjugationexperiments, and azide-resistant E. coli J53 was used as therecipient. The selective plates containing ampicillin (100 mg/L) and sodium azide (100 mg/L) were used to selecttransconjugants. Meanwhile, the donor and recipient strainswere separately plated onto identical LB plates as negativecontrols to rule out spontaneous mutation. PCR (984 bpamplicons) and DNA sequencing were performed to ensurethat blaNDM‑1 was transferred to the recipient. More details arelisted in SI-4. The transfer frequency ( f) was calculated usingthe formula f = NT (number of transconjugants, in colony-forming units per milliliter)/NR (number of recipients, incolony-forming units per milliliter).

■ RESULTS AND DISCUSSIONDischarge of blaNDM‑1 Genes from WWTPs. Our

previous study revealed that blaNDM‑1 genes were prevalent intwo wastewater treatment plants (WWTPs) and selectivelysurvived the disinfection treatment stage.27 Since our previousreport of the NDM-1 gene in that WWTP, the abundance ofblaNDM‑1 genes has remained at a level of 103 copies/mL in theWWTP effluent. After a lengthy discharging period, theblaNDM‑1 gene now occurs in the downstream WWTP effluentreach of the receiving water, and the blaNDM‑1 sequences are99.8% identical (983 bp match) to the blaNDM‑1 gene fromAcinetobacter calcoaceticus (Gene ID KF951475.1). The

Figure 1. Geographical location of the sampling sites. The map showsthe locations where wastewater effluents are discharged to a receivingriver as well as locations along the river where samples were collectedfor this study.

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blaNDM‑1 gene level of the disinfected effluent was (2.71 ± 0.13)× 103 copies/mL, which is more enriched than the level in thereceiving water (i.e., 7.4−77.5% of the effluent).To further determine the prevalence of blaNDM‑1 genes and

their potential contribution to the discharge of wastewater intothe receiving river, culture-dependent approaches were used toinvestigate multidrug-resistant bacteria in the WWTP effluent.A total of 12 NDM-1-producing bacteria were isolated from theeffluent, and they were all identified as Enterococcus faecium, E.coli, Shigella sonnei, A. seohaensis, andW. falsenii. Compared witha previous study in the same WWTP in which only one species(Achromobacter sp.) was identified,27 more bacterial speciespossessing the blaNDM‑1 gene occurred in the WWTP. Mostworryingly, these NDM-1-producing isolates occurring in theWWTP are pathogenic and opportunistic pathogens, and theycould acquire antibiotic resistance, therefore increasing the riskof dissemination of blaNDM‑1 to human pathogens. As aconsequence, the occurrence of these NDM-1-producingpathogens in the environment has serious implications forhuman health.Trends in NDM-1 Gene Levels in the Receiving Water

Influenced by WWTP. The blaNDM‑1 and 16S rRNA (toevaluate the abundance of the total bacterial community) geneswere quantified with qPCR to understand the fate of blaNDM‑1

genes in the receiving water. Figure 2A demonstrates that therewas a considerable concentration of blaNDM‑1 genes in thereceiving water located downstream of the WWTP [from (2.01± 0.95) × 102 to (2.10 ± 0.19) × 103 copies/mL].Simultaneously, the absolute abundance of blaNDM‑1 decreasedwith downstream distance, which may be related to the

“dilution effect” of river water, whereas upstream of the WWTPeffluent, blaNDM‑1 genes were below the limit of detection(LOD). Furthermore, there was no anomalous increasedownstream of the receiving river, indicating that no othersources contributed to the occurrence and proliferation ofblaNDM‑1 genes within the sampling range. These results impliedthat the emergence of blaNDM‑1 genes in the receiving watermay be associated with WWTP effluent discharges.Additionally, the abundance of 16S rRNA genes in the

discharge point of wastewater [(2.79 ± 0.20) × 107 copies/mL]was significantly higher than in other sampling locations alongthe river [(5.34 ± 1.27) × 106 to (1.96 ± 0.61) × 107 copies/mL] (Table S3), indicating that the WWTP dischargessignificantly increased the biomass loads of its receivingwater. This observation demonstrated that the treated waste-water discharges have a considerable influence on the bacterialcommunity in the downstream river. Nevertheless, Figure 2Billustrates that at distances of >400 m, the 16S rRNA geneabundance recovered to the upstream level, which illustrates thedilution in the river.Three NDM-1-producing strains, two strains of A. seohaensis

and one strain of Stenotrophomonas maltophilia, were isolated inthe river downstream of the WWTP. These NDM-1-producingisolates have multidrug-resistant phenotypes; the MIC values tovarious antibiotics are comparable to those of isolates fromWWTP effluent, as indicated in Table S4. Furthermore, A.seohaensis strains in receiving water possess the blaNDM‑1 genewith 100% identity to the gene from the same species inwastewater effluent. In addition, the blaNDM‑1 gene sequences inthe receiving water were 100% identical (983 bp match) to thatof the WWTP effluent. These facts indicated the propagation ofNDM-1 to the receiving water upon WWTP discharge.Moreover, as mentioned above, a considerable level of blaNDM-1genes was detected downstream of the receiving water and even1000 m from the WWTP effluent. These findings and thesequence similarity confirmed that blaNDM‑1 genes in thereceiving water were derived from the direct discharge of theWWTP. Therefore, the continuous discharge of wastewatersignificantly resulted in the occurrence and persistence ofNDM-1 in downstream receiving water.It is striking that the S. maltophilia harboring the blaNDM‑1

gene was observed only in the receiving river and was notdetected in WWTP effluent under the same experimentalconditions (Table 1). The blaNDM‑1 gene carried by S.

maltophilia was 100% identical (983 bp match) to that in A.seohaensis isolated from the receiving water. Furthermore, S.maltophilia is ubiquitous in aqueous environments, soil, andplants.36 These results suggest the transfer of NDM-1 genes toindigenous bacteria in the receiving water. This potentialblaNDM‑1 transfer to indigenous bacteria was also further verifiedby conjugation experiments.

Figure 2. Absolute concentration of blaNDM‑1 (A) and 16S rRNA (B)genes in different water samples along the receiving river. A total ofeight sampling locations were included, and triplicate samples weretaken from each sampling location (N = 24). The control (upstream)was sampled approximately 300 m upstream of the WWTP. The 0 mlocation is the point of discharge of WWTP effluent into the river;other samples were collected from locations 0, 100, 200, 300, 400, 500,and 1000 m from the WWTP discharge into the receiving water.

Table 1. NDM-1-Producing Isolates in the WWTP and theReceiving River

samplinglocation

S.maltophilia

A.seohaensis

En.faecium E. coli

Sh.sonnei

W.falsenii

WWTPeffluent

NDa 4 1 3 2 2

receivingriver

1 2 NDa NDa NDa NDa

aNot detected.

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Conjugative Transfer of IncA/C Plasmid Harboringthe blaNDM‑1 Gene. Two isolates, W. falsenii and A. seohaensisfrom the effluent and the receiving water, respectively, wereused as donors for conjugation experiments, and the azide-resistant E. coli J53 strain was used as recipient to assess thedissemination of blaNDM‑1-positive plasmids in the receivingwater. The blaNDM‑1 gene was detected in the E. coli J53transconjugants, and its sequence was completely consistentwith that of the donor. Moreover, the transconjugants exhibitedresistance to the tested antibiotics that was similar to that of thebacteria isolated from the donor (Table 2). The blaNDM‑1 genewas located on the plasmids extracted from the E. coli J53transconjugants; therefore, these findings demonstrate thetransferability of blaNDM‑1-positive plasmids among bacteria.Furthermore, The transfer frequency of NDM-1 plasmids[(6.65 ± 0.72) × 10−6 transconjugants/recipient] wascomparable to those values published previously [(3.33 ±0.67) × 10−6 and 0.15−2.0 × 10−6 for plasmid RP4;37,38 (9.57± 0.79) × 10−6 for intI139], implying a high risk of blaNDM‑1propagation.The blaNDM‑1-carrying plasmids belonged to the incompat-

ibility group A/C (IncA/C) based on a PCR-based replicontyping method.40 IncA/C-type plasmids are large, broad-host-range plasmids41−43 and are usually associated with otherextended-spectrum β-lactamase genes, e.g., blaCMY‑2,

44−46

blaTEM,47 blaVEB,

48 blaCTX‑M,49 and blaKPC‑2.

50 These IncA/Cplasmids could disseminate antibiotic resistance genes amongclinically relevant bacteria, although their conjugal transfercapacities are variable.51−53 As a consequence, the self-conjugative IncA/C plasmid-borne NDM-1 genes and theiremergence in the receiving water highlighted the risk ofblaNDM‑1 gene dissemination in the environment.This study demonstrates the occurrence and fate of blaNDM‑1

genes in a receiving river following WWTP discharges andconfirms that blaNDM‑1 genes occurring in the river are derivedfrom wastewater. This finding is important because it illustratesthat the dissemination of New Delhi metallo-β-lactamase genes(blaNDM‑1) derived from a wastewater treatment plant is asignificant pathway for NDM-1 gene proliferation to theenvironment. Considering the risk that the NDM-1 gene andits host bacteria pose to public health, more emphasis must befocused on the transferable vectors in facilitating thedissemination of NDM-1 in the environment.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.estlett.6b00036.

Detailed PCR, MIC protocols, conjugation experiment,PCR primers, DNA extraction recoveries, and supportingcalculations (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: mao@tju.edu.cn. Phone: +86 (22) 87402072.*E-mail: luoy@nankai.edu.cn. Phone: +86 (22) 85358553.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe are thankful to Professor Minggui Wang (Fudan UniversityShanghai Medical College, Shanghai, China) for providing E.coli J53. This work was supported by the National Science Fundfor Distinguished Young Scholars (41525013) and the NationalNatural Science Foundation of China (Grants 31470440 and41473085).

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Table 2. MICs of Transconjugant (E. coli J53 strain harboring the blaNDM‑1 gene) and the E. coli J53 Recipient Strain

MIC (mg/L)

antibiotic W. falsenii (WWTP) transconjugant-W E. coli J53 A. seohaensis (river) transconjugant-A E. coli J53 E. coli J53

ampicillin >256 256 >256 >256 4kanamycin 256 8 >256 16 0.06tetracycline >256 4 256 4 0.03chloromycetin 128 128 256 128 2imipenem 256 256 256 256 0.03meropenem 256 128 >256 >256 0.03streptomycin >256 128 >256 128 4ciprofloxacin 16 2 8 1 0.06ceftazidime >256 >256 >256 >256 0.06gentamicin 8 4 8 4 0.12

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