Propagation of New Delhi Metallo-β-lactamase Genes (blaNDM-1

Mar 10, 2016 - Phone: +86 (22) 87402072., *E-mail: [email protected]. ... NDM-1 (New Delhi metallo-β-lactamase-1) are of great concern to public hea...
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Propagation of New Delhi Metallo-β-lactamase Genes (blaNDM‑1) from a Wastewater Treatment Plant to Its Receiving River Fengxia 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 Environmental Criteria, Nankai University, Tianjin 300071, China

†

S Supporting Information *

ABSTRACT: The emergence and spread of NDM-1 (New Delhi metallo-β-lactamase-1) are of great concern to public health. Our previous study reported the occurrence and persistence of NDM-1 genes in wastewater treatment plants (WWTPs). In this study, the occurrence and fate of NDM-1 genes and host bacteria were investigated in a WWTP discharge-receiving river. A considerable level of NDM-1 genes occurred in the receiving river, whereas no NDM-1 genes were detected upstream of the WWTP. This finding together with the DNA sequencing of NDM-1 genes demonstrated that the river NDM-1 is derived from the WWTP. Opportunistic pathogens, like Shigella sonnei, Enterococcus faecium, and Wautersiella falsenii, were isolated from both the receiving water and the WWTP. This study underscores the need to mitigate the release of NDM-1 from WWTPs and indicates that more attention should to be paid to the propagation of these genes to the receiving environment to alleviate their worldwide dissemination.

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INTRODUCTION The rapid emergence and dissemination of carbapenemresistant bacteria pose a growing threat to human health.1 Carbapenemase-producing Enterobacteriaceae have caused an unprecedented public health crisis of global dimensions2 because of their ability to efficiently defend against a broad spectrum of β-lactams, including almost all carbapenems.3 NDM-1 (New Delhi metallo-β-lactamase-1), a newly emergent carbapenemase, was first identified in Klebsiella pneumoniae isolated from a Swedish patient who had been hospitalized in New Delhi, India.4 To date, NDM-1 has drawn worldwide public concern because of its resistance to all antimicrobial agents except polymyxins.1,5,6 Initially, most reported NDM-1positive isolates were found in India, Bangladesh, and Pakistan.7 However, numerous cases of clinical infection caused by NDM1 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 and the United States,23,24 which heralds a global distribution of the blaNDM‑1 genes. Multidrug-resistant bacteria harboring the blaNDM‑1 gene have their primary reservoir in the clinic, with an increasing number of reports of their detection and occurrence.25 Worryingly, NDM-1 genes have been found in some environmental compartments, including hospital sewage,26 municipal wastewater,27 seepage, and tap water,28,29 indicating the risk that NDM-1 producers could widely disseminate the NDM-1 gene from medical origins. The occurrence of NDM-1 genes in the © XXXX American Chemical Society

environment has serious implications for human health. For instance, in New Delhi, NDM-1-producing bacteria in an environmental setting (seepage and tap water) would most likely influence the people who are reliant on public water and sanitation facilities.28 Our recent study revealed that blaNDM‑1 genes occurred in and even survived the sanitizing processes of two wastewater treatment plants (WWTPs).27 The release of NDM-1 genes increases the opportunity for blaNDM‑1 horizontal transfer. Our previous study provided evidence of NDM-1 gene transfer to the indigenous bacteria from the river.27 Obviously, this increases the extent of dissemination of blaNDM‑1 genes into the natural environment. Furthermore, WWTPs link human activities with receiving water and may also facilitate the transfer of blaNDM‑1 genes from clinical pathogens to environmental microorganisms.30−33 Notably, the treated wastewater from WWTPs is often used as a recycled water resource in many countries where freshwater resources are limited. Tianjin is one of these cities with very limited fresh water resources, and the WWTP effluent is drained directly into a river primarily used for farm irrigation and aquaculture, which represents a potential 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, 2016 Revised: March 10, 2016 Accepted: March 10, 2016

A

DOI: 10.1021/acs.estlett.6b00036 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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PCR Assays and Quantification of blaNDM‑1 Genes. Qualitative PCR (qPCR) amplifications were performed using a Biometra T100 gradient (Biometra). Primers for blaNDM‑1, which amplified a 984 bp product, were used for qPCR to ensure accurate target genes, and it is good for indicating DNA damage. The quantification of blaNDM‑1 was accomplished with real-time PCR on a Bio-Rad iQ5 instrument (Bio-Rad, Hercules, CA), and the 16S rRNA gene was quantified to evaluate the abundance of the total bacterial community. Primers that amplified 214 and 126 bp products were used for qPCR assays of the blaNDM‑1 and 16S rRNA genes, respectively. The primer sequences are listed in Table S1. Calibration standard curves for positive controls were generated as described previously.34 Negative controls contained all the components of the PCR mixture without DNA template. The details of PCR and qPCR assays are described in SI-1 and SI-2 of the Supporting Information. Isolation of blaNDM‑1-Positive Bacteria. Selective LuriaBertani (LB) agar plates containing ampicillin (100 mg/L), kanamycin (25 mg/L), sulfamethoxazole (50 mg/L), erythromycin (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 water samples based on the criteria of the Clinical and Laboratory Standards Institute (CLSI). The blaNDM‑1 gene in isolates was assessed by PCR with specific primers listed in Table S1. Then NDM-1-producing isolates were identified by 16S rRNA sequencing combined with the Biolog Microbial Identification system (BIOLOG, Hayward, CA). The minimum inhibitory concentrations (MICs) of various antibiotics in these blaNDM‑1positive isolates were determined by the agar dilution method as set by the CLSI.35 The E. coli ATCC25922 strain was used as a control strain in this study. More specialized details are available in SI-3 of the Supporting Information. Conjugative Transfer. To assess the transferability of bla NDM‑1 genes, two isolates, Wautersiella falsenii and Acinetobacter seohaensis, isolated from the effluent and receiving river, respectively, were used as the donor of conjugation experiments, and azide-resistant E. coli J53 was used as the recipient. The selective plates containing ampicillin (100 mg/ L) and sodium azide (100 mg/L) were used to select transconjugants. Meanwhile, the donor and recipient strains were separately plated onto identical LB plates as negative controls to rule out spontaneous mutation. PCR (984 bp amplicons) and DNA sequencing were performed to ensure that blaNDM‑1 was transferred to the recipient. More details are listed in SI-4. The transfer frequency (f) was calculated using the formula f = NT (number of transconjugants, in colonyforming units per milliliter)/NR (number of recipients, in colony-forming units per milliliter).

blaNDM‑1 genes impacts its receiving water. Here we quantified blaNDM‑1 abundance, isolated NDM-1-producing bacteria, and tested the transferability of blaNDM‑1-positive plasmids to demonstrate the occurrence and fate of the blaNDM‑1 gene and its host bacteria in the receiving water and to verify that blaNDM‑1 genes occurring in the river are derived from wastewater. To the best of our knowledge, this is the first study to provide direct evidence that the emergence of NDM-1 genes in the river is derived from the discharges of the upstream WWTP.

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MATERIALS AND METHODS Study Site and Water Sampling. Nine sampling sites were selected in January 2015 in the effluent of a WWTP and its receiving river as shown in Figure 1. The WWTP with

Figure 1. Geographical location of the sampling sites. The map shows the locations where wastewater effluents are discharged to a receiving river as well as locations along the river where samples were collected for this study.

conventional activated sludge serves a large population of 1 million in Tianjin. The WWTP effluent and river water samples were collected. The effluent samples were collected after chlorinated disinfection. The river water samples were collected from 0, 100, 200, 300, 400, 500, and 1000 m along the WWTP discharges into the receiving water. The control, which is assumed not to be influenced by the WWTP effluent, was sampled approximately 300 m upstream of the WWTP. Water samples were collected in triplicate for each sample site. All water samples (2.5 L) from the top 0.5 m of the surface were collected in sterile polypropylene bottles and were transported to 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 vacuum filtration apparatus, and the filters were stored at 4 °C until DNA was extracted. DNA extraction was performed using an ultraclean water DNA kit (MoBio Laboratories Inc., Carlsbad, CA) according to the manufacturer’s instructions. Meanwhile, Escherichia coli DH5α cloned with the CESA9 gene as an internal standard was used to determine DNA extraction efficiency, as described previously.34 The extracted DNA was further purified using the DNA pure-spin kit (Vigorousbio, Beijing, China) to minimize polymerase chain reaction (PCR) inhibition. DNA extraction recoveries at different sites are listed in Table S2 of the Supporting Information. All the DNA samples were stored at −20 °C until further analysis.

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RESULTS AND DISCUSSION Discharge of blaNDM‑1 Genes from WWTPs. Our previous study revealed that blaNDM‑1 genes were prevalent in two wastewater treatment plants (WWTPs) and selectively survived the disinfection treatment stage.27 Since our previous report of the NDM-1 gene in that WWTP, the abundance of blaNDM‑1 genes has remained at a level of 103 copies/mL in the WWTP effluent. After a lengthy discharging period, the blaNDM‑1 gene now occurs in the downstream WWTP effluent reach of the receiving water, and the blaNDM‑1 sequences are 99.8% identical (983 bp match) to the blaNDM‑1 gene from Acinetobacter calcoaceticus (Gene ID KF951475.1). The B

DOI: 10.1021/acs.estlett.6b00036 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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“dilution effect” of river water, whereas upstream of the WWTP effluent, blaNDM‑1 genes were below the limit of detection (LOD). Furthermore, there was no anomalous increase downstream of the receiving river, indicating that no other sources contributed to the occurrence and proliferation of blaNDM‑1 genes within the sampling range. These results implied that the emergence of blaNDM‑1 genes in the receiving water may 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 along the river [(5.34 ± 1.27) × 106 to (1.96 ± 0.61) × 107 copies/ mL] (Table S3), indicating that the WWTP discharges significantly increased the biomass loads of its receiving water. This observation demonstrated that the treated wastewater discharges have a considerable influence on the bacterial community in the downstream river. Nevertheless, Figure 2B illustrates that at distances of >400 m, the 16S rRNA gene abundance recovered to the upstream level, which illustrates the dilution in the river. Three NDM-1-producing strains, two strains of A. seohaensis and one strain of Stenotrophomonas maltophilia, were isolated in the river downstream of the WWTP. These NDM-1-producing isolates have multidrug-resistant phenotypes; the MIC values to various antibiotics are comparable to those of isolates from WWTP effluent, as indicated in Table S4. Furthermore, A. seohaensis strains in receiving water possess the blaNDM‑1 gene with 100% identity to the gene from the same species in wastewater effluent. In addition, the blaNDM‑1 gene sequences in the receiving water were 100% identical (983 bp match) to that of the WWTP effluent. These facts indicated the propagation of NDM-1 to the receiving water upon WWTP discharge. Moreover, as mentioned above, a considerable level of blaNDM-1 genes was detected downstream of the receiving water and even 1000 m from the WWTP effluent. These findings and the sequence similarity confirmed that blaNDM‑1 genes in the receiving water were derived from the direct discharge of the WWTP. Therefore, the continuous discharge of wastewater significantly resulted in the occurrence and persistence of NDM-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 not detected in WWTP effluent under the same experimental conditions (Table 1). The blaNDM‑1 gene carried by S.

blaNDM‑1 gene level of the disinfected effluent was (2.71 ± 0.13) × 103 copies/mL, which is more enriched than the level in the receiving 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 into the receiving river, culture-dependent approaches were used to investigate multidrug-resistant bacteria in the WWTP effluent. A total of 12 NDM-1-producing bacteria were isolated from the effluent, and they were all identified as Enterococcus faecium, E. coli, Shigella sonnei, A. seohaensis, and W. falsenii. Compared with a previous study in the same WWTP in which only one species (Achromobacter sp.) was identified,27 more bacterial species possessing the blaNDM‑1 gene occurred in the WWTP. Most worryingly, these NDM-1-producing isolates occurring in the WWTP are pathogenic and opportunistic pathogens, and they could acquire antibiotic resistance, therefore increasing the risk of dissemination of blaNDM‑1 to human pathogens. As a consequence, the occurrence of these NDM-1-producing pathogens in the environment has serious implications for human health. Trends in NDM-1 Gene Levels in the Receiving Water Influenced by WWTP. The blaNDM‑1 and 16S rRNA (to evaluate the abundance of the total bacterial community) genes were quantified with qPCR to understand the fate of blaNDM‑1 genes in the receiving water. Figure 2A demonstrates that there was a considerable concentration of blaNDM‑1 genes in the receiving 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 decreased with downstream distance, which may be related to the

Table 1. NDM-1-Producing Isolates in the WWTP and the Receiving River sampling location

S. maltophilia

A. seohaensis

NDa

4

1

2

WWTP effluent receiving river a

Figure 2. Absolute concentration of blaNDM‑1 (A) and 16S rRNA (B) genes in different water samples along the receiving river. A total of eight sampling locations were included, and triplicate samples were taken from each sampling location (N = 24). The control (upstream) was sampled approximately 300 m upstream of the WWTP. The 0 m location 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.

En. faecium

E. coli

Sh. sonnei

W. falsenii

1

3

2

2

NDa

NDa

NDa

NDa

Not detected.

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, and plants.36 These results suggest the transfer of NDM-1 genes to indigenous bacteria in the receiving water. This potential blaNDM‑1 transfer to indigenous bacteria was also further verified by conjugation experiments. C

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Environmental Science & Technology Letters 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 kanamycin tetracycline chloromycetin imipenem meropenem streptomycin ciprofloxacin ceftazidime gentamicin

>256 256 >256 128 256 256 >256 16 >256 8

256 8 4 128 256 128 128 2 >256 4

>256 >256 256 256 256 >256 >256 8 >256 8

>256 16 4 128 256 >256 128 1 >256 4

4 0.06 0.03 2 0.03 0.03 4 0.06 0.06 0.12

Conjugative Transfer of IncA/C Plasmid Harboring the blaNDM‑1 Gene. Two isolates, W. falsenii and A. seohaensis from the effluent and the receiving water, respectively, were used as donors for conjugation experiments, and the azideresistant E. coli J53 strain was used as recipient to assess the dissemination of blaNDM‑1-positive plasmids in the receiving water. The blaNDM‑1 gene was detected in the E. coli J53 transconjugants, and its sequence was completely consistent with that of the donor. Moreover, the transconjugants exhibited resistance to the tested antibiotics that was similar to that of the bacteria isolated from the donor (Table 2). The blaNDM‑1 gene was located on the plasmids extracted from the E. coli J53 transconjugants; therefore, these findings demonstrate the transferability of blaNDM‑1-positive plasmids among bacteria. Furthermore, The transfer frequency of NDM-1 plasmids [(6.65 ± 0.72) × 10−6 transconjugants/recipient] was comparable 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‑1 propagation. The blaNDM‑1-carrying plasmids belonged to the incompatibility group A/C (IncA/C) based on a PCR-based replicon typing method.40 IncA/C-type plasmids are large, broad-hostrange plasmids41−43 and are usually associated with other extended-spectrum β-lactamase genes, e.g., blaCMY‑2,44−46 blaTEM,47 blaVEB,48 blaCTX‑M,49 and blaKPC‑2.50 These IncA/C plasmids could disseminate antibiotic resistance genes among clinically relevant bacteria, although their conjugal transfer capacities are variable.51−53 As a consequence, the selfconjugative IncA/C plasmid-borne NDM-1 genes and their emergence in the receiving water highlighted the risk of blaNDM‑1 gene dissemination in the environment. This study demonstrates the occurrence and fate of blaNDM‑1 genes in a receiving river following WWTP discharges and confirms that blaNDM‑1 genes occurring in the river are derived from wastewater. This finding is important because it illustrates that the dissemination of New Delhi metallo-β-lactamase genes (blaNDM‑1) derived from a wastewater treatment plant is a significant pathway for NDM-1 gene proliferation to the environment. Considering the risk that the NDM-1 gene and its host bacteria pose to public health, more emphasis must be focused on the transferable vectors in facilitating the dissemination of NDM-1 in the environment.

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

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86 (22) 87402072. *E-mail: [email protected]. Phone: +86 (22) 85358553. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Professor Minggui Wang (Fudan University Shanghai Medical College, Shanghai, China) for providing E. coli J53. This work was supported by the National Science Fund for Distinguished Young Scholars (41525013) and the National Natural Science Foundation of China (Grants 31470440 and 41473085).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.estlett.6b00036. D

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DOI: 10.1021/acs.estlett.6b00036 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX