The institutional review board of the Academy of Military Medical Sciences waived the need for written informed consent from the participants. This study is approved and authorized for each location by the Academy of Military Medical Sciences Review Board. There was no request for a specific permission according to Chinese law. A total of 119 water samples were gathered from river water (n = 20), treated drinking water samples (n = 50), sewage from 4 comprehensive hospitals (n = 20), raw water samples from the pools (n = 9) and community life sewage (n = 20) in Beijing from September to November 2010. For each sample, 500 ml of target water was collected and a 100 ml aliquot was centrifuged at 1000×g for 10 minutes at ambient temperature. After carefully decanting the supernatant, pellets were re-suspended with 1 ml Luria-Bertani (LB) liquid medium and 400 µl samples were seeded onto LB agar plates containing imipenem (10 µg/ml). All colonies on the culture plates were selected and identified by PCR as previously described [4] and was further sequenced for confirmation. All NDM-1 positive strains were identified using Vitek GNI+ cards (bioMérieux, France), and sequence analysis of the 16S rRNA gene. The primers used to amplify the 16s rRNA gene were 5′-TACCTTGTTACGACTT-3′ and 5′- AGAGTTTGATCITGGA-3′ [9]. The other A. baumannii isolates, as identified by Vitek GNI+ cards (bioMérieux, France) were isolated in our laboratory, between September 2009 and February 2010 (Table 1).

Bacterial susceptibility testing was carried out by the Kirby-Bauer method according to the Clinical Laboratory Standards Institute (CLSI) guidelines (2008) [10]. The antibiotic discs used were imipenem, meropenem, cefdazadime, cefotaxime, cefepime, gentamicin, tobramycin, tetracycline, ciprofloxacin, polymyxin B, chloramphenicol and tigecycline. The tested bacterium was picked up with sterile loop and suspended in peptone water and incubated at 37°C for 3 hours. The turbidity of the suspension was adjusted to 0.5 McFarland’s standard, and the suspension was then spread on the surface of a LB agar plates using sterile cotton swab. The antimicrobial susceptibility test disc was placed on the agar. The plates were incubated at 37°C overnight. The zone of inhibition was measured and interpreted as per the CLSI guidelines.

Conjugation transfer assay was performed in broth culture with E. coli J53 as the recipient. Donor was respectively mixed at a ratio of 1∶3 with ten A. baumannii containing the blaNDM-1 gene from the sewage. Transconjugants were selected on MacConkey medium containing sodium azide (100 mg/mL) and ceftazidime (16 mg/mL). Plasmid DNA was respectively extracted from ten A. baumannii containing the blaNDM-1 gene from the sewage, and was transformed by electroporation into competent E. coli JM109. The transformants were selected on LB agar plates containing ceftazidime (16 mg/mL). Transformants and transconjugants were further confirmed by Vitek GNI+ cards (bioMérieux, France) and tested for antimicrobial susceptibility by the Kirby-Bauer method according to CLSI (2008). Plasmids were extracted from donor strain, recipient strain (E. coli J53 and E. coli JM109), transconjugant and transformant by the QIAGEN Large-Construct Kit and were further analyzed by specific PCR and sequencing for blaDNM-1.

Pulsed-field gel electrophoresis (PFGE) of blaNDM-1 carrying A. baumannii isolates recovered from sewage, environmental sources, and clinical isolates with or without blaNDM-1 was carried out with a CHEF-Mapper XA PFGE system (Bio-Rad, USA) for 22 h at 6 V/cm and 14°C, with a pulse angle of 120° and pulse times from 5 to 20 s. The restriction endonuclease ApaI was used for in-situ digestion of intact A. baumannii genomic DNA. PFGE banding patterns were analyzed visually by using a Bio-Rad Gel Doc 2000 system. Genetic relationship was analyzed by the BioNumerics version 46.0 software.

Ten isolates containing the blaNDM-1 gene were identified in sewage prior to disinfect from the general hospitals; this included an effluent sample from a hospital after disinfecting by chlorination before discharge and community life sewage (Table 2). No NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water and community life sewage. Biochemical identification and sequencing of 16S rRNA demonstrated that the ten isolates were A. baumannii.

Antimicrobial susceptibility testing was performed to determine the drug resistance profile of the blaNDM-1 positive isolates. Previous studies indicated that most clinical NDM-1 producing bacteria were resistant to all antibiotics except colistin and tigecycline [1], [3]. Zhou et al [11] reported clinical isolates from a child patient in China were resistant to all β-lactams except aztreonam but sensitive to aminoglycosides and quinolones. In this study, they were sensitive to aminoglycosides, chloramphenicol, colistin and tigecycline that A. baumannii possessing blaNDM-1 were isolated from the hospital environment and the hospital sewage (Table 3), In addition, two isolates were sensitive to quinolones and six isolates were non-resistant (four susceptible and two intermediate) to tetracycline, respectively. The data indicates that sewage-associated isolates of A. baumannii have unique antibiotic resistance profiles from those reported for clinically-obtained A. baumannii harbouring blaNDM-1.

The conjugation and transformation experiments were performed to investigate whether the gene for NDM-1 in A. baumannii isolates recovered from sewage were located on plasmids and whether the transconjugant and transformant reduced susceptibility of the recipient strain towards antibiotics. Transconjugants and transformants were respectively randomly chosen, and the blaNDM-1 gene was determined by PCR amplification and sequencing (see in Fig. S1). The result showed that the plasmids carrying blaNDM-1 from A. baumannii from the sewage were successfully transferred to E. coli J53 and E. coli JM109. Susceptibility tests revealed that both the transconjugant and transformant decreased susceptibility to imipenem, cefepime, ciprofloxacin, cefdazadime, cefotaxime as compared with the recipients E. coli J53 and JM109 (Figure 1). Interestingly, all the transconjugants and transformants stably maintained the blaNDM-1-containing plasmid after seven passages in the absence of selection pressure.

To investigate the genetic relationship between blaNDM-1 carrying A. baumannii isolates recovered from sewage, environmental sources, and clinical isolates with or without blaNDM-1, PFGE was performed as previously described [12]. Among the 13 blaNDM-1 positive isolates recovered in this study, 11 PFGE patterns were observed (Figure 2). Two isolates, WJ3-2 and WJ3-5, had an indistinguishable Apa I-PFGE pattern among the ten sewage-recovered isolates of A. baumannii; two additional pairs (WJ0102, WJ0102-2; and WH0111 and WJ0102-1) were ≥94% similar. All six of these isolates were blaNDM-1-positive and had nearly identical phenotypic antimicrobial sensitivity profiles (both WJ0111 and WJ0102-1 were TE non-susceptible, WJ0111 was resistant and WJ0102-1 was recorded as intermediate). Collectively this data suggests these isolates came from a common source. Similarly, environmental isolates grouped into several distinct clusters. Furthermore, a large number of band variations were observed between the blaNDM-1 carrying isolates recovered from hospital sewage, and the blaNDM-1 carrying clinical and hospital environmental isolates. This variation was also evident in A. baumannii isolates without blaNDM-1. These results suggest that A. baumannii isolates in Beijing hospital system are genetically diverse overall, and that among the blaNDM-1 carrying A. baumannii isolates, several clones were detected.