“Roar” of blaNDM-1 and “silence” of blaOXA-58 co-exist in Acinetobacter pittii

Acinetobacter pittii 44551 was recovered from a patient with gout combined with tuberculosis and was found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 on two different plasmids pNDM-44551 and pOXA58-44551, respectively. pNDM-44551 displayed high self-transferability across multiple bacterial species, while pOXA58-44551 was likely co-transferable with pNDM-44551 into A. baumannii receipts. pNDM-44551 was a close variant of the previously characterized pNDM-BJ01, and the blaNDM-1 gene cluster was arranged sequentially as orfA, ISAba14, aphA6, ISAba125, blaNDM-1, bleMBL, ΔtrpF, dsbC, tnpR, and zeta. pOXA58-44551 was a repAci9-containing plasmid, and blaOXA-58 was embedded in a 372F-ISAba3-like-blaOXA-58-ISAba3 structure. The mobile genetic platforms of blaNDM-1 and blaOXA-58 herein showed some differences from their previously characterized variants. The production of NDM-1 in strain 44551 contributed the majority to its high resistance to carbapenems, while the blaOXA-58 stayed silent most likely due to the lack of an upstream promoter to drive its transcription. Increased surveillance of Acinetobacter co-harboring blaNDM-1 (active) and blaOXA-58 (either active or silent) is urgently needed.

contribute to drug resistance. The present study describes the genetic environment, transferability, and antibiotic susceptibility of bla NDM-1 and bla OXA-58 harbored on different plasmids in a single clinical A. pittii isolate from China.
Results and Discussion bla NDM-1 and bla OXA-58 on different plasmids in a single A. pittii isolate. A total of 31 carbapenem-resistant Acinetobacter isolates were collected at the Meizhou People's Hospital, Guangdong Province, China, from June 2010 to December 2012. They were classified into A. pittii (n 5 1, designated 44551), A. nosocomialis (n 5 2), and A. baumannii (n 5 28). PCR screening for the MBL and OXA carbapenemase genes in these strains indicated the presence of bla NDM-1 and bla OXA-58 in strain 44551 and the habitation of bla OXA-23 in 21 A. baumannii isolates (67.7%), while all the other PCR reactions gave negative results. DNA sequencing further confirmed the presence of bla NDM-1 and bla OXA-58 in 44551. PCR detection of the extended spectrum b-lactamase genes bla TEM , bla CTX-M , bla PER , bla SHV , bla DHA , and bla CMY [22][23][24][25] in 44551 showed negative results. The observation of bla OXA-23 as the most prevalent carbapenemase gene in A. baumannii is consistent with previous findings 26 .
A. pittii 44551 was isolated from the sputum of a 62-year-old male hospitalized for gouty arthritis with a skin soft tissue infection combined with pulmonary tuberculosis (TB) in August 2011. After the initiation of anti-tuberculosis treatment with HREZ (isoniazid, rifampicin, ethambutol hydrochloride, and pyrazinamide), the patient's sputum samples were screened weekly for Mycobacterium tuberculosis. The patient's pulmonary infection symptoms, including fever, weakness and coughing, were relieved in response to anti-TB therapy, and his sputum acid-fast stain gave negative results after two weeks of treatment. After one month of hospitalization, the patient improved perceptibly with stable vital signs. He was discharged with instructions to continue taking 3HREZ/9-12HRZ as prescribed and to undergo regular follow-up. Unfortunately, the patient did not return for regular checkup and has been out of touch with our clinic. S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) assay ( Fig. 1a) showed that A. pittii 44551 harbored four different plasmids at ,20 kb, ,40 kb, ,50 kb and ,90 kb. Subsequent Southern hybridization (Fig. 1b) with a probe specific to bla NDM-1 or bla OXA-58 revealed that these two genes were located on the ,40 kb (designated pNDM-44551) and ,50 kb (pOXA58-44551) plasmids, respectively. Using the PCR-based replicon typing scheme of A. baumannii 27 , repAci9 was detected in pOXA58-44551 but no classified type of replicase could be identified from pNDM-44551.
A weak PCR signal of bla OXA-58 was observed in a small portion of the bla NDM-1 -positive MZPB-44551 clones at a 1510 ratio, as well as in their passage cultures, which was further confirmed by sequencing. Meanwhile, Random Amplified Polymorphic DNA showed that these bla OXA-58 -and bla NDM-1 -positive conjugants were MZPB-originated species. The PCR results of one of the above conjugants, designated MZPB-44551 OXA581 , were shown in Fig. S1a and S1b. Nonetheless, the pOXA58-44551 signal was invisible by S1-PFGE/Southern hybridization in MZPB-44551 OXA581 (Fig. 1a), which was attributable to the very low copy number of pOXA58-44551. In addition, no PCR signal of bla OXA-58 was detected in all the bla NDM-1 -haboring J53 or EC600 conjugants tested, which is consistent with the fact that bla OXA-58 has only been found in Acinetobacter 28 . In all, it seemed that pOXA58-44551 could co-transfer with pNDM-44551 only into the Acinetobacter recipient.
It is worth noting that MZPB-44551 showed distinct antimicrobial susceptibility profile from 44551 across almost all the tested blactams (Table 1), while the pNDM-44551 copy number (Fig. 1a) or the bla NDM-1 mRNA abundance (Fig. S2) was comparable between these two strains. Based on the spontaneous PB-resistance of MZPB under PB selection, it is reasonable to speculate that this distinction may be mainly due to different species backgrounds.
Genetic surroundings of bla NDM-1 . pNDM-BJ01 was recovered from A. lwoffii in China in 2012 and harbored four regions encoding for conjugate transfer, plasmid replication and stability, a type IV secretion system, and the bla NDM-1 gene cluster 18 . Primer walking combined PCR/sequencing indicated that pNDM-44551 contained 41 of the 46 CDSs annotated in pNDM-BJ01 with .99% sequence identity and the missing five CDSs were located in a tandem manner within the bla NDM-1 gene cluster.
The aphA6 gene was usually found adjacently upstream of the ISAba125-bla NDM-1 structure in the pNDM-BJ01-like plasmids (Table S1). It has been postulated that a Tn125 transposon structure inserts into the non-coding region downstream of aphA6, which is evidenced by the 3-bp GTT target site duplication at the point of insertion as shown in pNDM-BJ01 18 . A 64 bp direct repeat was observed flanking the ISAba125-bla NDM-1 insertion in pNDM-44551, and each monomer was composed of the first 20 bp of the CDS of aphA6 or bla NDM-1 together with its upstream 44 bp sequence ( Fig. 2a), which was consistent with the previous report that bla NDM-1 is a chimeric gene resulted from the in-frame fusion of a preexisting bla NDM-1 with aphA6 29 .
A total of 11 single nucleotide polymorphism (SNP) sites were present in the aphA6 CDS of pNDM-44551 relative to pNDM-BJ01, resulting in three amino acid (a.a.) changes of L84F, A156T, and R163K. In addition, a 67 bp deletion occurred within the 39 flanking region of aphA6 of pNDM-44551 compared with pNDM-BJ01 (Fig. 2b). The aphA6 CDS together with its 39 flanking region of pNDM-44551 was almost identical to the counterpart of TnaphA6 (accession number JF343537, located in the chromosome of A. baumannii from Australia) and that of pWH8144 (accession number JG241792, in A. baumannii from China), with only one SNP mismatch (T to C) at the 59 end of aphA6. By contrast, this kind of aphA6 gene considerably differed from the counterpart of pNDM-BJ01 (Fig. 2b). aphA6 has been proposed to be an ancestral gene in Acinetobacter and possesses considerable nucleotide polymorphism 30 . There might be two possible explanations for the aforesaid aphA6 sequence difference: 1) the original recombination of aphA6 with bla NDM-1 may be two independent events in pNDM-44551 and pNDM-BJ01, and 2) homologous recombination may occur to induce an aphA6 swap upon the spread of the aphA6-ISAba125bla NDM-1 -containing plasmid into an aphA6-carrying Acinetobacter strain.
The above observations strongly suggest that pNDM-44551 represents a close derivate of pNDM-BJ01 although pNDM-44551 might have undergone multiple evolutionary events especially within the Table 1 (Table S1). It should be noted that the ISAba125 element upstream of bla NDM-1 is usually intact in Acinetobacter but often truncated in Enterobacteriaceae (Table S1), suggesting the probable spread of the bla NDM-1 genetic platforms from Acinetobacter to Enterobacteriaceae 13,18,29,34 Genetic surroundings of silent bla OXA-58 . In general, bla OXA-58 is embedded in a conserved platform ISAba3-like-bla OXA-58 -ISAba3 in Acinetobacter, and the upstream ISAba3-like element is often interrupted by other insertion sequence (IS) elements, which in turn provide the promoters enhancing the bla OXA-58 expression to mediate higher degrees of drug resistance compared with the parent intact ISAba3-like [35][36][37][38][39] . Sequence analysis revealed that the bla OXA-58 gene of pOXA58-44551 was located between a downstream ISAba3 and an upstream ISAba3-like, both of which were intact; the downstream ISAba3 was followed by a gene cluster aaC3 (aminoglycoside N39-acetyltransferase III)-ATPase (ATPase protein) (Fig. 3), which differed from the previously reported common pattern araC1 (transcription regulator)-lysE (threonine efflux protein) in bla OXA-58 genetic contexts in Acinetobacter spp. from China 40 and other countries 39,41 . The C-terminal 23 a.a. sequence of the transposase of the upstream ISAba3-like element in pOXA58-44551 was replaced with an unknown 26 a.a. fragment, and moreover the 39-untranslated region (39-UTR) and the right inverted repeat (IRR) were lost (Fig. 3). This entire upstream ISAba3-like element was essentially the same as the counterpart in the bla OXA-58 genetic structure in an A. pittii isolate AP04 from China 40 and some unpublished bla OXA-58 -containing sequences (accession numbers JX101647, FJ195389 and FJ200187), as well as in a bla OXA-97 genetic structure 36 . In addition, a 372 bp DNA fragment (named 372F) as the left neighbor of the upstream ISAba3-like element in pOXA58-44551 (Fig. 3) showed 100% sequence identity to that in the genome of the insect Dendroctonus ponderosae (APGK01007886.1). In all, bla OXA-58 was mobilized into the genetic platform 372F-ISAba3-like-bla OXA-58 -ISAba3 (flanked by the 28 bp direct repeat; having a GC content much lower than those of the surrounding regions), which was further inserted into the backbone of pOXA58-44551 most likely through IS-mediated transposition. The evidences described below from different aspects denoted that bla OXA-58 was expressed either very weakly or not at all in 44551. First, the addition of EDTA (the inhibitor of MBLs including NDM1) in the agar plates for bacterial cultivation made 44551 lose almost the entire resistance to the tested b-lactams, but the addition of excess NaCl (the inhibitor of OXA-58) had no effect on the resistance profile of 44551 (Table 1), indicating that the production of NDM-1 enzyme contributed the majority to b-lactams resistance while bla OXA-58 contributed little to the resistance. Second, the ISAba3-like element upstream of bla OXA-58 in pOXA58-44551 was intact, suggesting a lack of the bla OXA-58 -driven promoter which was usually provided by other inserted IS elements in the typical OXA-58encoding genetic structures [35][36][37][38][39] (Fig. 3). This postulation was further supported by a recent surveillance study, which showed that as many as 25 of the total of 32 bla OXA-58 -positive Acinetobacter isolates recovered from 23 Chinese provinces remained susceptible to carbapenems, and all of them contained an intact ISAba3-like element upstream of bla OXA-58 , while the remaining non-susceptible isolates showed an insertion of additional IS element into the upstream ISAba3-like element 42 . Third, repeated attempts to transform the plasmid pBBRIMCS3-OXA-58 (containing the ISAba3-like-bla OXA-58 fragment cloned from pOXA58-44551) into E. coli DH5a failed to generate an ampicilin-resistant, bla OXA-58 -haboring clone. Finally, northern blot with a probe specific to bla OXA-58 showed very weak or almost invisible signals in A. pittii 44551, but a very strong band could be detected in the BL21-OXA58 strain, which was an E. coli BL21 strain carrying the plasmid pET-28a-bla OXA58 (Fig. 4a and 4b). Consistent with the northern blot results, RT-qPCR assay revealed that the relative mRNA abundance of bla OXA-58 in strain 44551 was about 30-fold lower than that in BL21-OXA58, with the 16S rRNA genes being the internal control (Fig. 4c).
Concluding remarks. In the present study, bla NDM-1 and bla OXA-58 were found to be harbored on two different plasmids in a single clinical A. pittii isolate named 44551, with the former gene expressing well and the latter one silent. Each of these two resistance genes was embedded in a genetic structure differing partly from its previously characterized variants. The bla NDM-1 -carrying plasmid in 44551, conferring a high level of carbapenem resistance, showed a strong ability of horizontal transfer into Acinetobacter and Enterobacteriaceae. The silent bla OXA-58 has the potential to evolve into the active form due to additional IS element insertion driven by environmental pressures such as the presence of carbapenems. The fact that A. pittii 44551 colonized in the lower respiratory tract of the indicated hospitalized patient increased the possibility of the strain 44551 dissemination into hospital settings. Since the Acinetobacter strains co-harboring bla NDM-1 (active) and bla OXA-58 (either active or silent) have the potential to widely spread in China 42 , increased surveillance of these kinds of bacteria in hospital and community settings is urgently needed.

Methods
Clinical Acinetobacter isolates. Each clinical sample was inoculated onto MacConkey agar plates and the dominant strain was recovered and identified using Vitek II (BioMérieux, Durham). Discrimination of Acinetobacter was performed by one-tube multiplex PCR specific for A. baumannii identification 43 and by 16S-23S rRNA intergenic spacer sequencing for other types of Acinetobacter 44 . All the primers used in this study are listed in Table S2.
Detection of carbapenemase genes and their genetic contexts. The MBL genes bla IMP , bla VIM , bla SIM , and bla NDM were screened by multiplex PCR 45 . The OXA genes bla OXA-23 , bla OXA-24 , and bla OXA-58 were detected individually by PCR. The plasmid sample was prepared from 44551 using a BAC/PAC DNA Isolation Maxi Kit (Omega Bio-Tek), and the flanking regions were sequenced by primer walking from both ends of bla NDM-1 or bla OXA-58 . All PCR amplicons were subjected to DNA sequencing with an ABI 3700 sequencer.  Plasmid construction and electrotransformation. The bla NDM-1 or bla OXA-58 coding region together with its immediately upstream IS element was amplified from 44551 and then cloned into the cloning vector pBBRIMCS3 46 , generating the recombinant plasmid pBBRIMCS3-NDM-1 or pBBRIMCS3-OXA-58, respectively. The entire coding region of bla OXA-58 was cloned into the expression vector pET-28a, generating pET-28a-bla OXA-58 . pBBRIMCS3-NDM-1 or pBBRIMCS3-OXA-58 was transformed into E. coli DH5a through electrotransformation, while pET-28abla OXA-58 was transformed into E. coli BL21, with an attempt to obtain the E. coli clone expressing the corresponding carbapenemase enzyme.
Conjugal transfer. A. pittii 44551 harboring bla NDM-1 and bla OXA-58 was used as the donor, and E. coli J53 (NaN 3 resistant), EC600 (rifampin resistant), and A. baumannii MZPB were used as the recipients. MZPB is a homemade polymyxin-resistant derivate of a clinical isolate A. baumannii MZ and remains susceptible to b-lactams.
Membrane mating experiments were performed on Mueller-Hinton (MH) agar 47 . After 18 h of incubation, the mixed cultures were suspended in MH broth, and plated onto MH agar containing ampicillin (50 mg/ml) and NaN 3 (200 mg/ml) for J53, ampicillin (50 mg/ml) and rifampin (500 mg/ml) for EC600, and ampicillin (50 mg/ ml) and polymyxin (20 mg/ml) for MZPB. Conjugants were picked randomly from the original selective plates and inoculated into the selective LB broth, these initial cultures were used for PCR-based screening for the presence of bla NDM-1 and bla OXA-58 . To exclude the donor strain contamination, the initial culture of positive conjugants were spread onto the Amp 1 /PB 1 plate, then the second-passage colonies were randomly picked for each strain and subjected for PCR detection of bla NDM-1 and bla OXA-58 . The strain species were differentiated by Random Amplified Polymorphic DNA with two short primers M13 and AP2 48 .
Antimicrobial susceptibility testing. The MIC values for each indicated strain cultured on the MH plates were measured using Etest (AB bioMérieux, Solna, Sweden). The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and the British Society for Antimicrobial Chemotherapy (SAC) breakpoints 49,50 . S1-PFGE and Southern blot. Bacterial genomic DNA was prepared in agarose plugs and digested with S1 nuclease (Takara). The linearized plasmids and partially digested genomic DNA were separated through the CHEF-Mapper XA PFGE system (Bio-Rad). The DNA fragments were stained with ethidium bromide (EtBr), transferred to a Hybond N1 membrane (GE Amersham Biosciences) and hybridized with the DIG-labeled probe specific to bla NDM-1 or bla OXA-58 . Probe labeling and signal detection were carried out with DIG high primer DNA labeling and detection starter kit II according to the manufacturer's instructions (Roche Diagnostics).
RNA extraction and Northern blot. By using Trizol Reagent (Life Technologies), total RNA was isolated from the overnight culture of each indicated strain with or without addition of 1 mM IPTG. The RNA samples were analyzed on a formaldehyde-containing 1.2% agarose gel. Subsequent EtBr staining, membrane transfer, probe labeling and hybridization, and signal detection were carried out as above.
RT-qPCR. 5 mg of RNA treated with DNase I (Promega) was subjected to reverse transcription by using random hexamers and PrimeScript RT reagents (Takara). Simultaneously, a control reverse transcription reaction without reverse transcriptase was performed to rule out genomic DNA contamination. The cDNA reactions were diluted 155 in water as the template for PCR detection of bla NDM-1 or bla OXA-58 , and diluted 15500 for amplification of the 16S rRNA genes. Each PCR reaction contained 2 ml of cDNA, 8 ml of forward and reverse primers (each at 0.75 mM), and 10 ml of SYBR green PCR Supermix (Bio-Rad). Three independent bacterial cultures (total RNA samples) were employed as biological replicates, and each RNA sample was analyzed in triplicate in PCR. The PCR parameters were 95uC for 10 min, followed by 40 cycles of 95uC for 5 s and 60uC for 30 s, using Bio-Rad CFX96 thermocycler. The detecting mRNA levels of bla NDM-1 or bla OXA-58 were normalized to those of the 16S rRNA genes.
Plasmid replicon typing. Plasmid pOXA58-44551 was recovered from the S1-PFGE gel and used as the template for PCR detection of replicase genes using the A. baumannii PCR-based replicon typing scheme 27 . The variants belonging to the same group of replicases were recognized by further PCR amplicon sequencing.
Nucleotide sequence accession numbers. The bla NDM-1 or bla OXA-58 gene cluster reported herein was deposited in GenBank with the accession number KF208467 or KF208466, respectively.