Clin Microbiol Infect
The sporadic emergence of New Delhi metallo-β-lactamase-1 (NDM-1)-producing Acinetobacter spp. has been reported in China; however, NDM-1-positive bacteria epidemics are rarely reported in intensive care units (ICUs) in China, or even in the world. During 15 months’ surveillance Acinetobacter spp. isolated from patients, heathcare workers and surfaces of a Chinese ICU were screened for the blaNDM-1 gene. A total of 27 of 3114 Acinetobacter spp. strains were NDM-1 positive and identified as A. pittii with sequence type 63 (ST63) by multilocus sequence typing. Of the 27 NDM-1-positive A. pittii strains, 22 were isolated from the ICU surfaces and grouped into a major clone A using pulsed-field gel electrophoresis typing, while the other five strains isolated from the patients were classified into three clones (A, B and C). The blaNDM-1 gene was located on a 45-kb plasmid for all three A. pittii clones. The plasmid could be transferred to A. pittii and A. baumannii recipients at both 30 and 37°C but not to Escherichia coli J53. The plasmid could not be classified into any of the known plasmid incompatibility groups. The blaNDM-1 region in the plasmid was flanked by two insertion sequence elements, ISAba125 and ISAba11, and no other carbapenemase gene was present in this NDM-1-positive A. pittii isolate. Thus, we present the first report on the transmission and characterization of NDM-1-producing A. pittii in an ICU in China as well as a novel blaNDM-1 gene-bearing plasmid.
The emergence and worldwide dissemination of New Delhi metallo-β-lactamase-1 (NDM-1)-producing pathogens in both humans and the environment have created a major therapeutic challenge for clinicians and attracted significant public health attention [1–4]. As nosocomial pathogens, Acinetobacter spp. are becoming a major concern because of their rapid development of resistance to a wide range of antimicrobials. The emergence of NDM-1-producing Acinetobacter spp. has been recently reported in many countries, such as India, Israel, Egypt, Germany, Spain, Switzerland, the United Arab Emirates and China [5–12]. Interestingly, blaNDM-1 has been shown to be a chimeric gene constructed by the fusion of the aminoglycoside resistance gene aphA6 with a mannin-binding lectin (MBL) gene, an event that probably occurred in Acinetobacter spp. ; hence, Acinetobacter spp. are the likely origin of this gene. A. baumannii was the most common NDM-1-producing Acinetobacter spp. and the blaNDM-1 gene was mostly chromosome located [8,14]. NDM-1-producing A. lwoffii isolates were recently reported in China, and two novel blaNDM-1 gene-bearing plasmids were sequenced and identified . The blaNDM-1 gene is located within a composite transposon flanked by two insertion elements of ISAba125 in the pNDM-BJ01 plasmid .
Acinetobacter pittii, previously called Acinetobacter genomic species (gen. sp.) 3, is increasingly recognized as a clinically important taxa within the Acinetobacter calcoaceticus–A. baumannii complex . A. pittii is ecologically diverse and is found in food, soil, and both clinically ill and healthy individuals [17,18]. Although accumulating evidence has shown that it is closely associated with nosocomial infection, A. pittii has historically been less recognized due to its absence of a formal binomial name and clear-cut differentiating phenotypic characteristics . Several sequencing-based typing methods have recently been provided to help with discrimination between A. pittii and A. baumannii, including rpoB gene sequencing, multilocus sequence typing (MLST) and 16S–23S rRNA gene intergenic spacer sequencing [20,21].
Acinetobacter spp. are some of the most prevalent Gram-negative bacteria isolated in China, particularly in intensive care units (ICUs), and they share a high prevalence of extensive drug-resistance . Four A. baumannii isolates with the blaNDM-1 gene were identified in four different provinces in China in 2010 , shortly after the emergence of many NDM-1-producing isolates within Enterobacteriaceae in India, Pakistan and the UK . The blaNDM-1 gene has been increasingly detected in clinical non-baumannii Acinetobacter spp. such as Acinetobacter lwoffii, Acinetobacter Junii and A. pittii, as well as A. lwoffii of food animal origin [15,23–25]. A recent study showed that nine of 726 non-baumannii Acinetobacter spp. collected from 28 provinces in China contained the blaNDM-1 gene, while three of the nine NDM-1-producing isolates were A. pittii . These findings have raised awareness of the emergence and spread of NDM-1-carrying bacteria in China. However, little is known about the epidemiology and transmission of NDM-1-producing Acinetobacter spp. in clinical hospital wards in China. Here we examined the transmission of multidrug-resistant Acinetobacter spp. in an ICU of a tertiary care hospital in Beijing, China, from April 2008 to July 2009 and then investigated and analysed the dissemination and characterization of NDM-1-producing Acinetobacter spp. isolates.
The study was approved by the Institutional Ethic Committees of the Academy of Military Medical Sciences and Chinese PLA General Hospital. Written consent was obtained from all patients before the study commenced.
Settings and patients
The ICU was located within a 3000-bed tertiary care hospital in Beijing, China, and consisted of 18 beds in one large room and two single-bed rooms. Most critically ill patients in this ICU had been transferred from the surgical departments of this hospital. ICU surfaces, including floors and tables, were disinfected with electrolyzed acid water thrice daily.
Sample collection and strain identification
The samples included the clinical specimens from potentially infected sites, such as sputum, urine and blood, and swabs from the anterior nares, forehead, groins and axillae of all patients in the ICU were collected within 24 h of admission and every other day thereafter. Swab samples were also collected from the surfaces near patients and work areas (e.g. bedrails, equipment buttons, bed sheets, chart files, water taps, drawer handles, dispensing tables and nurses’ station desks) once every 4 days. An open blood plate was used to collect samples from the air in a designated ward location at a height of 1.5 m for half an hour. In the meantime, healthcare workers were also screened for Acinetobacter spp. using swab samples from the anterior nares and hands once every 2 days.
Screened swabs and clinical specimens were inoculated on blood plates. Colonies resembling Acinetobacter spp. on the plates were isolated and transferred onto a China-Blue lactose agar plate (Luqiao, Beijing, China) to select Gram-negative bacteria. The Acinetobacter spp. were further identified according to their morphological and growth characteristics using the oxidase, triple sugar iron and citrate tests . To discriminate A. pittii from A. baumannii, the RNA polymerase β-subunit (rpoB) gene of the isolates was sequenced according to the method described previously .
Molecular detection of NDM-1 and other resistance determinants
Bacterial DNA was extracted using the Wizard7® genomic DNA purification kit. The blaNDM-1 gene was detected by polymerase chain reaction (PCR) using the primers NDM-1F: 5′-GAAGCTGAGCACCGCATTAG and NDM-1R: 5′-GGGCCGTATGAGTGATTGC. The primers were designed using the PRIMER3 web site (http://frodo.wi.mit.edu/primer3) according to GenBank database entry FN396876. PCR conditions were as follows: 95°C for 5 min; 30 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 40 s, followed by 72°C for 5 min. Carbapenemase determinants including blaOXA-51-like, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, blaIMP-like and blaVIM-like were detected according to the method described previously [29,30]. All PCR products were confirmed by sequencing and comparison with GenBank entries.
Antimicrobial susceptibility testing
The minimum inhibitory concentrations were measured using the agar dilution method or the Etest (AB bioMérieux, Solna, Sweden). The results were interpreted according to the interpretive standards of the Clinical Laboratory Standards Institute (CLSI) . Imipenem-ethylenediaminetetraacetic acid synergy tests were performed using the MBL E-test to screen MBL production according to the manufacturer’s instructions. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as the control strains for antimicrobial susceptibility testing.
Multilocus sequence typing was performed as described on the INSTITUT PASTEUR web site (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Abaumannii). The DNA sequences were uploaded into the MLST database, and the allelic numbers and sequence types (STs) were obtained online. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the Acinetobacter spp. isolates as previously described .
Plasmid analysis and Southern blot
Whole genomic DNA including the plasmid of the isolates was digested with 20 U of S1 nuclease at 37°C for 20 min and then separated by PFGE. The DNA fragments were then transferred to positively charged nylon membranes (Roche Applied Sciences, Penzberg, Germany). Hybridization was conducted using DIG Easy Hyb Granules (Roche Applied Sciences) at 64°C, while detection was performed using a DIG Nucleic Acid Detection Kit (Roche Applied Sciences).
NDM-1-positive A. pittii D186 (clone A by PFGE typing), H699 (clone B) and H924 (clone C) strains were used as the donors. Sodium azide-resistant E. coli J53, NDM-1-negative A. pittii D484 and A. baumannii H775 strains (sensitive to meropenem but resistant to ciprofloxacin) were used as the recipients, respectively. To select the transconjugants on China-Blue lactose agar plate, meropenem (20 mg/L) in combination with sodium azide (170 mg/L) was used for the E. coli J53 strain or meropenem (20 mg/L) in combination with ciprofloxacin (10 mg/L) was used for the D484 and H775 strains.
Genome sequencing, plasmid analysis
Whole genome sequencing of NDM-1-positive A. pittii D499 was performed using Illumina HiSeq 2000 (Accession: AGFH00000000.1; Illumina Inc., San Diego, CA, USA). The procedures for functional annotation were performed as described previously . The plasmid sequences were annotated by the RAST Server , and each predicted protein was further compared against the NCBI non-redundant protein database using BLASTP . To identify the plasmid type, the complete sequences of the pAP-D499 plasmid were investigated using BLASTX analysis and in silico PCR using the system developed for the PCR-based replicon typing method of A. baumannii .
From 7 April 2008 to 7 July 2009, 3114 Acinetobacter spp. were isolated: 1655 strains from patients, 104 strains from healthcare workers, and 1355 strains from the ICU surfaces. A total of 1149 of the 3114 isolates (36.9%) were imipenem resistant, and 27 of these 1149 isolates (2.3%) were NDM-1 positive, including two strains isolated from patient foreheads, three strains isolated from patient groins, and 22 strains isolated from ICU surfaces (Table 1). All 27 of these strains were identified as sequence type 63 (ST63) by MLST and A. pittii using a VITEK 2 GN card and rpoB gene sequencing. Remarkably, NDM-1-positive A. pittii strains were never isolated from the healthcare workers’ swab samples or the patients’ sputum, urine or blood samples.
|Order of isolating||Strains no.||Date of isolating||Source of sample||PFGE type||Minimal inhibitory concentration (mg/L)|
|1||D039||2008-6-11||Bed 7 Button||A||512||>32||>32||16||2||1||<0.125|
|3b||D151||2008-6-23||Bed 6 Button||A||512||>32||>32||16||2||2||<0.125|
|3c||D153||2008-6-23||Bed 7 Button||A||512||>32||>32||16||1||4||<0.125|
|9c||D293||2008-7-7||Bed 6 Chart file||A||512||>32||>32||8||1||2||<0.125|
|9d||D294||2008-7-7||Bed 7 Chart file||A||512||>32||>32||8||2||2||<0.125|
|9e||D298||2008-7-7||Bed 4 Chart file||A||512||>32||>32||8||2||2||<0.125|
|11b||D335||2008-7-11||Nurse’s station desk||A||512||>32||>32||16||2||2||<0.125|
|13||D424||2008-8-6||Patient 1 Forehead||A||512||>32||>32||8||1||4||<0.125|
|14||D499||2008-8-12||Patient 2 Forehead||A||>512||>32||>32||8||1||2||<0.125|
|15||H699||2009-5-6||Patient 3 Groin||B||512||>32||>32||8||2||4||<0.125|
|16||H924||2009-6-13||Patient 4 Groin||C||512||>32||>32||4||0.25||4||0.25|
|17||H944||2009-6-17||Patient 4 Groin||C||512||>32||>32||8||0.25||4||0.25|
As shown in Figs 1 and 2, the A. pittii clone A (PFGE typing) existed at the beginning of the surveillance in April 2008. This clone somehow became NDM-1 positive on June 11 2008 and disseminated within this ICU, affecting the surfaces of buttons, tables and water taps, and even the air (Table 1). After 2 months’ dissemination in the environment, A. pittii clone A was detected on the foreheads of the two patients who had stayed sequentially in the same bed, indicating a possible vector-based transmission of NDM-1-positive A. pittii (Table 1); however, this strain was never detected again. Nine months later, NDM-1-producing A. pittii clone B (strain H699) emerged on a patient’s groin on 6 May 2009. One month later, another NDM-1-positive A. pittii clone C was isolated twice from the fourth patient’s groin during his ICU stay from 5 May to 18 June 2009 (Table 1; Fig. 2).
Of the total of 518 patients who stayed in the ICU for at least 2 days during the 15-month study period, 116 (22.4%) received carbapenems. Meropenem was most frequently used (81 patients; 15.6% of the total), followed by imipenem (37 patients; 7.1% of the total). The mean age of the 518 patients was 59 years (range, 1–97 years) and 184 (35.5%) of them were female. The median length of stay in the ICU was 6 days (range, 2–277 days). A total of 450 patients (86.9%) underwent surgery after their hospital admission, while 63 patients (12.2%) underwent ventilator support during their ICU hospitalization. The ages of the four patients who carried NDM-1-positive A. pittii were 60–82 years. The first three patients stayed in the ICU for around a week, but the last patient stayed much longer, approximately 45 days. Remarkably, none of these patients received carbapenems. The underlying diseases of these four patients included urinary retention, cervical cancer, gastric cancer, sepsis and multiple organ failure.
According to CLSI 2012 , all of the NDM-1-positive isolates were resistant to cefotaxime, imipenem, meropenem and ertapenem and susceptible to amikacin, levofloxacin and minocycline. Interestingly, nearly half of the NDM-1-positive isolates (n = 13) showed colistin resistance (Table 1). The OXA-type and other MBL carbapenemase genes, such as blaOXA-51-like, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like,blaVIM-like and blaIMP-like, were not detected in any of the NDM-1-positive A. pittii isolates.
Plasmid analysis and southern blot showed that the blaNDM-1 gene was located on an approximate 45-kb plasmid. This blaNDM-1-carrying plasmid was considered unstable in the host because it was lost when the plasmid-carrying strain was cultured on a Mueller-Hinton agar plate for only one passage. This plasmid could be transferred to an NDM-1-negative A. pittii recipient (D484) and an A. baumannii recipient (H775) by in vitro conjugation at both 30 and 37°C, but it could not be transferred to E. coli J53. All transconjugants exhibited high-level resistance to carbapenems after plasmid acquisition (Table 2).
|Strain||Species||Grow at 44°C||PFGE type||blaNDM-1||Conjugation temperature||Minimal inhibitory concentration (mg/L)|
|D484–D186||A. pittii||−||D||+||30 & 37°C||>32||>32||64|
|D484–H699||A. pittii||−||D||+||30 & 37°C||>32||>32||64|
|D484–H924||A. pittii||−||D||+||30 & 37°C||>32||>32||64|
|H775–D186||A. baumannii||+||E||+||30 & 37°C||>32||>32||64|
|H775–H699||A. baumannii||+||E||+||30 & 37°C||>32||>32||64|
|H775–H924||A. baumannii||+||E||+||30 & 37°C||>32||>32||64|
The draft genome sequence of the NDM-1-positive A. pittii D499 strain included an approximate 4-mb chromosome and an approximate 45-kb NDM-1-carrying plasmid (named pAP-D499) . In silico analysis showed that the plasmid pAP-D499 contained 55 open reading frames (ORFs) with an average 40.8% GC content. The pAP-D499 plasmid possessed a putative transfer and replication region containing the traA and traC plasmid transfer genes, the plasmid-partitioning parA gene, and a type IV secretion system (T4SS) gene cluster region. It could not be classified according to replicase typing using in silico PCR . None of the 55 ORFs, including 28 hypothetical proteins, was similar to any of the current identified replicases by BLASTP. Only aminoglycoside resistance gene aphA6 and bleomycin resistance gene bleMBL were identified in this blaNDM-1-bearing plasmid. No carbapenemase resistance genes other than blaNDM-1 were found in the D499 plasmid or chromosomes.
The blaNDM-1 region in the pAP-D499 plasmid was flanked by two insertion sequence (IS) elements (ISAba125 and ISAba11), a finding that differed from those of earlier reports of A. lwoffii, A. pittii and A. baumannii [14,15,26]. The insertion sequence element ISAba11, which had a length of 1101 bp, represents an emerging insertion sequence family that encodes transposases . ISAba11 was bracketed by identical 5-bp direct repeats (attta), indicating target site duplication, and contained matching 13-bp terminal inverted repeat (TIR). It shared 99% sequence identity with that identified in A. baumannii strain ATCC 19606 and 98% sequence identity with a hypothetical protein encoded by A. baumannii ATCC 17978 [36,37].
The genetic content in the blaNDM-1 region of the pAP-D499 plasmid was nearly the same as described previously (Fig. 3) [14,15], including the bleMBL gene, genes encoding the GroES and GroEL chaperonin proteins, and the ISCR element ISCR27 located downstream of blaNDM-1. The GroEL protein (547 amino acids) and ISCR27 (399 amino acids) shared 100% identity with that encoded by the pNDM-BJ01 plasmid from A. lwoffii .
In China, scattered emergence of NDM-1-positive Acinetobacter spp. has been reported, including A. baumanii, A. lwoffii and A. pittii [9,15,24,26]. In this study, NDM-1-positive A. pittii clone A disseminated predominantly within the ICU and was detected in the air a remarkable seven times. This result indicated that airborne transmission might contribute to the spread of NDM-1-positive A. pittii, a finding that is in line with those of earlier studies suggesting that airborne transmission may be important in the spread of some Acinetobacter species [38–40]. The lack of isolation of NDM-1-positive A. pittii from the hands and anterior nares of healthcare workers hinted that healthcare workers in this ICU might not be vectors of NDM-1-positive A. pittii dissemination. Stricter infection control practices such as environmental cleaning and air hygiene may be required to reduce the spread of NDM-1-positive A. pittii within the ICU.
The origin and spread of NDM-1-positive A. pittii in this ICU remains vague. Compared with the report on the emergence of NDM-1-positive bacteria in China, the NDM-1-positive A. pittii that emerged on 11 June 2008 was confirmed as the first NDM-1-positive strain in China. All of the patients and healthcare workers in the ICU during this period of time were questioned, and no direct evidence showed any contact with southwest Asia, especially India or Pakistan. The recently reported dissemination of NDM-1-positive bacteria in New Delhi and the detection of NDM-1 in the seepage and tap water indicated that the natural reservoir of the NDM-1 gene may play a critical role in the dissemination of the NDM-1 gene throughout different bacteria . Our preliminary data also showed the existence of the NDM-1 gene in the sewage effluent samples in this hospital (data not shown). Therefore, a detailed investigation into the origin and spread of NDM-1-positive A. pittii in this ICU in the near future would be of great interest.
It is known that multidrug-resistant A. baumannii is highly prevalent in Chinese hospitals , whereas A. pittii epidemics are rarely reported in ICUs in China . This is likely to be due to the fact that few studies have completely speciated Acinetobacter spp. However, it is quite interesting that all NDM-1-positive Acinetobacter spp. in the present study were the A. pittii ST63 strain, whereas 126 non-ST63 type Acinetobacter spp. were totally NDM-1 negative (data not shown). These data suggested that NDM-1 was prevalent in A. pittii but not A. baumannii during our surveillance, although A. baumannii was the most prevalent multidrug-resistant clinical microorganism during the same period and the blaNDM-1-carrying plasmids could also be transferred to A. baumannii recipients using an in vitro conjugation test (Table 2). It is difficult to explain the low incidence of NDM-1 in A. baumannii, and the complicated clinical situation herein may contribute to this phenomenon. Further detailed investigations into this issue are needed.
The blaNDM-1-carrying plasmid in A. pittii was approximately 45 kb, much smaller than the blaNDM-1-carrying plasmids in Enterobacteriaceae [1,2,9,15,42] but similar to the blaNDM-1-carrying plasmids in A. baumannii strains isolated in China in 2009 (30–50 kb) . Although a relatively high transfer frequency had been reported from NDM-1-positive A. lwoffii to E. coli in a conjugation test , the inability of this blaNDM-1-carrying plasmid to transfer into E. coli in our study was in line with earlier reports in which no blaNDM-1-carrying plasmids from A. baumannii could be transferred to E. coli recipients and no plasmids of Enterobacteriaceae were detected among NDM-1-producing A. baumannii .
Tn125 were recently shown to be the main vehicle for the dissemination of blaNDM-1 genes in A. baumanii and non-baumannii Acinetobacter spp. [14,26]. In our study, the composite transposon containing blaNDM-1 in the plasmid pAP-D499 was nearly identical to its counterpart in either of the A. lwoffii plasmids isolated from China or chromosomes of A. baumannii isolated from many European countries [14,15], except that the insertion sequence ISAba11 instead of ISAba125 located downstream of blaNDM-1 (Fig. 3). It was recently shown that the ISAba11 elements belong to a novel family that encodes transposases with a signature HHEK motif and had been found in many kinds of Acinetobacter spp. . The ISAba11 in the pAP-D499 was associated with typical 5-bp DR and possessed 13-bp TIR as previously reported . ISAba11 movement can result in inactivation of the A. baumannii lipid A biosynthesis genes lpxA and lpxC, resulting in the loss of lipopolysaccharide production and high-level colistin resistance. However, no lpxA or lpxC elements were found in pAP-D499, so the evolution of ISAba11 might differ from that found in A. baumannii isolates . The composite transposon containing blaNDM-1 in pAP-D499 was probably derived via recombination events between Tn125 and Tn11.
In a word, we characterized the dissemination of NDM-1-positive A. pittii strains in an ICU in China and identified a novel genetic structure containing the blaNDM-1 gene in Acinetobacter spp. Further investigations are required to track the transmission pathways of blaNDM-1-carrying plasmids and to understand the disseminative regularity of the blaNDM-1 gene in Acinetobacter spp. isolates.
JY performed the molecular genetic analyses and plasmid profiling, YC and XJ performed the blaNDM-1 and MLST analysis and bioinformatic analysis. YC, YL, WZ, YW, DZ, YX, RY, XH, GJ, YZ and XH collected the strains and clinical details and performed the minimum inhibitory concentration determinations. YL, QS, LL and LH designed and organized the study and proofread the manuscript. LH drafted and revised the manuscript. All of the authors read and approved the final manuscript.
We thank Tom D. Y. Chin, MD, of the University of Kansas Medical Center, Kansas City, Kansas, USA, for his critical review of this manuscript and thank Dr Yajun Song and Ruifu Yang for their kind advice regarding the study design. The study was supported by the National Key Program for Infectious Diseases of China (2008ZX10004-001-C) from the Ministry of Science and Technology, China, and a grant from the National Natural Scientific Foundation of China (No. 81102168).
The authors have no conflicts of interest to declare.