Carbapenems are of choice antibiotics to many infections, especially those triggered by milti-drug resistant gram-negative bacteria. Therefore, carbapenemase in clinical gram-negative organisms which can hydrolyze carbapenems are an important threat to public health. What is more wrose, New Delhi metallo-β-lactamase 1(NDM-1), a new type of carbapenemase, can hydrolyze almost all antimicrobials except colistin, tigecycline and sometimes aztreonam, and was thus referred to “superbug” by media. This article will focus on the problem of carbapenem resistance mediated by NDM-1.

NDM-1, a new type of Ambler classer B metallo-β-lactamases (MBLs), encoded by bla_NDM-1, was first reported in K. pneumoniae and Escherichia coli derived from a Swedish patient of Indian origin who was admitted to hospital in New Delhi, India in 2009 [1]. Since then, bla_NDM-1-positive bacteria have disseminated worldwide, including almost all seven continents except the Antarctica [2]. Indian subcontinent and China were the major reservoirs, Balkan states like Serbia, Montenegro and Bosnia–Herzegovina may be considered as a ‘secondary’ reservoir area while the Middle East (Morocco, Algeria, Libya, Egypt, Iraq, Kuwait, Oman, Lebanon and Afghanistan), southeast Asia (South Korea, Indonesia, Vietnam and Thailand) and parts of Europe (France, Italy]) may be additional reservoir areas. The bla_NDM-1 gene was identified in K. pneumoniae, E. coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Proteus spp., Citrobacter freundii, Morganella morganii, Providencia spp., Acinetobacter spp. and Raoultella ornithinolytica [3-23]. The bla_NDM-1 gene was mostly on different large plasmids and partly on chromosome [24]. Those plasmids carrying bla_NDM-1 were mostly transferable and coexisted with many other resistant determinants [9,11,17], making treatment of NDM-1-producing bacteria a further complication.

This study retrospectively survey the nationwide epidemiology of bla_NDM-1 in Enterobacteriaceae and ABCs strains derived from 18 tertiary hospitals presenting different provinces in China from July 1, 2011 to June 30, 2012.

Bacterial strains

The species of the family Enterobacteriaceae and ABCs were collected from 18 tertiary hospitals in different provinces in China from July 1, 2011 to June 30, 2012. 338 Enterobacteriaceae and 395 ABCs which were nonsusceptible to carbepenem were selected from 2170 Enterobacteriaceae and 600 ABCs clinical isolates. Standard strains for antimicrobial susceptibility were E. coli ATCC25922, E. coli ATCC35218 and Pseudomonas aeruginosa ATCC27853. Salmonella serotype Braenderup strain H9812 was used as the marker for PFGE.

PCR amplication

The DNA extraction was performed from fresh culture using boiling techniques. The primers used in this study were based on primers published by the Chinese Center For Disease Control and Prevention (CDC), F:TCG CAT AAA ACG CCT CTG; R:GAA ACT GTC GCA CCT CAT. The reaction mixtures were 20 μl: 2x Tap PCR MIX (TaKaRa, Dalian, China) 10 μl; 20 μM each primer 1 μl; DNA sample 2 μl and ddH2O 6 μl. Amplification was carried out under the following thermal cycling conditions: 5 min at 94°C; 30 cycles of amplification consisting of 15 s at 94°C, 30 s at 51°C, and 30 s at 72°C; and 10 min at 72°C for the final extension. The amplicon were analyzed by electrophoresis in a 1.5% agarose gel and were sequenced.

Species confirmation

The bla_NDM-1-positive organisms were affirmed for bacterial genus by the sequence analysis of the 16S rRNA, using the universal primers of 27F-AGAGTTTGATCCTGGCTCAG and 1492R-GGCTACCTTGTTACGACTT [25]. The thermal cycling conditions were: 5 min at 94°C; 30 cycles of amplification consisting of 60 s at 95°C, 60 s at 45°C, and 90 s at 72°C; and 10 min at 72°C for the final extension. DNA fragments were visualized by electrophoresis in a 1.5% agarose gel at 110 V for 45 min in 0.5xTBE (45 mM Tris- OH [pH 8.0], 45 mM boric acid, 1mM EDTA) followed by ethidium bromide staining. The PCR products were sequenced and compared with the published sequence in the Genbank database by multiple sequence alignment. To make sure the species, a Kligler Iron Agar assay was carried out first to detect whether the bacteria were ferment or not. The distinction determined using API (bioMe´rieux, Craponne, France) 20E or 20NE to further identify the bacterial species. The nonfermentative bacteria used API 20NE while the rest 20E. The ABCs were distinguished by PCR detection of bla_OXA-51-like which is intrinsic in A. baumannii using primers previously reported as F:TAA TGC TTT GAT CGG CCT TG;R:TGG ATT GCA CTT CAT CTT GG [26].

Antimicrobial susceptibility

Susceptibility testing for bla_NDM-1-positive isolates was performed by determining MICs by two-folder agar dilution test on Mueller-Hinton agar plates at 37°C. The results were interpreted according to the CLSI2013 M100-S23 guidelines [27]. The breakpoints of imipenem and meropenem for family Enterobacteriaceae were as follows: susceptible (S), ≤ 1 μg/ml; resistant(R), ≥ 4 μg/ml; for ertapenem were as follows: S, ≤ 0.5 μg/ml; R, ≥ 2 μg/ml. Likewise, the breakpoints of imipenem and meropenem for A.baumannii were: S, ≤ 4 μg/ml; R, ≥ 16 μg/ml. Both of the two species, the breakpoints of meropenem were used for panipenem.

PFGE

Bacterial DNA was prepared in agarose blocks and digested with restrict enzyme XbaI (four K. pneumonia and Salmonella serotype Braenderup strain H9812) and ApaI (two A. calcoaceticus). The DNA fragments were separated by use of a CHEF-Mapper XA PFGE system (Bio-Rad, USA) at 6 V/cm and 14°C, with a pulse angle of 120°, for 23 h and a switch time from 4 to 40 s in Enterobacteriaceae while 24 h and a switch time from 5 to 20s in A. calcoaceticus. The gel was stained with ethidium bromide to make the PFGE banding patterns visual.

S1-PFGE and southern hybridization

Refer to the literature published early [28], bacterial DNA was prepared in agarose blocks and digested with S1 nuclease, and then separated by PFGE as above with conditions of 14 h at 6 V/cm and 14°C, with a pulse angle of 120° and a switch time from 1 to 10 s. The gel was stained with ethidium bromide to make the bands visual. After that, the DNA fragments were transferred to nylon membranes (GE, China), hybridized with digoxigenin-labelled bla_NDM-1-specific probes and detected using an NBT/BCIP colour detection kit (Roche, Switzerland).

Plasmid analysis and southern hybridization

Plasmids were extracted according to Molecular Cloning: a laboratory manual then digested with EcoR I and agarose gel electrophoresis at 90v 45 min after prepared in agarose holes. The gel was stained with ethidium bromide to make the plasmid profiles visual. The plasmid fragments were then transferred to nylon membranes hybridized with digoxigenin-labelled bla_NDM-1-specific probes and detected using an NBT/BCIP colour detection kit as above.

The identification of bla_NDM-1-positive bacteria

All PCR detection for bla_NDM-1 results was positive. The sequencing results of the amplicons showed all were 100% identity with K. pneumoniae strain 05-506 (Genbank accession number: FN396876). 16S rRNA sequencing and biochemical API strips revealed that four were K. pneumoniae (M186, M187, M194, U091), two were ABCs (G113, X231), one was Enterobacter cloacae (Q297), one Enterobacter aerogenes (Q442) and one Citrobacter freundii (X122), respectively. The bla_OXA-51-like detection of the two ABCs was negative, indicating that both G113 and X231 were A. calcoaceticus.

The MICs of bla_NDM-1-positive bacteria

All nine strains showed highly resistant to broad spectrum penicillin, cephalosporins, β-lactamase inhibitor combinations, most carbapenem and nitrofurantoin, but showed variable susceptibilities to aminoglycosides and tetracyclines. The good news was that most strains show susceptible to fluoroquinolones and tigycycline (Table 1).

Table 1: The MICs of bla_NDM-1-positive bacteria.

PFGE

The three K. pneumonia from the same provinces (M186, M187, M194) had the same PFGE profiles while the two A. calcoaceticus showed different profiles (Figure 1).

Figure 1: PFGE profiles of four K.pneumonia(a) and two A. calcoaceticus(b).

Plasmid analysis of bla_NDM-1-positive bacteria

Except U091 on chromosome and X122 not succeed, the other seven strains all displayed the bla_NDM-1 gene were on plasmid, with size ranging from ~23 to ~96 kb (Figure 2).

Figure 2: Results of S1-PFGE(left) and Southern blot hybridization(right).M:Low Range PFG Marker.

Plasmid analysis and southern hybridization

Since S1-PFGE results weren’t good and repeated results weren’t stable, we conducted plasmid extraction and Southern blot to make sure whether the bla_NDM-1 was on plasmid or not. U091 didn’t have plasmid, which could further explain the above S1-PFGE & Southern blot result that why its bla_NDM-1 gene was on chromosome. Other eight strains all had plasmids. The sizes of plasmids were consistent with S1-PFGE results above. Southern blot hybridation showed the bla_NDM-1 gene were all on plasmids in the eight strains that haboured plasmids (Figure 3).

Figure 3: Plasmid profiles and Southern blot results.M:λDNA\HindIII..

In the past few decades, an alarming increase in the prevalence of antimicrobial resistant pathogens of serious community- and hospitalacquired infections has been shown worldwide. The increase in carbapenem resistance in Gram-negative bacteria has become a major concern. Bacteria producing NDM-1 had ever caused global panic because they can hydrolyze almost all antimicrobial agents except few, which were referred to “Superbug” by media. To further complicate matters, the bla_NDM-1 gene encoding NDM-1 has disseminated rapidly over distantly related geographical areas around the world [8,12,18,23]. In terms of human hosts, there are three major routes to acquire an NDM-1 producing organism: nosocomial, personal travel and community acquisition. The bla_NDM-1-carrying bacteria have been reported as gut colonizers in human with or without clinical symptoms, they can survive in the local environment as well, which may result in human acquiring the bla_NDM-1-positive bacteria unconciously. Hence, the bacteria possessing the bla_NDM-1 gene constitute tremendous health threat to human.

There are three main resistant mechanisms against β-lactam antibiotic: 1.the production of β-lactamases which cleave the amide bond of the β-lactam ring; 2.the possession of an altered or acquired penicillin binding protein with low affinity for β-lactams; 3.over expression of efflux pump mechanism [29]. The most common mechanism of resistance in carbapenem is the production of carbapenemases (one of β-lactamases), including enzymes of Ambler classes A, D and B (MBLs). NDM-1 is one of MBLs that mediates carbapenem-resistant. In this study, we confirmed those bla_NDM-1-bearing strains were resistant if not intermediate to carbapenem.

China has reported bla_NDM-1-producing bacteria since 2010 by Chinese CDC. Since then, many researches have been on this issue. Chen et al. [13] reported four A. baumannii at the mainland of China, Ho et al. [17] reported one E.coli in Hong Kong, Wu et al. [20] reported K. pneumoniae in Tai Wan. By now, a number of bla_NDM-1-positive bacteria have been reported. So far, the species China has discovered have E. coli, K. pneumoniae, K. oxytoca, K. ozaenae, E. cloacae, E. aerogen, C. freundii, Salmonella enteritidis, Morganella morganii, Providencia spp., Alcaligenes faecalis, Kocuria varians, Moraxella group, Comamonas testosteroni, Stenotrophomonas maltophilia, Staphylococcus capitis, Methylobacterium species, Raoultella ornithinolytica, Acinetobacter spp. and E. faecium. Though there are several molecular level researches on the genetic context of bla_NDM-1, there are limited studies about epidemiology of bla_NDM-1-containing isolates [6,10,13-15,17]. Compared with previously reports [10,13], this study demonstrates that the numbers and species of bla_NDM-1 in family Enterobacteriaceae have been significantly increased in China ,including Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogen and Citrobacter freundii. This situation should draw intensive attention since Enterobacteriaceae are the main cause of nosocomil infection. It will be undoubtedly much troublesome once they acquire bla_NDM-1.

Our study reported nine bla_NDM-1-producing strains in all. Except the U091 strain the bla_NDM-1 gene was on chromosome, all other eight Southern blot hybridization results showed that bla_NDM-1 were all on plasmid, which may result in horizontal transmission rapidly. Further studies are being done to elucidate the transmissibility and the background of resistance determinants.