Complete sequencing of the bla(NDM-1)-positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens

The complete sequence of the plasmid pNDM-1_Dok01 carrying New Delhi metallo-β-lactamase (NDM-1) was determined by whole genome shotgun sequencing using Escherichia coli strain NDM-1_Dok01 (multilocus sequence typing type: ST38) and the transconjugant E. coli DH10B. The plasmid is an IncA/C incompatibility type composed of 225 predicted coding sequences in 195.5 kb and partially shares a sequence with bla(CMY-2)-positive IncA/C plasmids such as E. coli AR060302 pAR060302 (166.5 kb) and Salmonella enterica serovar Newport pSN254 (176.4 kb). The bla(NDM-1) gene in pNDM-1_Dok01 is terminally flanked by two IS903 elements that are distinct from those of the other characterized NDM-1 plasmids, suggesting that the bla(NDM-1) gene has been broadly transposed, together with various mobile elements, as a cassette gene. The chaperonin groES and groEL genes were identified in the bla(NDM-1)-related composite transposon, and phylogenetic analysis and guanine-cytosine content (GC) percentage showed similarities to the homologs of plant pathogens such as Pseudoxanthomonas and Xanthomonas spp., implying that plant pathogens are the potential source of the bla(NDM-1) gene. The complete sequence of pNDM-1_Dok01 suggests that the bla(NDM-1) gene was acquired by a novel composite transposon on an extensively disseminated IncA/C plasmid and transferred to the E. coli ST38 isolate.     

泛耐药基因NDM-1在大肠杆菌中的克隆表达及耐药性检测

目的:构建bla(NDM-1)基因重组质粒,表达新德里金属β内酰胺酶1(NDM-1),并检测携带bla(NDM-1)基因重组质粒的大肠杆菌的耐药状况。方法:PCR扩增编码NDM-1的基因bla(NDM-1),构建表达载体pGEX4T-1-NDM-1,并转化至大肠杆菌,转化子经PCR后测序,以确认构建和转化成功;用Western印迹验证重组蛋白的表达;用药敏纸片法检测含重组质粒pGEX4T-1-NDM-1的大肠杆菌的耐药谱;用E-test法测定其最低抑菌浓度(MIC)。结果:PCR及测序结果显示载体构建和转化成功;含重组质粒pGEX4T-1-NDM-1的大肠杆菌在37℃时,经1 mmol/LIPTG诱导5 h后,SDS-PAGE可见目的条带;除对替加环素和粘菌素敏感外,该重组子对多种碳青霉烯类抗生素耐药,E-test检测其对亚胺培南的MIC为64μg/mL。结论:构建了含泛耐药基因bla(NDM-1)的重组质粒,转入大肠杆菌后表达了融合蛋白,并对多种碳青霉烯类抗生素耐药。为进一步研究bla(NDM-1)基因和蛋白的功能奠定了基础。     

Complete Genome Sequence of Klebsiella oxytoca E718, a New Delhi Metallo-β-Lactamase-1-Producing Nosocomial Strain

We report the complete genome sequence of Klebsiella oxytoca E718, a New Delhi metallo-β-lactamase-1 (NDM-1)-producing strain isolated from a renal transplant patient. The genome contains a 6,097,032-bp chromosome and two multidrug resistance plasmids with sizes of 324,906 bp and 110,781 bp.     

Proteomic Analysis of Clinical Isolate of Stenotrophomonas maltophilia with bla(NDM-1,)bla(L1) and bla(L2) β-Lactamase Genes under Imipenem Treatment

The co-occurrence of L1 and AmpR-L2 with bla(NDM-1) gene with an upstream 250-bp promoter was detected in a clinical isolate of Stenotrophomonas maltophilia DCPS-01, which was resistant to all β-lactams and sensitive only to colistin and fluoroquinolones. To investigate expression of resistance genes and the molecular mechanisms of bacteria resistance to carbapenems, proteomic profiles of the isolate was passaged with and without the drug by using 2D-PAGE. The results showed that 33 genes exhibiting a ≥3-fold change were identified as candidates that may help S. maltophilia survive drug selection. Strikingly, L1 was expressed more highly in cells grown with imipenem, and the abundant NDM-1 further increased, while very little L2 was detected even following induction. Specific activities for β-lactamase revealed that L2 remained at constitutive low levels (10.6 U/mg), while L1 and NDM-1 showed clear activity (69.8 U/mg). Our data support that imipenem could specifically and reversibly induce L1 and NDM-1, which together played key roles in drug resistance in DCPS-01. Although NDM-1 mediated resistance to carbapenems has been found in very few cases, to our knowledge, this is the first proteomics research of S. maltophilia with NDM-1, giving very broad-spectrum antibiotic resistance profiles.     

Molecular basis of NDM-1, a new antibiotic resistance determinant

The New Delhi Metallo-β-lactamase (NDM-1) was first reported in 2009 in a Swedish patient. A recent study reported that Klebsiella pneumonia NDM-1 positive strain or Escherichia coli NDM-1 positive strain was highly resistant to all antibiotics tested except tigecycline and colistin. These can no longer be relied on to treat infections and therefore, NDM-1 now becomes potentially a major global health threat.In this study, we performed modeling studies to obtain its 3D structure and NDM-1/antibiotics complex. It revealed that the hydrolytic mechanisms are highly conserved. In addition, the detailed analysis indicates that the more flexible and hydrophobic loop1, together with the evolution of more positive-charged loop2 leads to NDM-1 positive strain more potent and extensive in antibiotics resistance compared with other MBLs. Furthermore, through biological experiments, we revealed the molecular basis for antibiotics catalysis of NDM-1 on the enzymatic level. We found that NDM-1 enzyme was highly potent to degrade carbapenem antibiotics, while mostly susceptible to tigecycline, which had the ability to slow down the hydrolysis velocity of meropenem by NDM-1. Meanwhile, the mutagenesis experiments, including D124A, C208A, K211A and K211E, which displayed down-regulation on meropenem catalysis, proved the accuracy of our model.At present, there are no effective antibiotics against NDM-1 positive pathogen. Our study will provide clues to investigate the molecular basis of extended antibiotics resistance of NDM-1 and then accelerate the search for new antibiotics against NDM-1 positive strain in clinical studies.     

Genome sequence of a dominant, multidrug-resistant Acinetobacter baumannii strain, TCDC-AB0715

Acinetobacter baumannii has emerged as a significant nosocomial pathogen worldwide. The increasing trend of carbapenem and fluoroquinolone resistance in A. baumannii severely limits the usage of therapeutic antimicrobial agents. Here we report the genome sequence of a multidrug-resistant A. baumannii strain, TCDC-AB0715, harboring both bla(OXA-23) and bla(OXA-66).     

A novel sequence framework (bla(TEM-1G)) encoding the parental TEM-1 beta-lactamase

A novel parental bla(TEM) gene (bla(TEM-1G)), encoding a TEM-1 beta-lactamase (pI of 5.4) produced by the uropathogenic Escherichia coli strain FMV194 was isolated from a dog. We report PCR-restriction fragment length polymorphism analysis and nucleotide sequencing of this gene. The bla(TEM-1G) sequence was identical to the bla(TEM-1C) gene framework in the coding and promoter (P3) regions, except for a silent G(604)-->T mutation in the coding region. Molecular phylogenetic analysis of parental bla(TEM) genes indicated two distinct groups, one comprising bla(TEM-1F) and bla(TEM-2). The other group comprises bla(TEM-1C) which is the probable ancestor of bla(TEM-1A), bla(TEM-1D) and bla(TEM-1G). The bla(TEM-1G) gene has the same framework as a gene encoding an inhibitor-resistant TEM beta-lactamase produced by an E. coli strain of human origin. Thus, parental bla(TEM) genes encoding beta-lactamases in E. coli strains isolated from different host species, in this case human and canine, may be phylogenetically very close.     

Genetic determinants of antibacterial resistance among nosocomial Escherichia coli, Klebsiella spp., and Enterobacter spp. isolates collected in Russia within 2003-2007

Nosocomial bacterial isolates collected within 2003-2004 (n=411) and 2005-2007 (n=422) were highly resistant to cephalosporins III-IV and antibacterials of other groups (aminoglycosides, fluoroquinolons, chloramphenicol, and co-trimoxazole). Genes encoding TEM, SHV, CTX-M, OXA-2, and AmpC types of beta-lactamases (BLs) in the E. coli, Klebsiella spp., and Enterobacter spp. isolates were detected using polymerase chain reaction (PCR). Prevalent CTX-M-type BLs were detected in 85% of the E. coli, 87% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the first strain collection and in 94% of the E. coli, 91% of the Klebsiella spp., and 38% of the Enterobacter spp. isolates of the second one. Genes belonging to three subtypes of blacTx-M genes were identified: bla(CTX-M-1) (228 bla(CTX-M-15) and six bla(CTX-M-3) of the first strain collection; 275 bla(CTX-M-15), three bla(CTX-M-3), and one bla(CTX-M-22) of the second one), bla(CTX-M-2) (one bla(CTX-M-5) of the first strain collection and one bla(CTX-M-2) of the second one), bla(CTX-M-9) (17 bla(CTX-M-14) and one bla(CTX-M-9) of the first strain collection; seven bla(CTX-M-14) and one bla(CTX-M-9) of the second one). Three isolates of the first strain collection and one isolate of the second one carried two genes belonging to two different subtypes, i.e., bla(CTX-M-15) and bla(CTX-M-14) simultaneously. The bacterial isolates had high levels of associative resistance to ciprofloxacin, co-trimoxazole, gentamicin, amikacin, and chloramphenicol associated with the resistance gene cassettes aadA1, aadA2, aadA5, aadB, aacA4, aac(6')Ib; dfrA1, dfrA5, dfrA12, dfrA17, cmlA1, ereA2, and catB8 in the class 1 integrons and the resistance gene cassettes dfrA1, sat1, and aadA1 in the class 2 integrons.     

Non-radioactive PCR-SSCP with a single PCR step for detection of inhibitor resistant beta-lactamases in Escherichia coli

A method based on PCR-SSCP has been developed to detect presumptive Inhibitor-Resistant TEM (IRT) beta-lactamases in Escherichia coli. The capacity of this technique to differentiate genes from 11 control strains encoding IRT beta-lactamases was evaluated with PCR products digested with RsaI. All the bla(TEM) genes studied could be distinguished by their electrophoretic mobilities. Applied to 29 epidemiologically unrelated clinical isolates of E. coli resistant to amoxicillin-clavulanate (MIC, > or =32 microg/ml), the electrophoretic mobilities of the digested bla(TEM) PCR products were identical to those of the reference bla(TEM-1A) (6 strains) and bla(TEM-1B) (18 strains) genes. The remaining five bla(TEM) PCR products displayed SSCP profiles different from those of the reference bla(TEM) genes and their nucleotide sequence identified them as bla(TEM-1C) in one strain, bla(TEM-30/IRT-2) in two strains, bla(TEM-37/IRT-8) in one strain, and bla(TEM-40/IRT-11) in one isolate. Overexpression of the wild-type bla(TEM-1) gene, as detected by high-level resistance to beta-lactams and enzyme assay, accounted for resistance in the 24 E. coli containing bla(TEM-1). We report a simple one PCR step SSCP that can be used in epidemiological studies for rapid preliminary detection of IRT beta-lactamases; identification should be confirmed by sequence data.     

Plasmid-mediated transfer of the bla(NDM-1) gene in Gram-negative rods

The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase NDM-1 (New Delhi metallo-β-lactamase) producers, mostly in Enterobacteriacae. Five bla(NDM) (-1) -positive plasmids of different incompatibility groups (IncL/M, FII, A/C and two untypeable plasmids) from clinical Enterobacteriaceae were evaluated for conjugation properties and host specificity. Successful conjugative transfers were obtained using all tested enterobacterial species as recipients (Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Proteus mirabilis) and all plasmid types. Conjugation frequencies varied from 1 × 10(-4) to 6 × 10(-8) transconjugants per donor. Higher conjugation rates were obtained for two plasmids at 30 °C compared with that observed at 25 and 37 °C. Carbapenems used as selector did not lead to higher conjugation frequencies. None of the five plasmids was transferable to Acinetobacter baumannii or Pseudomonas aeruginosa by conjugation. This work underlines how efficient the spread of the carbapenemase bla(NDM) (-1) gene could be among Enterobacteriaceae.     

Simple and reliable multiplex PCR assay for detection of blaTEM,bla(SHV) and blaOXA-1 genes in Enterobacteriaceae

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.     

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6'')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.     

Draft genome sequences of three NDM-1-producing Enterobacteriaceae species isolated from Brazil

The emergence of multidrug-resistant Enterobacteriaceae strains producing carbapenemases, such as NDM-1, has become a major public health issue due to a high dissemination capacity and limited treatment options. Here we describe the draft genome of three NDM-1-producing isolates: Providencia rettgeri (CCBH11880), Enterobacter hormaechei subsp. oharae (CCBH10892) and Klebsiella pneumoniae (CCBH13327), isolated in Brazil. Besides bla^NDM-1, resistance genes to aminoglycosides [aadA1, aadA2, aac(6’)-Ib-cr] and quinolones (qnrA1, qnrB4) were observed which contributed to the multidrug resistance profile. The element ISAba125 was found associated to the bla^NDM-1 gene in all strains.     

An unexpected similarity between antibiotic-resistant NDM-1 and beta-lactamase II from Erythrobacter litoralis

NDM-1 (New Delhi metallo-beta-lactamase) gene encodes a metallo-beta-lactamase (MBL) with high carbapenemase activity, which makes the host bacterial strain easily dispatch the last-resort antibiotics known as carbapenems and cause global concern. Here we present the bioinformatics data showing an unexpected similarity between NDM-1 and beta-lactamase II from Erythrobacter litoralis, a marine microbial isolate. We have further expressed these two mature proteins in E. coli cells, both of which present as a monomer with a molecular mass of 25 kDa. Antimicrobial susceptibility assay reveals that they share similar substrate specificities and are sensitive to aztreonam and tigecycline. The conformational change accompanied with the zinc binding visualized by nuclear magnetic resonance, Zn²⁺-bound NDM-1, adopts at least some stable tertiary structure in contrast to the metal-free protein. Our work implies a close evolutionary relationship between antibiotic resistance genes in environmental reservoir and in the clinic, challenging the antimicrobial resistance monitoring.     

Copy Number Change of the NDM-1 Sequence in a Multidrug-Resistant Klebsiella pneumoniae Clinical Isolate

The genetic features of the antimicrobial resistance of a multidrug resistant Klebsiella pneumoniae strain harboring bla _NDM-1 were investigated to increase our understanding of the evolution of NDM-1. The strain, KPX, came from a Taiwanese patient with a hospitalization history in New Delhi. Complete DNA sequencing was performed; and the genes responsible for antimicrobial resistance were systematically examined and isolated by library screening. KPX harbored two resistance plasmids, pKPX-1 and pKPX-2, which are 250-kb and 141-kb in size, respectively, with bla _NDM-1 present on pKPX-1. The plasmid pKPX-1 contained genes associated with the IncR and IncF groups, while pKPX-2 belonged to the IncF family. Each plasmid carried multiple antimicrobial resistance genetic determinants. The gene responsible for resistance to carbapenems was found on pKPX-1 and that for resistance to aztreonam was found on pKPX-2. To our surprise, we discovered that bla _NDM-1 exists on pKPX-1 as multiple copies in the form of tandem repeats. Amplification of bla _NDM-1 was found to occur by duplication of an 8.6-kb unit, with the copy number of the repeat varying from colony to colony. This repeat sequence is identical to that of the pNDM-MAR except for two base substitutions. The copy number of bla _NDM-1 of colonies under different conditions was assessed by Southern blotting and quantitative PCR. The bla _NDM-1 sequence was maintained in the presence of the antimicrobial selection; however, removal of antimicrobial selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla _NDM-1 sequence. The dynamic nature of the NDM-1 sequence provides a strong argument for judicious use of the broad-spectrum antimicrobials in order to reduce the development and spread of antimicrobial resistance among pathogens.     

A structural view of the antibiotic degradation enzyme NDM-1 from a superbug

Gram-negative Enterobacteriaceae with resistance to carbapenem conferred by New Delhi metallo-β-lactamase 1 (NDM-1) are a type of newly discovered antibiotic-resistant bacteria. The rapid pandemic spread of NDM-1 bacteria worldwide (spreading to India, Pakistan, Europe, America, and Chinese Taiwan) in less than 2 months characterizes these microbes as a potentially major global health problem. The drug resistance of NDM-1 bacteria is largely due to plasmids containing the blaNDM-1 gene shuttling through bacterial populations. The NDM-1 enzyme encoded by the blaNDM-1 gene hydrolyzes β-lactam antibiotics, allowing the bacteria to escape the action of antibiotics. Although the biological functions and structural features of NDM-1 have been proposed according to results from functional and structural investigation of its homologues, the precise molecular characteristics and mechanism of action of NDM-1 have not been clarified. Here, we report the three-dimensional structure of NDM-1 with two catalytic zinc ions in its active site. Biological and mass spectroscopy results revealed that D-captopril can effectively inhibit the enzymatic activity of NDM-1 by binding to its active site with high binding affinity. The unique features concerning the primary sequence and structural conformation of the active site distinguish NDM-1 from other reported metallo-β-lactamases (MBLs) and implicate its role in wide spectrum drug resistance. We also discuss the molecular mechanism of NDM-1 action and its essential role in the pandemic of drug-resistant NDM-1 bacteria. Our results will provide helpful information for future drug discovery targeting drug resistance caused by NDM-1 and related metallo-β-lactamases.     

Sequence of Closely Related Plasmids Encoding bla NDM-1 in Two Unrelated Klebsiella pneumoniae Isolates in Singapore

Background

Spread of the bla _NDM-1 gene that encodes the New Delhi metallo-β-lactamase (NDM-1) in Enterobacteriaceae is a major global health problem. Plasmids carrying bla _NDM-1 from two different multi-drug resistant Klebsiella pneumonia isolates collected in Singapore were completely sequenced and compared to known plasmids carrying bla _NDM-1.

Methodology/Principal Findings

The two plasmids, pTR3 and pTR4, were transferred to Escherichia coli recipient strain J53 and completely sequenced by a shotgun approach using 3-kb paired-end libraries on 454. Although the K. pneumoniae strains were unrelated by molecular typing using PFGE and MLST, complete sequencing revealed that pTR3 and pTR4 are identical. The plasmid sequence is similar to the E. coli NDM-1-encoding plasmid p271A, which was isolated in Australia from a patient returning from Bangladesh. The immediate regions of the bla _NDM-1 gene in pTR3/4 are identical to that of p271A, but the backbone of our plasmid is much more similar to another IncN2 plasmid reported recently, pJIE137, which contained an additional 5.2-kb CUP (conserved upstream repeat) regulon region in comparison to p271A. A 257-bp element bounded by imperfect 39-bp inverted repeats (IR) and an incomplete version of this element flanking the 3.6-kb NDM-1-encoding region were identified in these plasmids and are likely to be the vestiges of an unknown IS.

Conclusions

Although the hosts are not epidemiologically linked, we found that the plasmids bearing the bla _NDM-1 gene are identical. Comparative analyses of the conserved NDM-1-encoding region among different plasmids from K. pneumoniae and E. coli suggested that the transposable elements and the two unknown IR-associated elements flanking the NDM-1-encoding region might have aided the spreading of this worrisome resistance determinant.     

CTX-M-14型超广谱β-内酰胺酶的序列分析与原核表达

目的对大肠埃希菌所产CTX-M-14型超广谱β-内酰胺酶（ESBLs）进行基因克隆和重组表达,探讨其特性。方法以产CTX-M-14型超广谱β-内酰胺酶大肠埃希菌12号菌总基因组DNA为模板,PCR扩增CTX-M-14,将其克隆入pUCm-T Vector载体后测定该核苷酸序列;再将基因编码区克隆到原核表达载体pET-28α,构建含CTX-M-14基因的重组表达质粒,转化到大肠埃希菌BL21中进行IPTG诱导表达。SDS-PAGE电泳鉴定表达的酶蛋白后再过Ni-NTA柱纯化。结果PCR扩增出大小为876bp的基因片段,与GenBank上同类酶的基因序列同源性为100％。大肠埃希菌BL21转化pET-28a／CTX-M-14重组质粒后,ESBLs试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示蛋白分子质量大约为30KD。结论成功表达重组的CTX-M-14型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定基础。     

Whole-genome sequence of a multidrug-resistant clinical isolate of Acinetobacter lwoffii

Acinetobacter lwoffii has been considered an opportunistic pathogen that can cause nosocomial infections in humans. Here, we present the genome sequence of A. lwoffii WJ10621, a multidrug-resistant clinical isolate that carries a plasmid with the NDM-1 resistance gene.