Biochemical Analysis of Metallo-β-Lactamase NDM-3 from a Multidrug-Resistant Escherichia coli Strain Isolated in Japan

New Delhi metallo-β-lactamase-3 (NDM-3) was identified in a multidrug-resistant Escherichia coli isolate, NCGM77, obtained from the feces of a patient in Japan. The enzymatic activities of NDM-3 against β-lactams were similar to those of NDM-1, although NDM-3 showed slightly lower k_cat/K_m ratios for all the β-lactams tested except for doripenem. The genetic context for bla_NDM-3 was tnpA-bla_NDM-3-ble_MBL-trpF-dsbC-tnpA-sulI-qacEdeltaI-aadA2-dfrA1, which was present on an approximately 250-kb plasmid.     

NDM-12, a Novel New Delhi Metallo-β-Lactamase Variant from a Carbapenem-Resistant Escherichia coli Clinical Isolate in Nepal

A novel New Delhi metallo-β-lactamase variant, NDM-12, was identified in a carbapenem-resistant Escherichia coli clinical isolate obtained from a urine sample from a patient in Nepal. NDM-12 differed from NDM-1 by two amino acid substitutions (M154L and G222D). The enzymatic activities of NDM-12 against β-lactams were similar to those of NDM-1, although NDM-12 showed lower k_cat/K_m ratios for all β-lactams tested except doripenem. The bla_NDM-12 gene was located in a plasmid of 160 kb.     

Semi-rational screening of the inhibitors and β-lactam antibiotics against the New Delhi metallo-β-lactamase 1 (NDM-1) producing E. coli

Bacteria containing bla_NDM-1 gene are a growing threat to almost all clinically β-lactam antibiotics. Especially, the New Delhi metallo-β-lactamase (NDM-1) has become a potential public survival risk. In this study, a novel and efficient strategy for inhibitors and β-lactam antibiotics screening using recombinant New Delhi metallo-beta-lactamase (NDM-1) was developed. First, the gene of bla_NDM-1 were identified and cloned from multi-drug resistance of Acinetobacter baumannii isolate; by the means of protein expression and purification, recombinant NDM-1 activity was up to 68.5 U ml⁻¹, and high purity NDM-1 protein with activity of 347.4 U mg⁻¹ was obtained. Finally, for NDM-1, the inhibitors (aspergillomarasmine A (AMA) and EDTA) with high affinity (HI) and the β-lactam antibiotics (imipenem) with low affinity (LA) were screened out. Surprisingly, the inhibition of the NDM-1 was enhanced by the use of inhibitor combinations (AMA–EDTA (1 : 2)), where the IC₅₀ of AMA–EDTA was reduced by 88% and 95%, respectively, comparing to the AMA and EDTA alone. More interesting, AMA–EDTA could restore the activity of imipenem when tested against NDM-1 expressing strains (E. coli and Acinetobacter baumannii), with a working time of 120 min and 330 min, respectively. This method is expected to be used in high-throughput screening, drug redesign (including new inhibitors and drugs) and “old drug new use”.

Bacteria containing bla_NDM-1 gene are a growing threat to almost all clinically β-lactam antibiotics. A semi-rational screening of the inhibitors and antibiotics against the New Delhi metallo-β-lactamase 1 has been developed in this study.     

Complete Sequences of Two Plasmids in a blaNDM-1-Positive Klebsiella oxytoca Isolate from Taiwan

Genetic determinants of a bla_NDM-1-positive, multidrug-resistant bacterial isolate that caused active infection was investigated by DNA sequencing. Two plasmids, pKOX_NDM1 and pKOX-R1, were identified for the Klebsiella oxytoca strain E718. Sequence annotation revealed a bla_NDM-1 gene in pKOX_NDM1 and two extended-spectrum β-lactamase producers (bla_CTX-M-3 and bla_SHV-12) and a wide array of resistance genes in pKOX-R1. These findings highlight the difficulty in treating multidrug-resistant bacterial infections and the potential danger of emerging resistant enterobacteria.     

High Incidence and Endemic Spread of NDM-1-Positive Enterobacteriaceae in Henan Province,China

The emergence and spread of New Delhi metallo-β-lactamase 1 (NDM-1)-producing carbapenem-resistant Enterobacteriaceae (CRE) present an urgent threat to human health. In China, the bla_NDM-1 gene has been reported mostly in Acinetobacter spp. but is rarely found in Enterobacteriaceae. Here, we report a high incidence and endemic spread of NDM-1-producing CRE in Henan Province in China. Sixteen (33.3%) of the 48 CRE isolates obtained from patients during June 2011 to July 2012 were positive for bla_NDM-1, and the gene was found to be carried on plasmids of various sizes (∼55 to ∼360 kb). These plasmids were readily transferrable to recipient Escherichia coli by conjugation, conferred resistance to multiple antibiotics, and belonged to multiple replicon types. The bla_NDM-1-positive CRE isolates were genetically diverse, and six new multilocus sequence typing (MLST) sequence types were linked to the carriage of NDM-1. Five of the isolates were classified as extensively drug-resistant (XDR) isolates, four of which also carried the fosA3 gene conferring resistance to fosfomycin, an alternative drug for treating infections by CRE. In each bla_NDM-1-positive CRE isolate, the bla_NDM-1 gene was downstream of an intact ISAba125 element and upstream of the ble_MBL gene. Furthermore, gene environment analysis suggested the possible transmission of bla_NDM-1-containing sequences from Acinetobacter spp. to Klebsiella pneumoniae and Klebsiella oxytoca. These findings reveal the emergence and active transmission of NDM-1-positive CRE in China and underscore the need for heightened measures to control their further spread.     

Genomic Characterization of Enterobacter cloacae Isolates from China That Coproduce KPC-3 and NDM-1 Carbapenemases

Here, we report two Enterobacter cloacae sequence type 231 isolates coproducing KPC-3 and NDM-1 that have caused lethal infections in a tertiary hospital in China. The bla_NDM-1-harboring plasmids carry IncA/C₂ and IncR replicons, showing a mosaic plasmid structure, and the bla_NDM-1 is harbored on a novel class I integron-like element. bla_KPC-3 is located on a Tn3-Δbla_TEM-1-bla_KPC-3-ΔTn1722 element, flanked by two 9-bp direct-repeat sequences and harbored on an IncX6 plasmid.     

Novel chimeric beta-lactamase CTX-M-64, a hybrid of CTX-M-15-like and CTX-M-14 beta-lactamases, found in a Shigella sonnei strain resistant to various oxyimino-cephalosporins, including ceftazidime

The plasmid-mediated novel β-lactamase CTX-M-64 was first identified in Shigella sonnei strain UIH-1, which exhibited resistance to cefotaxime (MIC, 1,024 μg/ml) and ceftazidime (MIC, 32 μg/ml). The amino acid sequence of CTX-M-64 showed a chimeric structure of a CTX-M-15-like β-lactamase (N- and C-terminal moieties) and a CTX-M-14-like β-lactamase (central portion, amino acids 63 to 226), suggesting that it originated by homologous recombination between the corresponding genes. The introduction of a recombinant plasmid carrying bla_CTX-M-64 conferred resistance to cefotaxime in Escherichia coli, and the activities of cefotaxime and ceftazidime were restored in the presence of clavulanic acid. Of note, CTX-M-64 production could also confer consistent resistance to ceftazidime, which differs from the majority of CTX-M-type enzymes, which poorly hydrolyze ceftazidime. These results were consistent with the kinetic parameters determined with the purified CTX-M-64 enzyme. The bla_CTX-M-64 gene was flanked upstream by an ISEcp1 sequence and downstream by an orf477 sequence. The sequence of the 45-bp spacer region between the right inverted repeat (IRR) of ISEcp1 and bla_CTX-M-64 was exactly identical to that of ISEcp1-bla_{CTX-M-15-like}. Moreover, the presence of a putative IRR of ISEcp1 at the right end of truncated orf477 is indicative of an ISEcp1-mediated transposition event in the bla_CTX-M-64 gene. The emergence of CTX-M-64 by probable homologous recombination would suggest the natural potential of an alternative mechanism for the diversification of CTX-M-type β-lactamases.     

Development of a Novel Real-Time PCR Assay with High-Resolution Melt Analysis To Detect and Differentiate OXA-48-Like β-Lactamases in Carbapenem-Resistant Enterobacteriaceae

The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla_OXA-48-like-specific primers coupled with an unlabeled 3′-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla_KPC, bla_SME, bla_IMP, bla_NDM-1, bla_VIM, bla_OXA-48, bla_OXA-162, bla_OXA-181, bla_OXA-204, bla_OXA-244, bla_OXA-245, and bla_OXA-232. Our assay identified the presence of bla_OXA-48-like β-lactamase genes and clearly distinguished between bla_OXA-48 and its variants in control strains, including between bla_OXA-181 and bla_OXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes.     

NDM-5 Carbapenemase-Encoding Gene in Multidrug-Resistant Clinical Isolates of Escherichia coli from Algeria

Here, we report the first autochthonous cases of infections caused by bla_NDM-5 New Delhi metallo-β-lactamase-producing Escherichia coli strains recovered from urine and blood specimens of three patients from Algeria between January 2012 and February 2013. The three isolates belong to sequence type 2659 and they coexpress bla_CTX-M-15 with the bla_TEM-1 and bla_aadA2 genes.     

KHM-1, a novel plasmid-mediated metallo-beta-lactamase from a Citrobacter freundii clinical isolate

A novel gene, bla_KHM-1, encoding a metallo-β-lactamase, KHM-1, was cloned from a clinical isolate of Citrobacter freundii resistant to most β-lactam antibiotics. Escherichia coli expressing bla_KHM-1 was resistant to all broad-spectrum β-lactams except for monobactams and showed reduced susceptibility to carbapenems. Recombinant KHM-1 exhibited EDTA-inhibitable hydrolytic activity against most β-lactams, with an overall preference for cephalosporins.     

The First NDM Metallo-β-Lactamase-Producing Enterobacteriaceae Isolate in Poland: Evolution of IncFII-Type Plasmids Carrying the blaNDM-1 Gene

Poland''s first Enterobacteriaceae isolate producing the New Delhi metallo-β-lactamase (NDM) was identified in August 2011. Escherichia coli sequence type ST410 NDM-1 was cultured from a critically ill patient who had been transferred directly from the Congo. The bla_NDM-1 gene was carried by conjugative IncFII-type plasmid pMC-NDM (87,619 bp), which showed structural similarity to plasmid pGUE-NDM, which was identified earlier in France in an E. coli ST131 isolate of Indian origin.     

blaNDM-5 Carried by an IncX3 Plasmid in Escherichia coli Sequence Type 167

bla_NDM-5 was found in Escherichia coli strain 0215 from a Chinese patient without travel history. Genomic sequencing and conjugation experiments were performed. Strain 0215 belonged to sequence type 167 (ST167) and had other resistance determinants, including bla_TEM-135, bla_CTX-M-14, and aac(6′)-Ib. bla_NDM-5 was carried by a 47-kb self-transmissible IncX3 plasmid and was in a complex genetic context similar to that of bla_NDM-1 on IncX3 plasmids. IncX3 plasmids might have emerged as a common vehicle mediating the spread of bla_NDM.     

In Vitro Activities of Ceftazidime-Avibactam and Aztreonam-Avibactam against 372 Gram-Negative Bacilli Collected in 2011 and 2012 from 11 Teaching Hospitals in China

Ceftazidime-avibactam, aztreonam-avibactam, and comparators were tested by reference broth microdilution against 372 nonrepetitive Gram-negative bacilli (346 unselected plus 26 selected meropenem-nonsusceptible Enterobacteriaceae isolates) collected from 11 teaching hospitals in China in 2011 and 2012. Meropenem-nonsusceptible isolates produced extended-spectrum β-lactamases (ESBLs; e.g., CTX-M-14/3), AmpCs (e.g., CMY-2), and/or carbapenemases (e.g., KPC-2 and NDM-1). Avibactam potentiated the activity of ceftazidime against organisms with combinations of ESBLs, AmpCs, and KPC-2. Aztreonam-avibactam was active against all β-lactamase producers (including producers of NDM-1 and IMP-4/8) except bla_OXA-containing Acinetobacter baumannii and some Pseudomonas aeruginosa isolates.     

Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

New Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members of Enterobacteriaceae as a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We cloned bla_NDM-1 from an Enterobacter cloacae clinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (the K_m was reduced, while the k_cat was similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔG_D^o and T_m of Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.     

Outbreak Caused by Enterobacteriaceae Harboring NDM-1 Metallo-β-Lactamase Carried in an IncFII Plasmid in a Tertiary Care Hospital in Mexico City

Carbapenem-resistant Enterobacteriaceae carrying New Delhi metallo-β-lactamase 1 (NDM-1) have rarely been reported in Latin America. We report of an outbreak caused by a bla_NDM-1-harboring plasmid spread through different bacterial species, including Escherichia coli (ST617) and Enterobacter cloacae (ST182) isolates from the same patient and three Klebsiella pneumoniae isolates (ST22) derived from three epidemiologically related patients. IncFII plasmids were found in all strains. Measures to control the outbreak were applied successfully.     

IncA/C Plasmid Carrying blaNDM-1, blaCMY-16, and fosA3 in a Salmonella enterica Serovar Corvallis Strain Isolated from a Migratory Wild Bird in Germany

A Salmonella enterica serovar Corvallis strain was isolated from a wild bird in Germany. This strain carried the IncA/C₂ pRH-1238 plasmid. Complete sequencing of the plasmid was performed, identifying the bla_NDM-1, bla_CMY-16, fosA3, sul1, sul2, strA, strB, aac(6′)-Ib, aadA5, aphA6, tetA(A), mphA, floR, dfrA7, and merA genes, which confer clinically relevant resistance to most of the antimicrobial classes, including β-lactams with carbapenems, fosfomycin, aminoglycosides, co-trimoxazole, tetracyclines, and macrolides. The strain likely originated from the Asiatic region and was transferred to Germany through the Milvus migrans migratory route.     

Electron microscopic structures, serum resistance, and plasmid restructuring of New Delhi metallo-β-lactamase-1 (NDM-1)-producing ST42 Klebsiella pneumoniae emerging in Japan

Enterobacteriaceae, carrying the New Delhi metallo-β-lactamase-1 (NDM-1) gene (bla _NDM-1), have emerged and posed a threat since 2006. In Japan, bla _NDM-1-carrying Escherichia coli was first described in 2010. In this study, we characterized NDM-1-positive Klebsiella pneumoniae strain 419 in Japan, which was isolated from the urine of a 90-year-old Japanese patient who had never been to the Indian subcontinent. K. pneumoniae 419 belonged to ST42. It possessed a surface capsule (with untypeable capsular PCR types) and was resistant to serum killing. K. pneumoniae 419 cells were occasionally flagellated or piliated and autoaggregated. K. pneumoniae 419 was resistant to β-lactams (including carbapenems), aminoglycosides, and fluoroquinolones, and was susceptible to imipenem (or biapenem), aztreonam, polymixin B, and colistin. It possessed at least eight plasmids; of those, a 74-kb plasmid (pKPJ1) of the replicon FIIA carried bla _NDM-1 and was conjugally transferred to E. coli strains, with a 71-kb transferable azithromycin-resistant (mphA ⁺) plasmid of the replicon F (pKPJ2), as a large (145-kb) plasmid (pKPJF100) through a transposition event. In addition to bla _NDM-1, pKPJ1 carried arr-2, pKPJ2 carried mphA, and pKPJF100 carried both. They were negative for the 16S rRNA methylase gene, e.g., which is frequently associated with bla _NDM-1. The data demonstrate that K. pneumoniae 419 possessed virulence- and fitness-associated surface structures, was resistant to serum killing, and possessed a unique (or rare) genetic background in terms of ST type and bla _NDM-1-carrying plasmid.     

Contemporary Diversity of β-Lactamases among Enterobacteriaceae in the Nine U.S. Census Regions and Ceftazidime-Avibactam Activity Tested against Isolates Producing the Most Prevalent β-Lactamase Groups

Escherichia coli (328 isolates), Klebsiella pneumoniae (296), Klebsiella oxytoca (44), and Proteus mirabilis (33) isolates collected during 2012 from the nine U.S. census regions and displaying extended-spectrum-β-lactamase (ESBL) phenotypes were evaluated for the presence of β-lactamase genes, and antimicrobial susceptibility profiles were analyzed. The highest ESBL rates were noted for K. pneumoniae (16.0%, versus 4.8 to 11.9% for the other species) and in the Mid-Atlantic and West South Central census regions. CTX-M group 1 (including CTX-M-15) was detected in 303 strains and was widespread throughout the United States but was more prevalent in the West South Central, Mid-Atlantic, and East North Central regions. KPC producers (118 strains [112 K. pneumoniae strains]) were detected in all regions and were most frequent in the Mid-Atlantic region (58 strains). Thirteen KPC producers also carried bla_CTX-M. SHV genes encoding ESBL activity were detected among 176 isolates. Other β-lactamase genes observed were CTX-M group 9 (72 isolates), FOX (10), TEM ESBL (9), DHA (7), CTX-M group 2 (3), NDM-1 (2 [Colorado]), and CTX-M groups 8 and 25 (1). Additionally, 62.9% of isolates carried ≥2 β-lactamase genes. KPC producers were highly resistant to multiple agents, but ceftazidime-avibactam (MIC_50/90, 0.5/2 μg/ml) and tigecycline (MIC_50/90, 0.5/1 μg/ml) were the most active agents tested. Overall, meropenem (MIC₅₀, ≤0.06 μg/ml), ceftazidime-avibactam (MIC₅₀, 0.12 to 0.5 μg/ml), and tigecycline (MIC₅₀, 0.12 to 2 μg/ml) were the most active antimicrobials when tested against this collection. NDM-1 producers were resistant to all β-lactams tested. The diversity and increasing prevalence of β-lactamase-producing Enterobacteriaceae have been documented, and ceftazidime-avibactam was very active against the vast majority of β-lactamase-producing strains isolated from U.S. hospitals.     

Evaluation of enzymatic and magnetic properties of γ-glutamyl-[1-13C]glycine and its deuteration toward longer retention of the hyperpolarized state

Dynamic nuclear polarization (DNP) is an emerging cutting-edge method of acquiring metabolic and physiological information in vivo. We recently developed γ-glutamyl-[1-¹³C]glycine (γ-Glu-[1-¹³C]Gly) as a DNP nuclear magnetic resonance (NMR) molecular probe to detect γ-glutamyl transpeptidase (GGT) activity in vivo. However, the detailed enzymatic and magnetic properties of this probe remain unknown. Here, we evaluate a γ-Glu–Gly scaffold and develop a deuterated probe, γ-Glu-[1-¹³C]Gly-d₂, that can realize a longer lifetime of the hyperpolarized signal. We initially evaluated the GGT-mediated enzymatic conversion of γ-Glu–Gly and the magnetic properties of ¹³C-enriched γ-Glu–Gly (γ-Glu-[1-¹³C]Gly and γ-[5-¹³C]Glu–Gly) to support the validity of γ-Glu-[1-¹³C]Gly as a DNP NMR molecular probe for GGT. We then examined the spin-lattice relaxation time (T₁) of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d₂ under various conditions (D₂O, PBS, and serum) and confirmed that the T₁ of γ-Glu-[1-¹³C]Gly and γ-Glu-[1-¹³C]Gly-d₂ was maintained for 30 s (9.4 T) and 41 s (9.4 T), respectively, even in serum. Relaxation analysis of γ-Glu-[1-¹³C]Gly revealed a significant contribution of the dipole–dipole interaction and the chemical shift anisotropy relaxation pathway (71% of the total relaxation rate at 9.4 T), indicating the potential of deuteration and the use of a lower magnetic field for realizing a longer T₁. In fact, by using γ-Glu-[1-¹³C]Gly-d₂ as a DNP probe, we achieved longer retention of the hyperpolarized signal at 1.4 T.

By examining enzymatic and magnetic properties, γ-Glu-[1-¹³C]Gly-d₂ was developed as a long-lived DNP molecular probe for detecting γ-glutamyl transpeptidase.     

Characterization of an Enterobacter cloacae Strain Producing both KPC and NDM Carbapenemases by Whole-Genome Sequencing

A carbapenem-resistant Enterobacter cloacae strain, WCHECl-14653, causing a fatal bloodstream infection, was characterized by genome sequencing and conjugation experiments. The strain carried two carbapenemase genes, bla_NDM-1 and bla_KPC-2, on separate IncF plasmids. The coexistence of bla_NDM-1 and bla_KPC-2 conferred slightly higher-level carbapenem resistance compared with that of bla_NDM-1 or bla_KPC-2 alone, and the coexistence of two IncF plasmids may generate new platforms for spreading carbapenemase genes.