Xiang-Dang Du

Characterization of a multidrug resistance plasmid from Enterococcus faecium that harbours a mobilized bcrABDR locus

Sir, Over the past two decades, Enterococcus faecalis and Enterococcus faecium have developed to be the leading causes of life-threatening hospital-acquired infection worldwide. Bacitracin, a polypeptide antibiotic, is used as a topical application in human medicine and also for prophylaxis and therapy in food animals in China. Previous studies showed that an ABC transporter gene locus bcrABDR conferred bacitracin resistance in Enterococcus spp. and Clostridium perfringens. – 5 In this study, 86 enterococcal strains were isolated from 196 faeces samples in Henan Province, China. Of the strains, 29 were from humans (E. faecalis, n1⁄419; E. faecium, n1⁄410) and 57 were from pigs (E. faecalis, n1⁄422; E. faecium, n1⁄435). The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Antimicrobial susceptibility testing was performed as previously described. Bacitracin-resistant strains were subjected to PCR to identify the presence of the bcrB gene using the primers bcrB-F/ bcrB-R listed in Table S1 (available as Supplementary data at JAC Online). The results indicated that the bcrB gene was present in both human (E. faecalis, n1⁄42; E. faecium, n1⁄42) and swine (E. faecalis, n1⁄49; E. faecium, n1⁄418) strains. However, the presence of the bcrB gene in swine strains (47.4%, 27/57) is much higher than that in human strains (13.8%, 4/29), and this may be largely because bacitracin, especially because it is licensed as a feed additive in the swine industry, is more widely used in pigs than in human medicine. MLST (performed following the protocols detailed at http://www.mlst.net/) indicated that there was no genetic similarity between the bcrB-carrying strains derived from humans and pigs, with the exception of E. faecalis ST116, which was shared by the H7 (human) strain and the P33 (pig) strain. Using E. faecalis JH2-2 (Rif) as the recipient, conjugation and transformation experiments were performed as previously described. For screening of both transconjugants and transformants, brain heart infusion agar was supplemented with 50 mg/L rifampicin and 32 mg/L bacitracin. The randomly selected E. faecalis H9 (human) and E. faecium P21 (pig) strains were able to transfer their bacitracin resistance by conjugation, and transfer frequencies of (4.5+0.3)×10 and (6.0+0.5)×10, respectively, were observed. For swine E. faecium P26, which failed to transfer in the mating experiment, the plasmids were extracted and then transferred into E. faecalis JH2-2 by transformation. The MICs for both transconjugants and transformants are shown in Table S2. A bcrB-positive plasmid derived from the transformant TP26 was sequenced using the 454 Life Sciences (Roche) GS-FLX System and designated pXD5. The complete sequence of pXD5 revealed a size of 56 543 bp in length as shown in Figure S1, which has been deposited in GenBank (KJ645709). Interestingly, the bcrABDR locus was found to be bracketed by two copies of the insertion sequence ISEnfa1 located in the same orientation, and furthermore the adjacent zeta gene was completely disrupted due to the integration of the ISEnfa1bcrABDR-ISEnfa1 segment. The assembling sequence of this segment was confirmed by three individual PCRs using primers P1/P2, P3/P4 and P5/P6, respectively (Table S1 and Figure 1). The characteristic 8 bp target site duplications (5′-TTTAGGAA-3′) were found (Figure 1). Based on the knowledge of the ISEnfa5-related insertion sequence, the acquisition of the bcrABDR-carrying segment in E. faecium P26 may relate to either of the following two processes. The first option is a single-step process postulating that the ISEnfa1-bcrABDR-ISEnfa1 segment might form a composite transposon. The second option postulates a double-step process. First, a single copy of ISEnfa1 integrates into the zeta gene. Then a circular intermediate consisting of bcrABDR and another copy of ISEnfa1 undergoes recombination with the already present ISEnfa1. An inverse PCR was performed using the primers P7/P8 (shown in Table S1 and Figure 1). Sequence analysis of the amplicon revealed that it contained the bcrABDR and one intact copy of ISEnfa1, suggesting that the bcrABDR locus might mobilize by the ISEnfa1-mediated recombination. This inverse PCR was also applied to the remaining bcrB-carrying strains, with positive results for 14 of the 31 strains. Analysis of the antimicrobial resistance regions on the plasmid pXD5 also found two resistance gene clusters (Figure S1). One was erm(B)-aadE-spw-lsa(E)-lnu(B), which seemed to originate from

Novel lnu(G) gene conferring resistance to lincomycin by nucleotidylation, located on Tn6260 from Enterococcus faecalis E531.

Objectives To identify a novel putative lincosamide resistance gene determinant in a swine Enterococcus faecalis E531 exhibiting a lincosamide resistance/macrolide susceptibility (L R M S ) phenotype and to determine its location and genetic environment. Methods The whole genomic DNA of E. faecalis E531, which tested negative for the known lincosamide nucleotidyltransferase genes, was sequenced. A putative lincosamide resistance gene determinant was cloned into an Escherichia coli - E. faecalis shuttle vector (pAM401) and transformed into E. faecalis JH2-2. The MICs were determined by the microbroth dilution method. Inactivity of lincomycin was examined by UPLC-MS/MS. Inverse PCR and primer walking were used to explore the genetic environment based on the assembled sequence. Results A novel resistance gene, designated lnu (G), which encodes a putative lincosamide nucleotidyltransferase, was found in E. faecalis E531. The deduced Lnu(G) amino acid sequence displayed 76.0% identity to Lnu(B) in Enterococcus faecium . Both E. faecalis E531 and E. faecalis JH2-2 harbouring pAM401- lnu (G) showed a 4-fold increase in the MICs of lincomycin, compared with E. faecalis JH2-2 or E. faecalis JH2-2 harbouring empty vector pAM401 only. UPLC-MS/MS demonstrated that the Lnu(G) enzyme catalysed adenylylation of lincomycin. The genetic environment analysis revealed that the lnu (G) gene was embedded into a novel putative transposon, designated Tn 6260 , which was active. Conclusions A novel lincosamide nucleotidyltransferase gene lnu (G) was identified in E. faecalis . The location of the lnu (G) gene on a mobile element Tn 6260 makes it easy to disseminate.

Complete Sequence of pEC012, a Multidrug-Resistant IncI1 ST71 Plasmid Carrying blaCTX-M-65, rmtB, fosA3, floR, and oqxAB in an Avian Escherichia coli ST117 Strain

A 139,622-bp IncI1 ST71 conjugative plasmid pEC012 from an avian Escherichia coli D-ST117 strain was sequenced, which carried five IS26-bracketed resistance modules: IS26-fosA3-orf1-orf2-Δorf3-IS26, IS26-fip-ΔISEcp1-blaCTX-M-65-IS903D-iroN-IS26, IS26-ΔtnpR-blaTEM-1-rmtB-IS26, IS26-oqxAB-IS26, and IS26-floR-aac(3)-IV-IS26. The backbone of pEC012 was similar to that of several other IncI1 ST71 plasmids: pV408, pM105, and pC271, but these plasmids had different arrangements of multidrug resistance region. In addition, the novel ISEc57 element was identified, which is in the IS21 family. The stepwise emergence of multi-resistance regions demonstrated the accumulation of different resistance determinants through homologous recombination. To the best of our knowledge, this is the first study to identify a multidrug-resistant IncI1 ST71 plasmid carrying blaCTX-M-65, rmtB, fosA3, floR, and oqxAB in an avian E. coli ST117 strain.

The genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken

Sir, Methylation of 16S ribosomal RNA (rRNA) mediated by 16S rRNA methylase, which confers high-level aminoglycoside resistance (MICs .1024 mg/L) in Enterobacteriaceae, has recently emerged as a major medical problem worldwide. Until now, seven plasmid-encoded 16S rRNA methylases, ArmA, RmtA, RmtB, RmtC, RmtD, RmtE and NpmA, have been reported in various clinical Gram-negative isolates in multiple geographic regions. armA is one of the most prevalent 16S rRNA methylase genes found in several Gram-negative pathogens. Since it was initially identified in Klebsiella pneumoniae in 2003, two studies focusing on its genetic environment and dissemination in Enterobacteriaceae from both humans and animals have been conducted. Here, we identify a novel genetic environment of armA on pHNE, an IncN plasmid, in one Escherichia coli isolate from a chicken. Previously, we reported the emergence of 16S rRNA methylases encoded by armA and rmtB in E. coli isolates from chickens. In a routine survey of antimicrobial-resistant isolates from chickens in 2009, armA was detected in an E. coli isolate exhibiting high-level resistance to the aminoglycoside antibiotic amikacin (MICs .1024 mg/L) (n1⁄41/18) from a diseased flock in a typical traditional farm in Henan Province, China. Phylogenetic group analysis using multiplex PCR revealed that this armA E. coli isolate belonged to phylogenetic group D, which was associated with extra-intestinal virulence. In order to further elucidate the genetic environment of armA, plasmids extracted from this armA-positive isolate (using the Plasmid Midi Kit, Qiagen, Germany) were transferred to E. coli DH10B by electroporation to investigate whether the armA resistance determinant was localized on plasmids. The transformants were screened on a Luria–Bertani agar plate containing

{μ-N-[(Diphenyl­phosphino)meth­yl]pyridin-2-amine-κ2N1:P}bis­{[2-(2,2′-bipyridin-6-yl)phenyl-κ3N,N′,C1]platinum(II)} bis­(perchlorate)

The title compound, [Pt2(C16H11N2)2(C18H17N2P)](ClO4)2, contains two PtII atoms, bridged by an N-[(diphenylphosphino)methyl]pyridin-2-amine (dppmp) ligand. One Pt atom is coordinated by one P atom from the dppmp ligand, and one C atom and two N atoms from a 6-phenyl-2,2′-bipyridine (pbpy) ligand in a square-planar geometry. The other Pt atom is coordinated by one N atom from the dppmp ligand, and one C atom and two N atoms from another pbpy ligand in a square-planar geometry. There are intramolecular π–π interactions between the pbpy ligands, with a centroid–centroid distance of 3.62 (1) Å between two pyridyl rings. The oxygen atoms of both perchlorate anions are disordered, each over two different positions [occupanicies 0.49 (3)/0.51 (3) and 0.48 (2)/0.52 (2)].

2,2'-Dimethyl-7,7'-(methyl-enediimino)di-1,8-naphthyridin-1-ium bis-(perchlorate).

In the title salt, C19H20N6 2+·2ClO4 −, the two planar 1,8-naphthyridine systems are linked by a methylenediamine group with a dihedral angle of 60.6 (1)° between the two systems. The crystal structure involves extensive N—H⋯O and C—H⋯O hydrogen bonding.

Comparative genomics of the rmtB -carrying IncI1 ST136 plasmids in avian escherichia coli isolates from chickens in China

Eight rmtB-carrying avian Escherichia coli strains from a farm in China were characterised in our previous study, but little is known about the backbones and entire multiresistance regions (MRRs) of these plasmids. Here, three rmtB-carrying IncI1 ST136 plasmids were analysed by whole-plasmid sequencing and were compared. These plasmids were composed of an 83 470-bp IncI1 backbone carrying genes responsible for plasmid replication, transfer, maintenance and stability functions, as well as a 17 330-bp MRR for pEC006 and pEC007, and a 34 626-bp MRR for pEC008. Plasmid pEC006 was not transferable, thus truncation of the traI gene may explain the inability to conjugate. pEC008 harboured the blaTEM-1, rmtB, aacC2, tetA, floR and strAB genes as well as a class 1 integron cassette array (|dfrA12|orfF|aadA2|), which were interspersed with different mobile elements, including Tn2, Tn1721, Tn1722, Tn5393, ISCfr1, IS5057, ISCR1 and ISCR2, and three copies of IS26. The MRR of pEC008 may have resulted from transposition of Tn1722 into the plasmid backbone. Acquisition and rearrangement of MRRs demonstrated the accumulation of different resistance determinants.