Hebing Liu

Identification of New Delhi Metallo-β-lactamase 1 in Acinetobacter lwoffii of Food Animal Origin

Background To investigate the presence of metallo-β-lactamase (MBL) genes and the genetic environment of the New Delhi metallo-β-lactamase gene bla NDM-1 in bacteria of food animal origin. Methodology/Principal Findings Gram-negative bacteria with low susceptibility to imipenem (MIC>8 µg/mL) were isolated from swab samples collected from 15 animal farms and one slaughterhouse in eastern China. These bacteria were selected for phenotypic and molecular detection of known MBL genes and antimicrobial susceptibility testing. For the bla NDM-1 positive isolate, conjugation and transformation experiments were carried out to assess plasmid transfer. Southern blotting was conducted to localize the bla NDM-1 genes, and DNA sequencing was performed to determine the sequences of bla NDM-1 and the flanking genes. In total, nine Gram-negative bacteria of four different species presented a MBL phenotype. bla NDM-1 was identified on a mobile plasmid named pAL-01 in an Acinetobacter lwoffii isolate of chicken origin. Transfer of pAL-01 from this isolate to E. coli J53 and JM109 resulted in resistance to multiple β-lactams. Sequence analysis revealed that the bla NDM-1 gene is attached to an intact insertion element ISAba125, whose right inverted repeat (IR-R) overlaps with the promoter sequence of bla NDM-1. Thus, insertion of ISAba125 likely enhances the expression of bla NDM-1. Conclusion The identification of a bla NDM-1- carrying strain of A. lwoffii in chickens suggests the potential for zoonotic transmission of bla NDM-1 and has important implications for food safety.

First Report of the Multiresistance Gene cfr in Streptococcus suis

ABSTRACT The multiresistance gene cfr was identified for the first time in streptococci, namely, in porcine Streptococcus suis isolate S10. The cfr gene was detected on the ∼100-kb plasmid pStrcfr, where it was bracketed by two copies of the novel insertion sequence ISEnfa5, located in the same orientation. The detection of a cfr- and ISEnfa5-containing amplicon by inverse PCR suggests that ISEnfa5 may play a role in the dissemination of cfr.

A novel phenicol exporter gene, fexB, found in enterococci of animal origin

OBJECTIVES To investigate two porcine Enterococcus isolates for the genetic basis of phenicol resistance and to determine the location and the genetic environment of the novel resistance gene. METHODS A total of 391 isolates with reduced florfenicol susceptibility (MIC ≥ 16 mg/L), obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. Isolates that were negative in these PCRs were analysed for their species assignment and antimicrobial susceptibility. Plasmids were extracted and subjected to transformation and conjugation assays. Restriction fragments of the phenicol resistance plasmids were cloned and sequenced. The sequences obtained were analysed and compared with sequences deposited in the databases. RESULTS The two isolates, Enterococcus faecium EFM-1 and Enterococcus hirae EH-1, exhibited MICs of chloramphenicol and florfenicol of 64 mg/L and carried a new phenicol resistance gene, designated fexB. This gene codes for a phenicol exporter of 469 amino acids organized in 14 transmembrane domains. The fexB gene was located on the 35 kb pEFM-1 from E. faecium and on the 25.3 kb pEH-1 from E. hirae, respectively. Both plasmids were non-conjugative. The fexB gene was found to be embedded in virtually the same genetic environment of 14.8 kb in both plasmids. CONCLUSION To the best of our knowledge, this is the first report of the new florfenicol exporter gene fexB. Based on its plasmid location, horizontal transfer from the enterococci to other bacteria is possible.

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

Preparation of high affinity antibody for ribavirin with new haptens and residue analysis in chicken muscle, eggs and duck muscle

ABSTRACT In this work, high affinity polyclonal antibodies for ribavirin (RBV) from new haptens were prepared and were used to analyse RBV residues in chicken muscle, eggs and duck muscle. The new haptens were synthesised with different spacers, and the best antibody was obtained with an IC50 value as low as 0.61 ng/mL in indirect competitive enzyme-linked immunosorbent assay (ELISA). The cross-reactivities with another five antiviral drugs including amantadine, rimantadine, moroxydine, zanamivir and oseltamivir were less than 0.1%, which indicated the good specificity of the antibody. An ELISA was developed based on the antibody and applied to detect RBV in multi-food matrices. The sample preparation prior to detection only needed simple dilution after trichloroacetic acid extraction. The limits of detection were 1.07, 1.18 and 1.03 μg/kg in chicken muscle, eggs and duck muscle, respectively. Recoveries ranged from 89.0% to 112.7% with coefficients of variation below 13.0%. Ten blind samples of chicken muscle were analysed simultaneously by ELISA and liquid chromatography-tandem mass spectrometry, and a good correlation between the methods was observed. The results indicated that the high affinity antibody could be applied for the simple and fast detection of RBV in multi-food matrices.