Background BlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection. Methods We designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains. Results The lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8–100%) and 100% specificity (95% CI 98.4–100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection. Conclusions We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

Background Carbapenem non-susceptible Gram-negative bacilli (CNS-GNB) were dominant pathogen causing clinical infections. The human intestine was important reservoir of GNB, but there were few studies to analysis the prevalence of fecal colonization with them. Methods Fecal samples were collected from hosiptal screening test for GNB was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the automatic microbiology analyzer and drug-resistant genes were characterized by polymerase chain reaction assays and DNA sequencing. The whole genome sequencing were used to analysis the characteristic of genetic structure of the isolates. Results A total of 680 CNS-GNB were collected. Acinetobacter spp. were the dominant species (33.8%) of the 22 genera. Carbapenemase genes were identified in 307 isolates (45.1%), including 206 (30.3%) blaNDM; 51 (7.5%) blaVIM−2, 48 (7.1%) blaIMP, and seven (1.0%) blaKPC−2. The blaNDM genes were first detected in three isolates, Providencia vermicola, Achromobacter spp., and Cupriavidus gilardii. Co-existence of blaVIM and blaIMP genes was detected in five isolates; Achromobacter co-producing VIM and IMP has not been previously reported. The mcr-1 gene was identified in five strains of Acinetobacter and one strain of K. pneumoniae. In addition, we detected seven isolates harboring the blaAFM−1 gene, a novel metallo-β-lactamase gene. This was first genomic analysis of ST11 K. pneumoniae co-producing NDM-5 and mcr-1, which revealed that blaNDM-5 and mcr-1 are located on two different plasmids. The plasmid harboring blaNDM-5, which was composed of a typical IncX3-type backbone, and the mcr-1 gene, was located between an IS30-like element ISApl1 and a PAP2-like encoding gene in the IncHI2-type plasmid. Conclusions the overall prevalence of fecal carriage of CNS-GNB in 10,000 stool samples was 7.45%, and more than half of CNS-GNB produced carbapenemase. Most CNS-GNB cases were associated with infectious disease, multiple hospitalizations, or long-term care, and a high prevalence of underlying disease.

Background: Carbapenem non-susceptible Gram-negative bacilli (CNS-GNB) were dominant pathogen causing clinical infections. The human intestine was important reservoir of gram-negative bacilli (GNB), but there were few studies to analysis the prevalence of fecal colonization with them.Methods: Fecal samples were collected from hosiptal screening test for GNB was conducted by using home-made MacConkey agar. Antimicrobial susceptibility was determined by the automatic microbiology analyzer and drug-resistant genes were characterized by polymerase chain reaction assays and DNA sequencing. The whole genome sequencing were used to analysis the characteristic of genetic structure of the isolates.Results: A total of 680 CNS-GNB were collected. Acinetobacter spp. were the dominant genus (33.8%, 230/680) of the 22 genera. Carbapenemase genes were identified in 307 isolates (45.1%, 307/680), including 206 (30.3%, 206/680) blaNDM; 51 (7.5%, 51/680) blaVIM-2, 48 (7.1%, 48/680) blaIMP, and seven (1.0%, 7/680) blaKPC-2. The blaNDM genes were first detected in three isolates, Providencia vermicola, Achromobacter spp., and Cupriavidus gilardii. Co-existence of blaVIM and blaIMP genes was detected in five isolates; Achromobacter co-producing VIM and IMP has not been previously reported. The mcr-1 gene was identified in five strains of Acinetobacter and one strain of Klebsiella pneumoniae. In addition, we detected seven isolates harboring the blaAFM-1 gene, a novel metallo-β-lactamase gene. This was first genomic analysis of ST11 Klebsiella pneumoniae co-producing NDM-5 and mcr-1, which revealed that blaNDM-5 and mcr-1 are located on two different plasmids. The plasmid harboring blaNDM-5, which was composed of a typical IncX3-type backbone, and the mcr-1 gene, was located between an IS30-like element ISApl1 and a PAP2-like encoding gene in the IncHI2-type plasmid. Conclusions: the overall prevalence of fecal carriage of CNS-GNB in 10,000 stool samples was 7.45% (745/10000), and CNS-GNB producing carbapenemase were up to 45.1% (307/680). Most CNS-GNB cases were associated with infectious disease, multiple hospitalizations, or long-term care, and a high prevalence of underlying disease.

A carbapenem-resistant Klebsiella pneumoniae (CRKP) strain, NFYY0065, was isolated from a fecal sample obtained from hospitalized patients in Nanfang hospital. By performing whole genome sequencing (WGS), we revealed that NFYY0065 harbored blaNDM-5 and mcr-1 genes, which were located on the IncX3 plasmid (pAN65-3) and IncHI2 plasmid (pAN65-1), respectively. Transfer of the blaNDM-5-bearing plasmid and mcr-1-bearing plasmid from NFYY0065 to Escherichia coli J53 conferred resistance to common beta-lactams and colistin on the transconjugants, respectively. Outer membrane vesicles (OMVs) obtained from the NFYY0065 strain were confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Purified vesicles harboring the blaNDM-5 and mcr-1 genes were confirmed by PCR/sequencing, and these OMVs derived from the NFYY0065 strain were transformed into ATCC700603 strains. The transformants were grown on co-antimicrobial plates (4 µg/ml meropenem and 4 µg/ml colistin), and further PCR/sequencing demonstrated that transformants carried the IncX3 plasmid and IncHI2 plasmid consistent with the plasmids of the original strain. This study highlights two points. It is the first report of co-expression of the blaNDM-5-IncX3 plasmid and mcr-1-IncHI2 plasmid in CRKP, and of the transfer of plasmids containing blaNDM-5 and mcr-1 genes via OMVs.