BackgroundCarbapenem resistance among Acinetobacter species has become a life-threatening problem. As a last resort in the treatment of gram-negative bacteria infection, resistance to colistin is also a serious problem. The aim of study was to analyze the mechanism of resistance and perform genotyping of carbapenem-resistant Acinetobacter from clinical infection and fecal survey samples in Southern China.MethodsOne hundred seventy and 74 carbapenem-resistant Acinetobacter were isolated from clinical infection samples and fecal survey samples, respectively. We detected the related genes, including carbapenemase genes (blaKPC, blaIMP, blaSPM, blaVIM, blaNDM, blaOXA-23-like, blaOXA-24/40-like, blaOXA-51-like, and blaOXA-58-like), colistin resistance-related genes (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5), a porin gene (carO), efflux pump genes (adeA, adeB, adeC, adeI, adeJ, and adeK), mobile genetic element genes (intI1, intI2, intI3, tnpU, tnp513, IS26, ISAba1, and ISAba125), and the integron variable region. Genotyping was analyzed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and dendrogram cluster analysis.ResultsAmong the 244 carbapenem-resistant Acinetobacter, the common carbapenemase-positive genes included the following: blaOXA-51-like, 183 (75.00%); blaOXA-23-like, 174 (71.30%); blaNDM-1, 57 (23.40%); and blaOXA-58-like, 30 (12.30%). The coexistence of mcr-1 and blaNDM-1 in five strains of A. junii was found for the first time. Eleven distinct carO gene variants were detected in 164 (67.20%) strains, and ten novel variants, which shared 92–99% identity with sequences in the Genbank database, were first reported. Efflux system genes were present in approximately 70% of the isolates; adeABC and adeIJK were observed in 76.23 and 72.13%, respectively. Class 1 integrons were detected in 180 (73.80%) strains and revealed that four gene cassette arrays contained 11 distinct genes. The genotyping by ERIC-PCR demonstrated a high genetic diversity of non-baumannii Acinetobacter, and greater than 90% similarity to A. baumannii.ConclusionsThe blaNDM-1 gene was identified in up to 77% of the carbapenem-resistant Acinetobacter isolated from fecal survey samples, indicating that the gut might be a reservoir of resistant opportunistic bacteria. Intestinal bacteria can be transmitted through the fecal-hand, which is a clinical threat, thus, the monitoring of carbapenem-resistant bacteria from inpatients’ feces should be improved, especially for patients who have been using antibiotics for a long time.Electronic supplementary materialThe online version of this article (10.1186/s12879-019-4423-3) contains supplementary material, which is available to authorized users.

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.