Metagenomic next-generation sequencing (mNGS) and droplet digital PCR (ddPCR) have recently demonstrated a great potential for pathogen detection. However, few studies have been undertaken to compare these two nucleic acid detection methods for identifying pathogens in patients with bloodstream infections (BSIs). This prospective study was thus conducted to compare these two methods for diagnostic applications in a clinical setting for critically ill patients with suspected BSIs. Upon suspicion of BSIs, whole blood samples were simultaneously drawn for ddPCR covering 20 common isolated pathogens and four antimicrobial resistance (AMR) genes, mNGS, and blood culture. Then, a head-to-head comparison was performed between ddPCR and mNGS. A total of 60 episodes of suspected BSIs were investigated in 45 critically ill patients, and ddPCR was positive in 50 (83.3%), mNGS in 41 (68.3%, not including viruses), and blood culture in 10 (16.7%) episodes. Of the 10 positive blood cultures, nine were concordantly identified by both mNGS and ddPCR methods. The head-to-head comparison showed that ddPCR was more rapid (~4 h vs. ~2 days) and sensitive (88 vs. 53 detectable pathogens) than mNGS within the detection range of ddPCR, while mNGS detected a broader range of pathogens (126 vs. 88 detectable pathogens, including viruses) than ddPCR. In addition, a total of 17 AMR genes, including 14 bla_KPC and 3 mecA genes, were exclusively identified by ddPCR. Based on their respective limitations and strengths, the ddPCR method is more useful for rapid detection of common isolated pathogens as well as AMR genes in critically ill patients with suspected BSI, whereas mNGS testing is more appropriate for the diagnosis of BSI where classic microbiological or molecular diagnostic approaches fail to identify causative pathogens.

Objective: Eravacycline is a novel, fully synthetic fluorocycline antibiotic being developed for the treatment of serious infections, with a broad-spectrum antimicrobial activity, including against carbapenem-resistant gram-negative bacteria (CRGNB). However, the in vitro activity of eravacycline against CRGNB has not been well known in China. In this study, we analysed the antibacterial activity of eravacycline against CRGNB isolates in order to provide a theoretical basis for the clinical treatment. Methods: A total of 346 isolates of CRGNB were collected from two different tertiary care hospitals in Zhejiang, China. Carbapenem resistance genes of all isolates were detected by polymerase chain reaction. And we analysed the in vitro activity of eravacycline against CRGNB by antimicrobial susceptibility tests. In addition, the time-kill curves were generated to evaluate the antibacterial effect of tigecycline and eravacycline. Results: Four different types of carbapenem-resistant isolates were collected, including 50 Escherichia coli isolates, 160 Klebsiella pneumoniae isolates, 42 Enterobacter cloacae complex isolates, and 94 Acinetobacter baumannii isolates. The carbapenem resistance genes were identified in 346 isolates, including bla_KPC-2 (48.0%), bla_OXA-23 (27.2%), bla_NDM-1 (23.1%), and bla_NDM-16 (0.3%). The antimicrobial susceptibility testing results showed that the minimum inhibitory concentration (MIC) values of 346 isolates were within the sensitivity range (≤ 0.0625~16 mg/L) and that the MIC₅₀ or MIC₉₀ of eravacycline was generally approximately 2-fold lower than tigecycline. In addition, the time-kill curves showed that the bactericidal effect of eravacycline was stronger than that of tigecycline against four different types of isolates. Conclusion: Our research indicated that eravacycline had a good antibacterial effect on CRGNB, which could provide a theoretical basis for the clinical treatment of drug-resistant bacterial infections in the future.

Spread of the carbapenemase-encoding and mobilized colistin resistance (mcr) genes among Enterobacteriales poses a great threat to global public health, especially when the both genes are transferred by a single plasmid. Here, we identified a bla_NDM–1- and mcr-9-co-encoding plasmid harbored by a clinical isolate of Klebsiella pneumoniae (KPN710429). KPN710429 was recovered from a blood sample from an inpatient in a tertiary hospital in China, and antimicrobial susceptibility testing showed that it was multidrug-resistant and only susceptible to aztreonam, colistin, and tigecycline. KPN710429 belongs to sequence type (ST) 1308 and capsular serotype KL144. The string test of KPN710429 was negative, and this strain didn’t exhibit a hypervirulent phenotype according to serum-killing and Galleria mellonella lethality assessments. Whole-genome sequencing revealed the KPN710429 genome comprises a single chromosome and three plasmids. All virulence associated genes were harbored by chromosome. Most of its antimicrobial resistance genes, including bla_NDM–1 and mcr-9 were carried by plasmid pK701429_2, belonging to the incompatibility (Inc) HI2/HI2A group and ST1. Comparative genomics assays indicates that pK710429_2 could be a hybrid plasmid, formed by a Tn6696-like bla_NDM–1 region inserting into a mcr-9-positive-IncHI2/HI2A plasmid. pK710429_2 contained the conjugative transfer gene regions, Tra1 and Tra2, with some structural variations, and conjugation assays revealed that pK710429_2 was transferable. Although pK710429_2 lacked the qseB-qseC regulatory genes, mcr-9 expression was upregulated after pretreatment with colistin for 6 h, leading to colistin resistance in KPN710429. To our knowledge, this is the first report of a bla_NDM–1- and mcr-9-co-encoding transferable plasmid harbored by a bloodstream-infection-causing K. pneumoniae strain in China. Effective surveillance should be implemented to assess the prevalence of the plasmid co-harboring carbapenemase-encoding gene and mcr-9.

For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance. CZA resistance was mediated by a 2 amino acid insertion outside the KPC omega-loop region in CRPA.

Klebsiella pneumoniae carbapenemase (KPC) variants can contribute to resistance to ceftazidime–avibactam (CZA) in Klebsiella pneumoniae (KP). However, two-copy KPC variant-mediated resistance to CZA has rarely been reported to date. Here, we aimed to clarify the evolutionary trajectory of CZA resistance driven by mutations in double-copy bla _KPC-2 to bla _KPC-189 carried by the tandem core structure (IS Kpn6-bla _KPC -IS Kpn27-tnpR -IS 26 ) during treatment of ST11 carbapenem-resistant K. pneumoniae (CRKP). The CZA-resistant KP strain carried double-copy bla _KPC-189 , a variant with alanine–threonine and aspartate–tyrosine substitutions at Ambler amino acid positions 172 (A172T) and 179 (D179Y) of bla _KPC-2 . Clone experiments confirmed that, compared with that of the wild-type bla _KPC-2 clone strain, the minimum inhibitory concentration of CZA increased 16-fold in the bla _KPC-189 -mutant strain. Furthermore, protein structure analysis revealed the A172T and D179Y mutations of bla _KPC-189 can have a direct effect on the binding affinity of CAZ and AVI for KPC. Sequence comparison revealed that bla _KPC-189 was mutated in a double-copy format upon CZA exposure, which was carried by the IS 26 -mediated tandem core structure IS Kpn27-bla _KPC -IS Kpn6 . This tandem core structure apparently evolves in vivo during infection, although not by self-transferring, and multiple IS Kpn27-bla _KPC -IS Kpn6 copy numbers could mediate transferable CZA resistance upon mobilization. In addition, compared with the wild-type bla _KPC-2 gene, the bla _KPC-189 gene had no fitness cost. In summary, our study highlighted the emergence of CZA-resistant bla _KPC-189 variants in the ST11 clone and the presence of a double-copy bla _KPC-189 in the IncFII-type plasmid, which is carried by a tandem core structure (IS 26- IS Kpn6-bla _KPC-189 -IS Kpn27-tnpR -IS 26 ). IMPORTANCE: To date, ceftazidime–avibactam (CZA) resistance caused by double-copy Klebsiella pneumoniae carbapenemase (KPC) variants has not been elucidated. The multicopy forms of carbapenem resistance genes carried by the same plasmid are relatively rare in most carbapenem-resistant Enterobacteriaceae . In this study, we elucidate the evolutionary trajectory of CZA resistance in ST11 carbapenem-resistant K. pneumoniae harboring a double-copy blaKPC and provide new insights into the mechanisms of acquired resistance to CZA.