Carbapenem-resistant and hypervirulent strains have emerged recently. These strains are both hypervirulent and multidrug resistant and may also be highly transmissible and able to cause severe infections in both the hospital and the community. Clinical and public health needs require a rapid and comprehensive molecular detection assay to identify and track the spread of these strains and provide timely infection control information. Here, we develop a rapid multiplex PCR assay capable of distinguishing carbapenem-resistant isolates of sequence type 258 (ST258) and ST11, and hypervirulent ST23, ST65/ST375, and ST86 clones, as well as capsular types K1, K2, K locus type 47 (KL47), and KL64, and virulence genes ,, , and The assay demonstrated 100% concordance with 118 previously genotyped isolates and revealed different populations of carbapenem-resistant and hypervirulent strains in two collections in China and the United States. The results showed that carbapenem-resistant and hypervirulent strains are still rare in the United States, whereas in China, ∼50% of carbapenem-resistant strains carry and virulence genes, which are largely associated with the epidemic ST11 strains. Similarly, a high prevalence of hypervirulent strains was found in carbapenem-susceptible isolates in two Chinese hospitals, but these primarily belong to ST23, ST65/ST375, and ST86, which are distinct from the carbapenem-resistant strains. Taken together, our results demonstrated that this PCR assay can be a useful tool for molecular surveillance of carbapenem-resistant and hypervirulent strains.
The prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is increasing globally, with different molecular mechanisms described. Here we studied the molecular mechanisms of carbapenem resistance, including clonal and plasmid dissemination, of 67 CRE isolates collected between 2012 and 2016 from a tertiary hospital in Eastern China, an CRE endemic region. Species identification and susceptibility testing were performed using the BD Phoenix Automated Microbiology System. Isolates were characterized by PCR (for carbapenemases, ESBLs, AmpC and porin genes), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and conjugation transfer experiments. Selected blaKPC-2 -harboring plasmids were subjected to next-generation sequencing using the Illumina Miseq platform. Among the 67 CRE isolates, 42 Klebsiella pneumoniae, 10 Serratia marcescens, 6 Enterobacter cloacae, 2 Raoultella ornithinolytica, 2 K. oxytoca, 1 K. aerogenes, and 4 Escherichia coli isolates were identified. Six different carbapenemases were detected, including blaKPC-2 (n = 45), blaKPC-3 (n = 1), blaNDM-1 (n = 6), blaNDM-5 (n = 1), blaIMP-4 (n = 2), and blaVIM-1 (n = 2); blaOXA-48-like genes were not detected. One E. cloacae strain possessed both blaNDM-1 and blaKPC-3, while two E. cloacae isolates harbored blaNDM-1 and blaVIM-1. ESBLs (CTX-M, SHV, and TEM) and/or AmpC (CMY, DHA, and ACT/MIR) genes were also identified in 59 isolates, including 13 strains that lacked carbapenemases. Several insertions or stop codon mutations were found within porin genes of K. pneumoniae, E. coli and S. marcescens isolates, both with and without carbapenemases. The 42 K. pneumoniae isolates belonged to 12 different sequence types (ST), with ST11 being the most common, while the 6 E. cloacae isolates comprised 4 different STs. The 10 S. marcescens all shared the same PFGE pulsotype, suggestive of clonal spread. Complete plasmid sequencing and PCR screening revealed both intra-strain and inter-species spread of a common blaKPC-2-harboring plasmid in our hospital. Taken together, our study revealed extensive genetic diversity among CRE isolates form a single Chinese hospital. CRE isolates circulating in the hospital differ significantly in their species, STs, porin genes, carbapenemase genes, and their plasmid content, highlighting the complex dissemination of CRE in this endemic region.
Objectives To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance. Methods The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance. Results We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime. Conclusions Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.
BackgroundDue to the widespread abusage of antibiotics, antibiotic-resistance in Streptococcus pneumoniae (S. pneumoniae) has been increasing quickly in recent years, and it is obviously urgent to develop new types of antibiotics. Two-component systems (TCSs) are the major signal transduction pathways in bacteria and have emerged as potential targets for antibacterial drugs. Among the 13 pairs of TCSs proteins presenting in S. pneumoniae, VicR/K is the unique one essential for bacterium growth, and block agents to which, if can be found, may be developed as effective antibiotics against S. pneumoniae infection.ResultsUsing a structure-based virtual screening (SBVS) method, 105 compounds were computationally identified as potential inhibitors of the histidine kinase (HK) VicK protein from the compound library SPECS. Six of them were then validated in vitro to be active in inhibiting the growth of S. pneumoniae without obvious cytotoxicity to Vero cell. In mouse sepsis models, these compounds are still able to decrease the mortality of the mice infected by S. pneumoniae and one compound even has significant therapeutic effect.ConclusionTo our knowledge, these compounds are the first reported inhibitors of HK with antibacterial activity in vitro and in vivo, and are novel lead structures for developing new drugs to combat pneumococcal infection.
PurposeThis observational study aimed to identify the independent risk factors for both the acquisition and mortality of carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CP-CRE) bacteremia and further assess the in vitro antimicrobial activities of ceftazidime–avibactam (CAZ/AVI) and aztreonam–avibactam (ATM/AVI) against recent CRE bacteremic isolates.Patients and methodsThis observational study was conducted to reveal the risk factors and mortality rate for CP-CRE bacteremia between 2012 and 2018 and also evaluate the in vitro antimicrobial activities of CAZ/AVI and ATM/AVI against recent CRE bacteremic isolates from 2016 to 2018.ResultsA total of 81 non-repetitive isolates were collected from 2012 to 2018, with 67.90% (55/81) being CP-CRE. Old age (P = 0.01), transfusion [odds ratio (OR): 17.19; 95% CI: 3.15–93.72; P = 0.001], longer ICU stay (P = 0.02), cancer (OR: 15.91; 95% CI: 3.56–71.37; P < 0.001), and previous carbapenem exposure (OR: 27.86; 95% CI: 5.03–154.19; P = 0.001) were identified as independent risk factors for the acquisition of CP-CRE bacteremia compared with the ESBL bacteremia. The in vitro antimicrobial activities of CAZ/AVI and ATM/AVI against the CRE bacteremic isolates from 2016 to 2018 showed a respective susceptibility rate of 70.68% (41/58) and 100.00% (58/58).ConclusionThe findings indicated that both CP-CRE/non-CP-CRE stratification and CRE resistance mechanism determination were necessary for better guiding the clinical management of CRE bacteremia: ATM/AVI probably works with both non-CP-CRE and CP-CRE bacteremia, even the most notorious double-carbapenemase producer with porin loss/deficiency, whereas CAZ/AVI works with most of the non-CP-CRE and KPC-producers in the region.
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