BackgroundVentilator-associated pneumonia (VAP) is a common nosocomial infection associated with high morbidity due to multidrug-resistant (MDR) pathogens. The purpose of this study was to determine the occurrence of extended-spectrum β-lactamase (ESBL) genes, especially blaCTX-M-15, in Klebsiella pneumoniae (K. pneumoniae)-associated VAP and to investigate the antimicrobial resistance patterns and molecular epidemiological characteristics of K. pneumoniae strains.Materials and methodsFrom January 2013 to December 2015, we retrospectively collected 89 VAP-causing K. pneumoniae isolates from tertiary-care hospitals in China, among which ESBL-producing strains were assessed for antimicrobial susceptibility. Several antibiotic resistance genes of clinical relevance in K. pneumonia isolates producing ESBL were investigated. Polymerase chain reaction (PCR) and DNA sequencing were employed to characterize the genetic contexts of blaCTX-M-15. Conjugative plasmids carrying blaCTX-M-15 were obtained by mating and further subjected to replicon typing. The genetic relatedness of isolates was assessed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.ResultsAll of the 30 ESBL-producing isolates identified displayed MDR phenotype, with blaSHV, blaCTX-M, blaOXA, and blaTEM detected in 21, 21, 1, and 20 isolates, respectively. blaCTX-M-15 was the most prevalent ESBL gene (19/30, 63.33%), and ISEcp1 was detected 48 bp upstream of 15 blaCTX-M-15 genes. Based on S1-PFGE analyses, 25 isolates exhibited different plasmid profiles, ranging from ~70 to 320 kb. The blaCTX-M-15 with blaTEM and qnr genes and the ISEcp1 element from eight isolates were co-transferrable to recipients via conjugation, with IncFIB, IncFIC, and IncFII being the most prevalent replicons. Twenty different PFGE patterns and 11 sequence types were identified, with ST304 being dominant.ConclusionThis work reports the emergence of blaCTX-M-15 in K. pneumoniae-induced VAP in China. We showed that IncFIB, IncFIC, and/or IncFII plasmids carrying blaCTX-M-15 with blaTEM, qnr resistance genes, and the ISEcp1 element mediate the local prevalence in K. pneumoniae-associated VAP.

Antibiotic resistance is a growing health crisis that is further complicated by treatment failures caused by bacteria that exhibit heterogeneous susceptibility to antibiotics. The aim of this study was to describe imipenem (IPM)-heteroresistant strains among multidrug-resistant (MDR) ESBL/AmpC-producing Klebsiella pneumoniae clinical isolates, investigate their molecular phenotypic characteristics, and elucidate the outcome of antibiotic treatment in mice infected with the heteroresistant isolates. Materials and Methods: Antimicrobial susceptibility of K. pneumoniae isolates was determined by the disk diffusion and E-test methods. Heteroresistance to IPM was confirmed by population analysis profile (PAP) assays. PCR and sequencing were employed to detect MDR determinants. Molecular differences between the susceptible and resistant subpopulations were evaluated by sequencing and quantitative real-time reverse transcription PCR (qRT-PCR) analysis. The effect of the carbapenem-heteroresistant strains on antibiotic treatment was assessed using a mouse model of peritonitis with heteroresistant K. pneumoniae and subsequent treatment with IPM. Results: In total, 37 MDR ESBL/AmpC-producing clinical isolates of K. pneumoniae were identified between September 2018 and December 2019. These strains were notably resistant to conventional antimicrobials other than carbapenems. Among the isolates, three strains exhibited heteroresistance to IPM and carried several ESBL and/or AmpC genes. Mice infected with a lethal dose of any of the three heteroresistant isolates were unable to survive in the presence of IPM treatment, as the percentage of the IPM-resistant subpopulation of each strain was increased in the peritoneum of these mice at 24 h after infection. The resistant subpopulation of the strains presented pulsed-field gel electrophoresis (PFGE) profiles that were identical to those of the susceptible subpopulation, but ompK36 porin showed a reduction in gene expression (0.09-to 0.50-fold) in the resistant subpopulation. Conclusion: Carbapenem-heteroresistant strains were present among the MDR K. pneumoniae isolates producing ESBL/AmpC β-lactamases, and these heteroresistant strains failed IPM therapy in experimentally infected mice.

No abstract

Background: Klebsiella pneumoniae is a pathogen that frequently causes nosocomial urinary tract infection (UTI), and the prevalence of plasmid-mediated resistance determinants among clinical isolates of K. pneumoniae leads to the appearance of resistance to antibiotics. The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in acquired AmpC (ac-AmpC) β‑lactamase‑producing K. pneumoniae isolates from patients with nosocomial UTI and to characterize the transmissibility of plasmids co-harbouring blaAmpC and PMQR genes.Methods: From January 2017 to June 2018, we collected 46 AmpC-producing K. pneumoniae isolates causing nosocomial UTI from a tertiary care hospital in China. β-lactamase, PMQR and virulence genes were detected by PCR and sequencing. Clonal relatedness was assessed using ERIC-PCR and multilocus sequence typing (MLST). Plasmids carrying multiple blaAmpC and PMQR genes were characterized by PCR-based replicon typing (PBRT) and S1-PFGE. Conjugation and electroporation experiments were carried out to assess resistance transfer mediated by plasmids. Overlapping PCR was used to map the genetic context of the blaAmpC genes. Results: In the studied isolates, non-susceptibility of third-generation cephalosporin and fluoroquinolone was very high (>80%). blaCMY-2, blaDHA-1, and quinolone resistance gene (qnr) were detected in 11, 41 and 33 isolates, respectively. Among the isolates, 6 strains co-harboured multiple AmpC and qnrB genes. The blaAmpC and qnrB genes from these six isolates were co-transferrable to recipients via conjugation or electroporation, with IncFIA, IncFIB and IncA/C being the dominant replicons (sizes from ~78 to 217 kb). Forty-six isolates were categorized into 25 ERIC types, and the 6 isolates harbouring multiple blaAmpC and qnrB genes belonged to ST1/STnew1. The conserved genetic structures in blaCMY-2 and blaDHA-1 were identical to those described in the pNF4656 and pSAL-1 plasmids, respectively.Conclusion: This work reports that qnrB is highly prevalent in AmpC-producing K. pneumoniae isolates and illustrates the emergence of plasmids co-harbouring multiple acquired blaAmpC and qnrB genes in K. pneumoniae causing UTI in China. We determined that the IncFIA, IncFIB and IncA/C plasmids carrying blaAmpC with qnrB resistance genes and several mobile genetic elements mediate the local prevalence in K. pneumoniae UTI. The genetic context of blaAmpC was highly conserved.