Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Comparison of influenza- and COVID-19-associated pulmonary aspergillosis in China.

PURPOSE: We conducted a monocentric retrospective study using the latest definitions to compare the demographic, clinical, and biological characteristics of influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA). METHODS: The study retrospectively enrolled 180 patients, including 70 influenza/IPA patients (with positive influenza A/B and Aspergillus) and 110 COVID-19/IPA patients (with positive SARS-CoV-2 and Aspergillus). Among them, 42 (60%) and 30 (27.3%) patients fulfilled the definitions of IAPA and CAPA, respectively. RESULTS: The CAPA patients had significantly higher in-hospital mortality (13/31, 41.9%) than IAPA patients (8/42, 19%) with a P-value of 0.033. Kaplan-Meier survival curve also showed significantly higher 30-day mortality for CAPA patients (P = 0.025). Additionally, the CAPA patients were older, though insignificantly, than IAPA patients (70 (60-80) vs. 62 (52-72), P = 0.075). A lower percentage of chronic pulmonary disease (12.9 vs. 40.5%, P = 0.01) but higher corticosteroids use 7 days before and after ICU admission (22.6% vs. 0%, P = 0.002) were found in CAPA patients. Notably, there were no significant differences in the percentage of ICU admission or ICU mortality between the two groups. In addition, the time from observation to Aspergillus diagnosis was significantly longer in CAPA patients than in IAPA patients (7 (2-13) vs. 0 (0-4.5), P = 0.048). CONCLUSION: Patients infected with SARS-CoV-2 and Aspergillus during the concentrated outbreak of COVID-19 in China had generally higher in-hospital mortality but a lower percentage of chronic pulmonary disease than those infected with influenza and Aspergillus. For influenza-infected patients who require hospitalization, close attention should be paid to the risk of invasive aspergillosis upfront.

In vitro activity of cefiderocol, a siderophore cephalosporin, against carbapenem-resistant hypervirulent Klebsiella pneumoniae in China.

Cefiderocol is a siderophore cephalosporin that binds ferric iron and utilizes iron transporters to cross the cell membrane. Hypervirulent Klebsiella pneumoniae (hvKp) is known to produce more siderophores; in this case, the uptake of cefiderocol may be decreased. Therefore, the objective of this study was to evaluate the in vitro activity of cefiderocol against hvKp isolates. A total of 320 carbapenem-resistant K. pneumoniae (CRKp) isolates were collected in China between 2014 and 2022, including 171 carbapenem-resistant hvKp (CR-hvKp) and 149 carbapenem-resistant classical K. pneumoniae (CR-cKp). Quantitative detection of siderophores showed that the average siderophore production of CR-hvKp (234.6 mg/L) was significantly higher than that of CR-cKp (68.9 mg/L, P < 0.001). The overall cefiderocol resistance rate of CR-hvKp and CR-cKp was 5.8% (10/171) and 2.7% (4/149), respectively. The non-susceptible rates of both cefiderocol and siderophore production of CR-hvKp isolates were higher than those of CR-cKp in either NDM-1- or KPC-2-producing groups. The MIC90 and MIC50 for CR-hvKp and CR-cKp were 8 mg/L and 2 mg/L and 4 mg/L and 1 mg/L, respectively. The cumulative cefiderocol MIC distribution for CR-hvKp was significantly lower than that of CR-cKp isolates (P = 0.003). KL64 and KL47 consisted of 53.9% (83/154) and 75.7% (53/70) of the ST11 CR-hvKp and CR-cKp, respectively, and the former had significantly higher siderophore production. In summary, cefiderocol might be less effective against CR-hvKp compared with CR-cKp isolates, highlighting the need for caution regarding the prevalence of cefiderocol-resistant K. pneumoniae strains, particularly in CR-hvKp isolates.

"Superbugs" with hypervirulence and carbapenem resistance in Klebsiella pneumoniae: the rise of such emerging nosocomial pathogens in China.

Although hypervirulent Klebsiella pneumoniae (hvKP) can produce community-acquired infections that are fatal in young and adult hosts, such as pyogenic liver abscess, endophthalmitis, and meningitis, it has historically been susceptible to antibiotics. Carbapenem-resistant K. pneumoniae (CRKP) is usually associated with urinary tract infections acquired in hospitals, pneumonia, septicemias, and soft tissue infections. Outbreaks and quick spread of CRKP in hospitals have become a major challenge in public health due to the lack of effective antibacterial treatments. In the early stages of K. pneumoniae development, HvKP and CRKP first appear as distinct routes. However, the lines dividing the two pathotypes are vanishing currently, and the advent of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) is devastating as it is simultaneously multidrug-resistant, hypervirulent, and highly transmissible. Most CR-hvKP cases have been reported in Asian clinical settings, particularly in China. Typically, CR-hvKP develops when hvKP or CRKP acquires plasmids that carry either the carbapenem-resistance gene or the virulence gene. Alternatively, classic K. pneumoniae (cKP) may acquire a hybrid plasmid carrying both genes. In this review, we provide an overview of the key antimicrobial resistance mechanisms, virulence factors, clinical presentations, and outcomes associated with CR-hvKP infection. Additionally, we discuss the possible evolutionary processes and prevalence of CR-hvKP in China. Given the wide occurrence of CR-hvKP, continued surveillance and control measures of such organisms should be assigned a higher priority.

Within-host acquisition of colistin-resistance of an NDM-producing Klebsiella quasipneumoniae subsp. similipneumoniae strain through the insertion sequence-903B-mediated inactivation of mgrB gene in a lung transplant child in China.

Background: Colistin, as the antibiotic of "last resort" for carbapenem-resistant Klebsiella, develop resistance during administration of this antimicrobial agent. We identified an NDM-1-producing Klebsiella quasipneumonuae subsp. similipneumoniae (KQSS) strain KQ20605 recovered from a child, which developed resistance to colistin (KQ20786) through acquiring an IS903B element between the -27th and -26th bp of mgrB promoter region after 6-day colistin usage. Objectives: The aim of this study is to explore the source of IS903B in the disruptive mgrB gene and its underlying mechanisms. Materials and methods: Antibiotics susceptibility testing was conducted via microbroth dilution method. The in vitro colistin-induced experiment of KQ20605 was performed to mimic the in vivo transition from colistin-sensitive to resistant. Whole-genome sequencing was used to molecular identification of colistin resistance mechanism. Results: The IS903B element integrated into mgrB gene of KQ20786 had a 100% nucleotide identity and coverage match with one IS903B on plasmid IncR, and only 95.1% (1005/1057) identity to those on chromosome. In vitro, upon the pressure of colistin, KQ20605 could also switch its phenotype from colistin-sensitive to resistant with IS elements (e.g., IS903B and IS26) frequently inserted into mgrB gene at "hotspots", with the insertion site of IS903B nearly identical to that of KQ20786. Furthermore, IS26 elements in this isolate were only encoded by plasmids, including IncR and conjugative plasmid IncN harboring bla NDM. Conclusion: Mobilizable IS elements on plasmids tend to be activated and integrated into mgrB gene at "hotspots" in this KQSS, thereby causing the colistin resistance emergence and further dissemination.

Within-host resistance evolution of a fatal ST11 hypervirulent carbapenem-resistant Klebsiella pneumoniae.

OBJECTIVES: Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKp) has become a great threat to public health. This study reported an hv-CRKp-associated fatal infection and revealed its mechanisms of antimicrobial resistance and within-host evolution. METHODS: A carbapenem-susceptible K. pneumoniae (CSKp) and 11 KPC-producing CRKp strains were isolated from a lung transplant recipient receiving continual antimicrobial therapy for 1.5 years. Pulsed-field gel electrophoresis (PFGE) separated two clusters between CSKp and CRKp. RESULTS: Further whole genome sequencing analysis found that all 11 CRKp were ST11-KL64 clones, while the CSKp was ST412-KL57. Among these 11 CRKp strains, three and one were resistant to colistin and ceftazidime/avibactam (CAZ/AVI), respectively. Three different mechanisms were found to be responsible for the colistin resistance, including the insertions of two different IS (ISKpn74 and IS903B) into the same position of mgrB and one related to the efflux pump system. CAZ/AVI resistance was associated with blaKPC-2 mutation, and it was also found that increasing blaKPC-2 expression increased the MICs of CAZ/AVI, but not at the resistance level. All these 12 strains had iucABCDiutA virulence cluster and rmpA/rmpA2 genes, with higher siderophore production than a reference classic K. pneumoniae (cKp), which were thought to be hypervirulent K. pneumoniae (hvKp). However, only the CSKp showed higher mucoviscosity according to the mucoviscosity assay. Genomic analysis showed that the rmpA variation (interrupted by ISKpn26) existed in all CRKp strains except the CSKp strain, demonstrating that hypermucoviscous phenotype assays could not accurately identify hvKp. CONCLUSION: This study depicted a rapid and diverse within-host evolution of resistance in hv-CRKp of ST11-KL64 clone.

Development and evaluation of a multiplex quantitative polymerase chain reaction assay for detecting bacteria associated with lower respiratory tract infection.

OBJECTIVES: This study aimed to establish a multiplex quantitative polymerase chain reaction (MQ-PCR) assay for 12 bacterial pathogens found in lower respiratory tract infection (LRTI) and to evaluate its performance in a cohort of 211 patients with LRTI. METHODS: The study was divided into two stages: a pilot study to establish the methodology and a clinical validation study to evaluate its performance. In the pilot study, we established the MQ-PCR and analyzed its performance regarding limits of detection, reproducibility, specificity, and efficiency. In the clinical validation study, we obtained 211 sputum and/or bronchoalveolar lavage fluid (BALF) samples and detected pathogens by MQ-PCR. The MQ-PCR time was 3 h from sample collection to complete pathogen detection. RESULTS: The limit of detection was 1000 copies/ml, and the maximum efficiency was >95%. When cutoffs of ≥105 copies/ml in sputum and ≥104 copies/ml in BALF were applied, the sensitivity, specificity, and positive and negative predictive values of the MQ-PCR were 77% (95% confidence interval [CI] 67-88%), 94% (95% CI 93-95%), 25% (95% CI 19-31%), and 99% (95% CI 99-100%), respectively. CONCLUSIONS: This study demonstrates that the new MQ-PCR assay is time-saving, more effective and sensitive, and brings us closer to mainstream adoption of quantitative molecular detection of bacteria.