Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae.

ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the "mcsw" morphotype. Two distinct morphotypes ("ntrd" and "msdw") were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

Comparison of in vitro synergy between polymyxin B or colistin in combination with 16 antimicrobial agents against multidrug-resistant Acinetobacter baumannii isolates.

PURPOSES: This study determined the synergy of polymyxin B (POLB) and colistin (COL) with 16 other tested antimicrobial agents in the inhibition of multidrug-resistant Acinetobacter baumannii (MDR-AB). METHODS: We used chequerboard assays to determine synergy between the drugs against 50 clinical MDR-AB from a tertiary hospital in the Zhejiang province in 2019, classifying combinations as either antagonistic, independent, additive, or synergistic. The efficacy of hit combinations which showed highest synergistic rate were confirmed using time-kill assays. RESULTS: Both POLB and COL displayed similar bactericidal effects when used in combination with these 16 tested drugs. Antagonism was only observed for a few strains (2%) exposed to a combination of POLB and cefoperazone/sulbactam (CSL). A higher percentage of synergistic combinations with POLB and COL were observed with rifabutin (RFB; 90%/96%), rifampicin (RIF; 60%/78%) and rifapentine (RFP; 56%/76%). Time-kill assays also confirmed the synergistic effect of POLB and rifamycin class combinations. 1/2 MIC rifamycin exposure can achieve bacterial clearance when combined with 1/2 MIC POLB or COL. CONCLUSION: Nearly no antagonism was observed when combining polymyxins with other drugs by both chequerboard and time-kill assays, suggesting that polymyxins may be effective in combination therapy. The combinations of POLB/COL with RFB, RIF, and RFP displayed neat synergy, with RFB showing the greatest effect.

Comparison of the inoculum effect of in vitro antibacterial activity of Imipenem/relebactam and Ceftazidime/avibactam against ESBL-, KPC- and AmpC-producing Escherichia coli and Klebsiella pneumoniae.

OBJECTIVE: To evaluate effect of inoculum size of extended-spectrum ß-Lactamase (ESBL)-producing-, AmpC-producing-, and KPC-producing Escherichia coli and Klebsiella pneumoniae on the in vitro antibacterial effects of imipenem/relebactam (IMR) and ceftazidime/avibactam (CZA). METHODS: We compared the impact of inoculum size on IMR and CZA of sixteen clinical isolates and three standard isolates through antimicrobial susceptibility tests, time-kill assays and in vitro PK/PD studies. RESULTS: When inoculum size increased from 105 to 107 CFU/mL, an inoculum effect was observed for 26.3% (5/19) and 52.6% (10/19) of IMR and CZA, respectively; time-kill assays revealed that the concentration of CZA increased from ≥ 4 × MIC to 16 × MIC to reach 99.9% killing rate against K. pneumoniae ATCC-BAA 1705 (KPC-2-, OXA-9- and SHV-182-producing) and 60,700 (SHV-27- and DHA-1-producing). While for IMR, a concentration from 1 × MIC to 4 × MIC killed 99.9% of the four strains. When the inoculum size increased to 109 CFU/mL, neither IMR nor CZA showed a detectable antibacterial effect, even at a high concentration. An in vitro PK/PD study revealed a clear bactericidal effect when IMR administered as 1.25 g q6h when inoculum size increased. CONCLUSION: An inoculum effect on CZA was observed more frequent than that on IMR. Among the ß-lactamase-producing strains, the inoculum effect was most common for SHV-producing and KPC-producing strains.

Clinical, biological and genome-wide comparison of carbapenem-resistant Klebsiella pneumoniae with susceptibility transformation to polymyxin B during therapy.

OBJECTIVES: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major clinical concern, and polymyxin B (PMB) is a 'last resort' antibiotic for its treatment. Understanding the effects of drug susceptibility transformation in CRKP-infected patients undergoing PMB treatment would be beneficial to optimize PMB treatment strategies. METHODS: We retrospectively collected data from patients infected with CRKP and treated with PMB from January 2018 to December 2020. CRKPs were collected before and after PMB therapy, and patients were classified into the 'transformation' group (TG) and 'non-transformation' group (NTG) by the shift of susceptibility to PMB. We compared clinical characteristics between these groups, and further analysed the phenotypic and genome variation of CRKP after PMB susceptibility transformation. RESULTS: A total of 160 patients (37 in the TG and 123 in the NTG) were included in this study. The duration of PMB treatment before PMB-resistant K. pneumoniae (PRKP) appearance in TG was even longer than the whole duration of PMB treatment in NTG (8 [8] vs. 7 [6] days; p 0.0496). Compared with isogenic PMB-susceptible K. pneumoniae (PSKP), most PRKP strains had missense mutations in mgrB (12 isolates), yciC (10 isolates) and pmrB (7 isolates). The competition index of 82.4% (28/34) of PRKP/PSKP pairs was <67.6% (23/34), and 73.5% (25/34) of PRKP strains showed a higher 7-day lethality in Galleria mellonella and a greater ability to resist complement-dependent killing than their corresponding PSKP, respectively. CONCLUSION: Low dose with longer PMB treatment durations may be associated with the emergence of polymyxin resistance. The evolution of PRKP is predominantly mediated by an accumulation of mutations, including those in mgrB, yciC, and pmrB. Lastly, PRKP exhibited reduced growth and increased virulence compared with parental PSKP.

Mechanisms of ceftazidime/avibactam resistance in drug-naïve bacteraemic Enterobacterales strains without metallo-beta-lactamase production: Associated with ceftazidime impedance.

In order to investigate ceftazidime/avibactam (CZA) resistance characteristics and mechanisms of bacteraemic Enterobacterales strains that had not been treated previously with CZA, 9708 strains were collected from 43 hospitals in 18 provinces across China from January 2019 to June 2020. The minimum inhibitory concentration (MIC) values of CZA in 165 (1.70%) strains were ≥8/4 mg/L. Ten (6.06%) CZA-resistant strains without metallo-ß-lactamase production were obtained from the individuals without prior exposure to CZA, including six Escherichia coli isolates, three Klebsiella pneumoniae isolates and one Enterobacter cloacae isolate. Whole-genome sequencing revealed that ECB88611, ECB142593 and ECB144539 had encoded disrupted OmpF loss of function. OmpF of ECB126041 had a 2_9 MKRNILAV deletion; OmpK35 of three K. pneumoniae isolates harboured amino acid fragment deletions from positions 1 to 38; and ELB117287 had encoded disrupted OmpF. The G132D amino acid substitution of OmpC of ECB88611, ECB142593 and ECB144539, and the 134_135GD insertion of OmpK36 of three K. pneumoniae isolates were predicted to alter ceftazidime permeability. 333_334 YRIK or YRIN insertions occurred in PBP3 of six E. coli isolates. The relative expression of blaKPC-2 in KPB125108 was 4.527 ± 0.2166 times higher than the control strain, and the relative expression of acrF in six E. coli isolates was 2-3 times higher than the control strain. The addition of phenylalanine-arginine-ß-naphthylamine at 100 mg/L decreased the MIC values of CZA against nine strains significantly. In conclusion, the antimicrobial resistance mechanisms in 10 isolates included increased expression of blaKPC-2, non-functional OMPs, upregulation of efflux pump activity, and variants of PBP3. Most of these mechanisms affected the antimicrobial activity of CZA by impeding ceftazidime.

Evaluation of Piperacillin/Sulbactam, Piperacillin/Tazobactam and Cefoperazone/Sulbactam Dosages in Gram-Negative Bacterial Bloodstream Infections by Monte Carlo Simulation.

The optimal regimens of piperacillin/sulbactam (PIS 2:1), piperacillin/tazobactam (PTZ 8:1), and cefoperazone/sulbactam (CSL 2:1) are not well defined in patients based on renal function. This study was conducted to identify optimal regimens of BLBLIs in these patients. The antimicrobial sensitivity test was performed by a two-fold agar dilution method. Monte Carlo simulation (MCS) was used to simulate the probability of target attainment (PTA) and the cumulative fraction of response (CFR) for various dosing regimens in patients with different renal functions. For strains with an MIC ≤ 8/4 mg/L, PIS 4.5 g q6h achieved 99.03%PTA in the subset of patients with creatinine clearance (CrCL) > 90 mL/min. For patients with CrCL 60-90 mL/min, PIS 4.5 g q6h achieved 81.2% CFR; for those with CrCL 40-59 mL/min, PIS 4.5 g q8h achieved 80.25% CFR. However, for patients infected by ESBL-producing Enterobacteriaceae, PIS 4.5 g q6h achieved a CFR lower than 80%. For patients infected by A. baumannii with a CrCL of 31-60 mL/min, PIS 6.0 g q8h and 4.5 g q6h achieved 81.24% and 82.42% CFR, respectively. For those infected by P. aeruginosa, PIS 4.5 g q6h reached 90% CFR. PIS and PTZ achieved a similar CFR when piperacillin was at the same dose. The CFRs of CSL were much lower than those of the other two agents in Enterobacteriaceae and P. aeruginosa infections. The antibacterial spectrum of PIS is superior to that of PTZ and CSL. Higher dosages and dosing adjustment according to renal function should be considered to treat Gram-negative bacterial BSIs.

Comparison of clinical and microbiological characteristics of community-acquired hypervirulent Klebsiella pneumoniae liver abscess in diabetic and non-diabetic patients.

Invasive infection caused by hypervirulent Klebsiella pneumoniae (HvKP) has been reported worldwide. Most of the patients are community population, related to diabetes mellitus (DM), chronic liver disease and other basic diseases, which prone to systemic migratory infection. In this study, we collected 377 patients with community acquired Klebsiella pneumoniae liver abscess in our hospital from January 2013 to December 2018, 65.8% of whom were male, and 49.6% had DM. Patients with DM are prone to eye and central nervous system (CNS) infection, which need continuous local abscess drainage during treatment. Among them, patients with poor blood glucose control have a higher rate of blood stream infections (BSI). 219 strains of HvKP were obtained, with K1/K2 Serotype accounted for 81.7%. The incidence of BSI in K2 patients was higher than that in K1 patients. The PCR results indicate that the carrying rate of virulence genes (rmpA、areo、kfu、allS、iroN、magA、uge、wcaG) in K1/K2 type strains is significantly higher than that in non K1/K2 type strains. ST23 and ST65 are the most common multilocus sequence typing (MLST), which belong to K1 and K2 Serotype respectively. All of HvKP strains showed high sensitivity to commonly used clinical antibiotics other than ampicillin, with 54.3% of the strains exhibiting high viscosity characteristics. Meanwhile, 35 classic Klebsiella pneumoniae (cKP) strains were collected, and their serum typing is mainly non K1/K2. The carrying rate of virulence genes and viscosity degree in HvKP are significantly higher than those in cKP. Primary liver abscess caused by HvKP is prone to multiple tissue and organ infections, but it shows higher sensitivity to most commonly used antibiotics in clinical practice except for ampicillin. After effective treatment, the overall prognosis of patients is better. This study analyzes the pathogenic characteristics of HvKP and elaborates on the clinical characteristics of patients, which can provide reference for clinical and scientific research work.

Efficacy and safety of novel carbapenem-ß-lactamase inhibitor combinations: Results from phase II and III trials.

Objectives: The addition of novel ß-lactamase inhibitors to carbapenems restores the activity against multidrug-resistant Gram-negative bacteria. The aim of this study was to summarize the evidence on the efficacy and safety of novel carbapenem-ß-lactamase inhibitor combinations. Methods: We conducted a meta-analysis of clinical trials comparing novel carbapenem-ß-lactamase inhibitor combinations with comparators to assess the clinical and microbiological responses, mortality, and adverse events (AEs). Results: A total of 1,984 patients were included. The pooled risk ratios (RRs) of clinical cure, microbiological eradication, all-cause mortality, and 28-day mortality were 1.11 (95% CI: 0.98-1.26), 0.98 (95% CI: 0.82-1.16), 0.90 (95% CI: 0.49-0.94), and 0.68 (95% CI: 0.49-0.94) between the novel carbapenem-ß-lactamase inhibitor combinations and control groups. Sensitivity analysis revealed that the phase II trial of imipenem-cilastatin/relebactam (ICR) against complicated urinary tract infections could be the most important factor of heterogeneity for the microbiological response. The therapeutic effect of novel carbapenem-ß-lactamase inhibitor combinations was better in meropenem-vaborbactam (MEV), phase III trials, and number of patients less than 200. The RRs of AEs from any cause and serious adverse events (SAEs) for patients receiving novel carbapenem-ß-lactamase inhibitor combinations were 0.98 (95% CI: 0.93-1.04) and 1.01 (95% CI: 0.75-1.36), respectively. Conclusions: ICR and MEV were superior to comparators for clinical cure and survival rate in the treatment of complicated infections, and both were as tolerable as the comparators.

Small wards in the ICU: a favorable measure for controlling the transmission of carbapenem-resistant Klebsiella pneumoniae.

PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the leading causes of healthcare-associated infections (HAIs) and is particularly pervasive in intensive care units (ICUs). This study takes ICU layout as the research object, and integrates clinical data and bacterial genome analysis to clarify the role of separate, small wards within the ICU in controlling the transmission of CRKP. METHODS: This study prospectively observed the carriage and spread of CRKP from a long-term in-hospital patient (hereafter called the Patient) colonized with CRKP in the gut and located in a separate, small ward within the ICU. The study also retrospectively investigated CRKP-HAIs in the same ICU. The relationship and transmission between CRKP isolates from the Patient and HAI events in the ICU were explored with comparative genomics. RESULTS: In this study, 65 CRKP-HAI cases occurred during the investigation period. Seven CRKP-HAI outbreaks were also observed. A total of 95 nonrepetitive CRKP isolates were collected, including 32 strains from the Patient in the separate small ward. Phylogenetic analysis based on core genome single-nucleotide polymorphism (cgSNP) showed that there were five possible CRKP clonal transmission events and two clonal outbreaks (A1, A2) during the study. CRKP strains from the Patient did not cause CRKP between-patient transmission or outbreaks in the ICU during the 5-year study period. CONCLUSION: The presence of a long-term hospitalized patient carrying CRKP and positioned in a separate, small ward did not lead to CRKP transmission or infection outbreaks in the ICU. Combining a small-ward ICU layout with normative HAI control measures for multidrug-resistant pathogen infection was effective in reducing CRKP transmission.

Molecular mechanisms underlying bacterial resistance to ceftazidime/avibactam.

Ceftazidime/avibactam (CAZ/AVI), a combination of ceftazidime and a novel ß-lactamase inhibitor (avibactam) that has been approved by the U.S. Food and Drug Administration, the European Union, and the National Regulatory Administration in China. CAZ/AVI is used mainly to treat complicated urinary tract infections and complicated intra-abdominal infections in adults, as well as to treat patients infected with Carbapenem-resistant Enterobacteriaceae (CRE) susceptible to CAZ/AVI. However, increased clinical application of CAZ/AVI has resulted in the development of resistant strains. Mechanisms of resistance in most of these strains have been attributed to blaKPC mutations, which lead to amino acid substitutions in ß-lactamase and changes in gene expression. Resistance to CAZ/AVI is also associated with reduced expression and loss of outer membrane proteins or overexpression of efflux pumps. In this review, the prevalence of CAZ/AVI-resistance bacteria, resistance mechanisms, and selection of detection methods of CAZ/AVI are demonstrated, aiming to provide scientific evidence for the clinical prevention and treatment of CAZ/AVI resistant strains, and provide guidance for the development of new drugs. This article is categorized under: Infectious Diseases > Molecular and Cellular Physiology.

In Vitro Activity Comparison of Ceftazidime-Avibactam and Aztreonam-Avibactam Against Bloodstream Infections With Carbapenem-Resistant Organisms in China.

Objectives: The aim of this work was to investigate the activity of ceftazidime-avibactam (CZA) and aztreonam-avibactam (AZA) against bloodstream infections caused by carbapenem-resistant organisms (CROs). Methods: Non-duplicate CROs, including 56 carbapenem-resistant Escherichia coli (CR-Eco), 318 carbapenem-resistant Klebsiella pneumoniae (CR-Kpn), and 65 carbapenem-resistant Pseudomonas aeruginosa (CR-Pae), were collected using the Blood Bacterial Resistant Investigation Collaborative System (BRICS) program in China. The minimum inhibitory concentrations (MICs) of 24 antibiotics were tested. Carbapenemase genes were amplified for CZA-resistant CROs by PCR. The MICs of CZA and AZA were further determined with avibactam at 8 and 16 mg/L, respectively. Results: The resistance rate of polymyxin B against CROs was less than 5%. Only one CR-Kpn was resistant to tigecycline. The resistance rates of CZA against CR-Eco, CR-Kpn, and CR-Pae were 75.0%, 12.6%, and 18.5%, respectively. The MIC90 values of AZA against CR-Eco, CR-Kpn, and CR-Pae were 2/4, 1/4, and 64/4 mg/L, respectively. Among the CZA-resistant CROs, 42 (100%) CR-Eco, 24 (60%) CR-Kpn, and 1 (8.3%) CR-Pae isolates harbored metallo-ß-lactamase genes. The increase of avibactam concentration enhanced the susceptibility of CZA and AZA against CROs, especially for CR-Eco and CR-Kpn. Conclusions: The in vitro activity of AZA was superior to that of CZA against CR-Eco and CR-Kpn, whereas CZA showed better effect against CR-Pae.

Antibacterial Activity and Optimal Treatment of Ceftazidime-Avibactam and Aztreonam-Avibactam Against Bloodstream Infections Caused by Carbapenem-Resistant Klebsiella pneumoniae.

Objectives: This work was to investigate the activity and optimal treatments of ceftazidime-avibactam (CZA) and aztreonam-avibactam (AZA) against bloodstream infections caused by carbapenem resistant Klebsiella pneumoniae (BSIs-CRKP). Methods: A total of 318 nonduplicate BSIs-CRKP isolates were collected from Blood Bacterial Resistant Investigation Collaborative System (BRICS) program. The minimum inhibitory concentration (MIC) of CZA and AZA were determined by agar dilution method. Carbapenemase genes and multilocus sequence typing were amplified by PCR. Monte Carlo simulation (MCS) was conducted to calculate cumulative fraction of response (CFR) of different CZA or AZA administrations. Results: The MIC90 of CZA and AZA were 128/4 and 1/4 mg/L, respectively. There are 87.4 and 3.5% isolates carried bla KPC-2 and bla NDM-1. A total of 68 ST types were identified and 29 novel ST types. ST11 accounted for 66.6%. Further MCS showed CFR of CZA using two-step infusion therapy (rapid first-step 0.5 h infusion and slow second-step 3 h infusion, TSIT) (2.5 g 0.5 h, 3.75 g every 8 h with 3 h infusion and 3.75 g 0.5 h, 2.5 g every 8 h with 3 h infusion) was above 89%. The CFR of AZA with TSIT was above 96%. Conclusion: TSIT with sufficient pharmacokinetic conditions could be useful for enhancing the therapeutic efficacy of CZA and AZA against BSIs-CRKP.

Comparative Genomic Analysis Provides Insights into the Evolution and Genetic Diversity of Community-Genotype Sequence Type 72 Staphylococcus aureus Isolates.

Staphylococcus aureus sequence type (ST) 72, the predominant community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) lineage in South Korea, has emerged as a major cause of bloodstream infection in hospital settings. However, relatively little information is available regarding the genomic characteristics and dissemination of ST72. Here, we characterized the whole-genome sequence of 24 ST72 isolates from China, along with 83 ST72 genomes from global sources. Of these 107 ST72 isolates, 63 were MRSA and 44 were methicillin-susceptible S. aureus (MSSA). Phylogenetic analysis revealed four distinct clades (A, B, C, and D), of which clade D contained only MSSA isolates. By characterizing the evolutionary dynamics of the ST72 lineage, we found that the MRSA from China might not have developed from the MSSA in China. Furthermore, we observed both international transmission of ST72 isolates and interregional transmission within China. The distributions of the SCCmec and spa types of isolates differed among clades. Additionally, in silico analyses revealed that the distributions of resistance genes, virulence genes, and mobile genetic elements (MGEs) also differed among isolates of the four clades. This was especially true for clade D isolates, which had the lowest level of antimicrobial resistance and had obtained specific virulence genes such as tsst-1 by acquisition of specific MGEs. Notably, ST72 MRSA isolates were more antibiotic resistant than ST72 MSSA isolates, but comparably virulent. Our findings provide insight into the potential transmission and genotypic features of ST72 clones across the globe. IMPORTANCE Understanding the evolution and dissemination of community-genotype ST72 Staphylococcus aureus isolates is important, as isolates of this lineage have rapidly spread into hospital settings and caused serious health issues. In this study, we first carried out genome-wide analysis of 107 global ST72 isolates to characterize the evolution and genetic diversity of the ST72 lineage. We found that the MSSA lineage in China might have evolved independently from the MRSA isolates from China, and that ST72 isolates have the potential to undergo both international transmission and interregional transmission within China. The diversity of isolates correlated with distinct acquisitions of SCCmec elements, antibiotic resistance genes, virulence genes, and mobile genetic elements. The comprehensive information on the ST72 lineage emerging from this study will enable improved therapeutic approaches and rapid molecular diagnosis.