Optimization of an in vitro Pseudomonas aeruginosa Biofilm Model to Examine Antibiotic Pharmacodynamics at the Air-Liquid Interface.

Pseudomonas aeruginosa is an important cause of lower respiratory tract infections, such as ventilator-associated bacterial pneumonia (VABP). Using inhaled antibiotics to treat VABP can achieve high drug concentrations at the infection site while minimizing systemic toxicities. Despite the theoretical advantages, clinical trials have failed to show a benefit for inhaled antibiotic therapy in treating VABP. A potential reason for this discordance is the presence of biofilm-embedded bacteria in lower respiratory tract infections. Drug selection and dosing are often based on data from bacteria grown planktonically. In the present study, an in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model was optimized to evaluate the activity of simulated epithelial lining fluid exposures of inhaled and intravenous doses of polymyxin B and tobramycin against two P. aeruginosa strains. Antibiotic activity was also determined against the P. aeruginosa strains grown planktonically. Our study revealed that inhaled antibiotic exposures were more active than their intravenous counterparts across biofilm and planktonic populations. Inhaled exposures of polymyxin B and tobramycin exhibited comparable activity against planktonic P. aeruginosa. Although inhaled polymyxin B exposures were initially more active against P. aeruginosa biofilms (through 6 h), tobramycin was more active by the end of the experiment (48 h). Together, these data slightly favor the use of inhaled tobramycin for VABP caused by biofilm-forming P. aeruginosa that are not resistant to either antibiotic. The optimized in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model may be beneficial for the development of novel anti-biofilm agents or to optimize antibiotic dosing for infections such as VABP.

Improved characterization of aminoglycoside penetration into human lung epithelial lining fluid via population pharmacokinetics.

Aminoglycosides are important treatment options for serious lung infections, but modeling analyses to quantify their human lung epithelial lining fluid (ELF) penetration are lacking. We estimated the extent and rate of penetration for five aminoglycosides via population pharmacokinetics from eight published studies. The area under the curve in ELF vs plasma ranged from 50% to 100% and equilibration half-lives from 0.61 to 5.80 h, indicating extensive system hysteresis. Aminoglycoside ELF peak concentrations were blunted, but overall exposures were moderately high.

Assessment of antibiotic release and antibacterial efficacy from pendant glutathione hydrogels using ex vivo porcine skin.

Acute bacterial skin and skin structure infections (ABSSSIs) confer a substantial burden on the healthcare system. Local antibiotic delivery systems can provide controlled drug release directly to the site of infection to maximize efficacy and minimize systemic toxicity. The purpose of this study was to examine the antibacterial activity of antibiotic-loaded glutathione-conjugated poly(ethylene glycol) hydrogels (GSH-PEG) against ABSSSIs utilizing an ex vivo porcine dermal explant model. Vancomycin- or meropenem-loaded GSH-PEG hydrogels at 3 different dose levels were loaded over 1 h. Drug release was monitored in vitro under submerged conditions, by the Franz cell diffusion method, and ex vivo utilizing a porcine dermis model. Antibacterial activity was assessed ex vivo on porcine dermis explants inoculated with Staphylococcus aureus or Pseudomonas aeruginosa isolates treated with vancomycin- or meropenem-loaded GSH-PEG hydrogels, respectively. Histological assessment of the explants was conducted to evaluate tissue integrity and viability in the context of the experimental conditions. A dose-dependent release was observed from vancomycin and meropenem hydrogels, with in vitro Franz cell diffusion data closely representing ex vivo vancomycin release, but not high dose meropenem release. High dose vancomycin-loaded hydrogels resulted in a >3 log10 clearance against all S. aureus isolates at 48 h. High dose meropenem-loaded hydrogels achieved 6.5, 4, and 2 log10 reductions in CFU/ml against susceptible, intermediate, and resistant P. aeruginosa isolates, respectively. Our findings demonstrate the potential application of GSH-PEG hydrogels for flexible, local antibiotic delivery against bacterial skin infections.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence.

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.

Structural insights into the mechanism of overcoming Erm-mediated resistance by macrolides acting together with hygromycin-A.

The ever-growing rise of antibiotic resistance among bacterial pathogens is one of the top healthcare threats today. Although combination antibiotic therapies represent a potential approach to more efficiently combat infections caused by susceptible and drug-resistant bacteria, only a few known drug pairs exhibit synergy/cooperativity in killing bacteria. Here, we discover that well-known ribosomal antibiotics, hygromycin A (HygA) and macrolides, which target peptidyl transferase center and peptide exit tunnel, respectively, can act cooperatively against susceptible and drug-resistant bacteria. Remarkably, HygA slows down macrolide dissociation from the ribosome by 60-fold and enhances the otherwise weak antimicrobial activity of the newest-generation macrolide drugs known as ketolides against macrolide-resistant bacteria. By determining a set of high-resolution X-ray crystal structures of drug-sensitive wild-type and macrolide-resistant Erm-methylated 70S ribosomes in complex with three HygA-macrolide pairs, we provide a structural rationale for the binding cooperativity of these drugs and also uncover the molecular mechanism of overcoming Erm-type resistance by macrolides acting together with hygromycin A. Altogether our structural, biochemical, and microbiological findings lay the foundation for the subsequent development of synergistic antibiotic tandems with improved bactericidal properties against drug-resistant pathogens, including those expressing erm genes.

Pharmacokinetics and dialytic clearance of baricitinib during in vivo continuous venovenous haemodialysis in a patient with COVID-19.

OBJECTIVES: To date, there are no published pharmacokinetic (PK) data for baricitinib in critically ill patients requiring continuous renal replacement therapy. This paper describes in detail the plasma PK and dialytic clearance of baricitinib in a patient infected with coronavirus disease 2019 (COVID-19) requiring continuous renal replacement therapy in order to suggest dosing strategies in this population. METHODS: Baricitinib 2 mg daily was used for the treatment of COVID-19 in a critically ill patient on continuous venovenous haemodialysis (CVVHD). Prefiltration plasma drug concentrations of baricitinib were measured at hours 1, 2, 12, and 24 after drug administration. Postfiltration and ultrafiltrate concentrations were collected at hour 24. RESULTS: Plasma PK parameters of baricitinib in this patient were as follows: maximum plasma concentration (Cmax), 20.98 ng/mL; minimum plasma concentration (Cmin), 9.84 ng/mL; half-life (t1/2), 23.85 h; apparent volume of distribution at the steady state (Vss), 99.42 L; total clearance at the steady state (CLss), 2.89 L/h; and area under the concentration-time curve (AUC0-∞), 692.14 ng · h/mL. The saturation coefficient for baricitinib at 24 h after administration was 0.607. The transmembrane clearance of baricitinib by CVVHD running at a flow rate of 2 L/h was 1.21 L/h, representing 41.9% of the total clearance of baricitinib. CONCLUSIONS: In a critically ill COVID-19 patient on CVVHD, a 2-mg dose of baricitinib achieves a Cmax comparable with healthy subjects, but total clearance was reduced to about 20%. Larger studies exploring multiple patients and dialysis modes are needed to determine the optimal dosing strategy for baricitinib in this patient population.

Mechanistic Insights to Combating NDM- and CTX-M-Coproducing Klebsiella pneumoniae by Targeting Cell Wall Synthesis and Outer Membrane Integrity.

Metallo-ß-lactamase (MBL)-producing Gram-negative bacteria cause infections associated with high rates of morbidity and mortality. Currently, a leading regimen to treat infections caused by MBL-producing bacteria is aztreonam combined with ceftazidime-avibactam. The purpose of the present study was to evaluate and rationally optimize the combination of aztreonam and ceftazidime-avibactam with and without polymyxin B against a clinical Klebsiella pneumoniae isolate producing NDM-1 and CTX-M by use of the hollow fiber infection model (HFIM). A novel de-escalation approach to polymyxin B dosing was also explored, whereby a standard 0-h loading dose was followed by maintenance doses that were 50% of the typical clinical regimen. In the HFIM, the addition of polymyxin B to aztreonam plus ceftazidime-avibactam significantly improved bacterial killing, leading to eradication, including for the novel de-escalation dosing strategy. Serial samples from the growth control and monotherapies were explored in a Galleria mellonella virulence model to assess virulence changes. Weibull regression showed that low-level ceftazidime resistance and treatment with monotherapy resulted in increased G. mellonella mortality (P < 0.05). A neutropenic rabbit pneumonia model demonstrated that aztreonam plus ceftazidime-avibactam with or without polymyxin B resulted in similar bacterial killing, and these combination therapies were statistically significantly better than monotherapies (P < 0.05). However, only the polymyxin B-containing combination therapy produced a statistically significant decrease in lung weights (P < 0.05), indicating a decreased inflammatory process. Altogether, adding polymyxin B to the combination of aztreonam plus ceftazidime-avibactam for NDM- and CTX-M-producing K. pneumoniae improved bacterial killing effects, reduced lung inflammation, suppressed resistance amplification, and limited virulence changes.

Ceftazidime-avibactam based combinations against carbapenemase producing Klebsiella pneumoniae harboring hypervirulence plasmids.

The combination of carbapenem resistance and hypervirulence in Klebsiella pneumoniae is an emerging and urgent threat due to its potential to resist common antibiotics and cause life-threatening infections in healthy hosts. This study aimed to evaluate the activity of clinically relevant antibiotic regimens against carbapenem-resistant K. pneumoniae with hypervirulence plasmids and to identify pathways associated with antibiotic tolerance using transcriptomics. We studied two carbapenem-resistant K. pneumoniae isolates, CDI694 and CDI231, both harboring hypervirulence plasmids. Time-kill and dynamic one-compartment pharmacokinetic/pharmacodynamic assays were used to assess ceftazidime/avibactam-based therapies. RNAseq was performed following 48 h of antibiotic exposure. Closed genomes of CDI694 and CDI231 were obtained; each isolate harbored carbapenem-resistance and hypervirulence (containing rmpA/rmpA2 and iut genes) plasmids. Ceftazidime/avibactam-based regimens were bactericidal, though both isolates continued to grow in the presence of antibiotics despite no shifts in MIC. Transcriptomic analyses suggested that perturbations to cell respiration, carbohydrate transport, and stress-response pathways contributed to the antibiotic tolerance in CDI231. Genes associated with hypervirulence and antibiotic resistance were not strongly impacted by drug exposure except for ompW, which was significantly downregulated. Treatment of carbapenem-resistant K. pneumoniae harboring hypervirulence plasmids with ceftazidime/avibactam-based regimens may yield a tolerant population due to altered transcription of multiple key pathways.

Research priorities towards precision antibiotic therapy to improve patient care.

Antibiotic resistance presents an incessant threat to our drug armamentarium that necessitates novel approaches to therapy. Over the past several decades, investigation of pharmacokinetic and pharmacodynamic (PKPD) principles has substantially improved our understanding of the relationships between the antibiotic, pathogen, and infected patient. However, crucial gaps in our understanding of the pharmacology of antibacterials and their optimal use in the care of patients continue to exist; simply attaining antibiotic exposures that are considered adequate based on traditional targets can still result in treatment being unsuccessful and resistance proliferation for some infections. It is this salient paradox that points to key future directions for research in antibiotic therapeutics. This Personal View discusses six priority areas for antibiotic pharmacology research: (1) antibiotic-pathogen interactions, (2) antibiotic targets for combination therapy, (3) mechanistic models that describe the time-course of treatment response, (4) understanding and modelling of host response to infection, (5) personalised medicine through therapeutic drug management, and (6) application of these principles to support development of novel therapies. Innovative approaches that enhance our understanding of antibiotic pharmacology and facilitate more accurate predictions of treatment success, coupled with traditional pharmacology research, can be applied at the population level and to individual patients to improve outcomes.

Combatting Planktonic and Biofilm Populations of Carbapenem-Resistant Acinetobacter baumannii with Polymyxin-Based Combinations.

Carbapenem-resistant Acinetobacter baumannii (CRAB) can cause serious infections that are associated with high mortality rates. During the course of an infection, many CRAB isolates are able to form biofilms, which are recalcitrant to several antibiotics and can be difficult to treat. Polymyxin-based regimens are a first-line treatment option for CRAB infections, but they have not been optimized against both planktonic and biofilm phases of growth. The objective of this study was to identify polymyxin-based combinations that are active against planktonic and biofilm populations of CRAB. Four CRAB isolates (meropenem MICs: 8-256 mg/L) capable of forming biofilms were used in each experiment. The activities of polymyxin B alone and in combination with ampicillin/sulbactam, meropenem, minocycline, and rifampin were assessed using time-kill assays, with the CRAB isolates grown in planktonic and biofilm phases. Viable colony counts were used to detect the bactericidal activity and synergy of the antibiotic combinations. Against the planktonic populations, polymyxin B combined with meropenem, minocycline, ampicillin/sulbactam, and rifampin caused 3.78, -0.15, 4.38, and 3.23 mean log10 CFU/mL reductions against all isolates at 24 h, respectively. Polymyxin B combined with meropenem, ampicillin/sulbactam, or rifampin was synergistic against 75-100% (3/4 or 4/4) of CRAB isolates. Against biofilms, polymyxin B combined with meropenem, minocycline, ampicillin/sulbactam, and rifampin caused 1.86, 1.01, 0.66, and 3.55 mean log10 CFU/mL reductions against all isolates at 24 h, respectively. Only the combination of polymyxin B and rifampin retained bactericidal activity or synergy against any of the isolates when grown as biofilms (50% of isolates). The combination of polymyxin B and rifampin may be promising for CRAB infections that have planktonic and biofilm populations present.

Antibiotic susceptibility patterns of viridans group streptococci isolates in the United States from 2010 to 2020.

Background: Viridans group streptococci (VGS) are typically part of the commensal flora but can also cause severe invasive diseases such as infective endocarditis. There are limited data available showing antibiotic susceptibility over time for VGS. Objectives: To evaluate antibiotic susceptibility trends in VGS over time. Methods: In vitro susceptibility patterns for 33 antibiotics were examined for Streptococcus mitis, Streptococcus oralis, and non-speciated VGS isolates from patients in Veterans Affairs (VA) Medical Centers in the United States between 2010 and 2020. Susceptibility determinations were made by the individual clinical microbiology laboratories and data were retrospectively collected from the VA Corporate Data Warehouse. Susceptibility trends were analysed using Poisson regression. Results: A total of 14 981 VGS isolates were included of which 19.5%, 0.7% and 79.8% were S. mitis, S. oralis and non-speciated VGS isolates, respectively. Cumulative susceptibility rates across all years were similar between species for ceftriaxone (range: 96.0% to 100%), clindamycin (81.3% to 84.5%), and vancomycin (99.7% to 100%). For penicillin, susceptibility rates were 71.0%, 80.9% and 86.3% for S. mitis, S. oralis and non-speciated isolates, respectively. From 2010 to 2020, susceptibility of non-speciated VGS isolates decreased for erythromycin (P = 0.0674), penicillin (P = 0.0835), and tetracycline (P = 0.0994); though the decrease was only significant for clindamycin (P = 0.0033). For S. mitis, a significant susceptibility rate decrease was observed for erythromycin (P = 0.0112). Conclusions: Susceptibility rates for some clinically relevant antibiotics declined between 2010 and 2020. This worrisome trend highlights the need to improve antimicrobial stewardship efforts to limit unnecessary antibiotic use and preserve empirical treatment options.

Aminoglycoside-resistance gene signatures are predictive of aminoglycoside MICs for carbapenem-resistant Klebsiella pneumoniae.

BACKGROUND: Aminoglycoside-containing regimens may be an effective treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp), but aminoglycoside-resistance genes are common in these strains. The relationship between the aminoglycoside-resistance genes and aminoglycoside MICs remains poorly defined. OBJECTIVES: To identify genotypic signatures capable of predicting aminoglycoside MICs for CR-Kp. METHODS: Clinical CR-Kp isolates (n = 158) underwent WGS to detect aminoglycoside-resistance genes. MICs of amikacin, gentamicin, plazomicin and tobramycin were determined by broth microdilution (BMD). Principal component analysis was used to initially separate isolates based on genotype. Multiple linear regression was then used to generate models that predict aminoglycoside MICs based on the aminoglycoside-resistance genes. Last, the performance of the predictive models was tested against a validation cohort of 29 CR-Kp isolates. RESULTS: Among the original 158 CR-Kp isolates, 91.77% (145/158) had at least one clinically relevant aminoglycoside-resistance gene. As a group, 99.37%, 84.81%, 82.28% and 10.76% of the CR-Kp isolates were susceptible to plazomicin, amikacin, gentamicin and tobramycin, respectively. The first two principal components explained 72.23% of the total variance in aminoglycoside MICs and separated isolates into four groups with aac(6')-Ib, aac(6')-Ib', aac(6')-Ib+aac(6')-Ib' or no clinically relevant aminoglycoside-resistance genes. Regression models predicted aminoglycoside MICs with adjusted R2 values of 56%-99%. Within the validation cohort, the categorical agreement when comparing the observed BMD MICs with the predicated MICs was 96.55%, 89.66%, 86.21% and 82.76% for plazomicin, gentamicin, amikacin and tobramycin, respectively. CONCLUSIONS: Susceptibility to each aminoglycoside varies in CR-Kp. Detection of aminoglycoside-resistance genes may be useful to predict aminoglycoside MICs for CR-Kp.

Genomic Features Associated with the Degree of Phenotypic Resistance to Carbapenems in Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae strains cause severe infections that are difficult to treat. The production of carbapenemases such as the K. pneumoniae carbapenemase (KPC) is a common mechanism by which these strains resist killing by the carbapenems. However, the degree of phenotypic carbapenem resistance (MIC) may differ markedly between isolates with similar carbapenemase genes, suggesting that our understanding of the underlying mechanisms of carbapenem resistance remains incomplete. To address this problem, we determined the whole-genome sequences of 166 K. pneumoniae clinical isolates resistant to meropenem, imipenem, or ertapenem. Multiple linear regression analysis of this collection of largely blaKPC-3-containing sequence type 258 (ST258) isolates indicated that blaKPC copy number and some outer membrane porin gene mutations were associated with higher MICs to carbapenems. A trend toward higher MICs was also observed with those blaKPC genes carried by the d isoform of Tn4401. In contrast, ompK37 mutations were associated with lower carbapenem MICs, and extended spectrum ß-lactamase genes were not associated with higher or lower MICs in carbapenem-resistant K. pneumoniae. A machine learning approach based on the whole-genome sequences of these isolates did not result in a substantial improvement in prediction of isolates with high or low MICs. These results build upon previous findings suggesting that multiple factors influence the overall carbapenem resistance levels in carbapenem-resistant K. pneumoniae isolates. IMPORTANCE Klebsiella pneumoniae can cause severe infections in the blood, urinary tract, and lungs. Resistance to carbapenems in K. pneumoniae is an urgent public health threat, since it can make these isolates difficult to treat. While individual contributors to carbapenem resistance in K. pneumoniae have been studied, few reports explore their combined effects in clinical isolates. We sequenced 166 clinical carbapenem-resistant K. pneumoniae isolates to evaluate the contribution of known genes to carbapenem MICs and to try to identify novel genes associated with higher carbapenem MICs. The blaKPC copy number and some outer membrane porin gene mutations were associated with higher carbapenem MICs. In contrast, mutations in one specific porin, ompK37, were associated with lower carbapenem MICs. Machine learning did not result in a substantial improvement in the prediction of carbapenem resistance nor did it identify novel genes associated with carbapenem resistance. These findings enhance our understanding of the many contributors to carbapenem resistance in K. pneumoniae.

Capability of Enterococcus faecalis to shield Gram-negative pathogens from aminoglycoside exposure.

BACKGROUND: Enterococcus faecalis commonly produce aminoglycoside-modifying enzymes (AMEs) and are implicated in polymicrobial infections. OBJECTIVES: To determine if AME-producing E. faecalis is capable of protecting Enterobacteriaceae and Pseudomonas aeruginosa from gentamicin exposure. METHODS: Two Klebsiella pneumoniae isolates, two Escherichia coli isolates, and two Pseudomonas aeruginosa isolates were investigated in monoculture time-kill experiments, and each Gram-negative organism was also evaluated during co-culture with either AME-producing or AME-deficient E. faecalis. A pharmacokinetic/pharmacodynamics analysis that utilized Log Ratio Areas and a Hill-type mathematical model was used to determine if the maximal killing or potency of gentamicin against the Gram-negative organisms was altered by the presence of the E. faecalis. RESULTS: The maximal killing and potency of gentamicin was the same during monoculture and co-culture experiments for both K. pneumoniae isolates and one E. coli isolate (P > 0.05). In contrast, the maximal killing of gentamicin was attenuated against one E. coli isolate and both P. aeruginosa isolates during co-culture with E. faecalis (P < 0.05). The potency of gentamicin was variable against the three aforementioned isolates. Against the E. coli isolate, the potency of gentamicin was significantly reduced by the presence of either E. faecalis isolate (EC50 95% CI = 4.23-4.43 mg/L monoculture versus 3.86-4.19 mg/L and 3.55-3.96 mg/L during co-culture with AME-producing and AME-deficient E. faecalis, respectively). The potency of gentamicin increased or decreased for P. aeruginosa depending on which E. faecalis isolate was investigated. CONCLUSIONS: The AME-producing E. faecalis did not provide a consistent protective effect from aminoglycosides for the Gram-negative pathogens.

Generating Genotype-Specific Aminoglycoside Combinations with Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Antibiotic combinations, including ceftazidime/avibactam (CAZ/AVI), are frequently employed to combat KPC-producing Klebsiella pneumoniae (KPC-Kp), though such combinations have not been rationally optimized. Clinical KPC-Kp isolates with common genes encoding aminoglycoside-modifying enzymes (AMEs), aac(6')-Ib' or aac(6')-Ib, were used in static time-kill assays (n = 4 isolates) and the hollow-fiber infection model (HFIM; n = 2 isolates) to evaluate the activity of gentamicin, amikacin, and CAZ/AVI alone and in combinations. A short course, one-time aminoglycoside dose was also evaluated. Gentamicin plus CAZ/AVI was then tested in a mouse pneumonia model. Synergy with CAZ/AVI was more common with amikacin for aac(6')-Ib'-containing KPC-Kp but more common with gentamicin for aac(6')-Ib-containing isolates in time-kill assays. In the HFIM, although the isolates were aminoglycoside-susceptible at baseline, aminoglycoside monotherapies displayed variable initial killing, followed by regrowth and resistance emergence. CAZ/AVI combined with amikacin or gentamicin resulted in undetectable counts 50 h sooner than CAZ/AVI monotherapy against KPC-Kp with aac(6')-Ib'. CAZ/AVI monotherapy failed to eradicate KPC-Kp with aac(6')-Ib and a combination with gentamicin led to undetectable counts 70 h sooner than with amikacin. A one-time aminoglycoside dose with CAZ/AVI provided similar killing to aminoglycosides dosed for 7 days. In the mouse pneumonia model (n = 1 isolate), gentamicin and CAZ/AVI achieved a 6.0-log10 CFU/lung reduction at 24 h, which was significantly greater than either monotherapy (P < 0.005). Aminoglycosides in combination with CAZ/AVI were promising for KPC-Kp infections; this was true even for a one-time aminoglycoside dose. Selecting aminoglycosides based on AME genes or susceptibilities can improve the pharmacodynamic activity of the combination.

In vitro Optimization of Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Ceftazidime/avibactam is an important treatment option for infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp), however, resistance can emerge during treatment. The objective of the study was to define the ceftazidime/avibactam concentrations required to suppress bacterial regrowth in ceftazidime/avibactam susceptible isolates and identify active therapies against ceftazidime/avibactam-resistant KPC-Kp. Time-kill assays were performed against twelve ST258 KPC-Kp isolates that harbored bla KPC - 2 or bla KPC - 3. Nine KPC-Kp isolates (KPC-Kp 5A, 6A, 7A, 8A, 9A, 24A, 25A, 26A, and 27A) were susceptible to ceftazidime/avibactam, two (KPC-Kp 6B and 7B) were ceftazidime/avibactam resistant and meropenem susceptible, and one (KPC-Kp 1244) was resistant to both ceftazidime/avibactam and meropenem. Sequencing of the bla KPC genes revealed mutations in KPC-Kp 6B (D179Y substitution) and 7B (novel 21 base pair deletion) that both affected the Ω-loop encoding portion of the gene. Time-kill assays showed that against ceftazidime/avibactam-susceptible KPC-Kp, ceftazidime/avibactam concentrations ≥40/7.5 mg/L caused mean 5.42 log1 0CFU/mL killing and suppressed regrowth. However, regrowth occurred for some KPC-Kp isolates with a ceftazidime/avibactam concentration of 20/3.75 mg/L. Against ceftazidime/avibactam-resistant and meropenem-susceptible KPC-Kp 6B and 7B, bactericidal activity and synergy was observed for ceftazidime/avibactam in combination with meropenem ≤3.125 mg/L, while meropenem concentrations ≥50 mg/L were bactericidal as monotherapy. In contrast, clinically achievable concentrations of ceftazidime/avibactam were bactericidal against KPC-Kp 1244, which was ceftazidime/avibactam-resistant and meropenem-resistant due to outer membrane porin mutations and elevated bla KPC expression. Achieving high ceftazidime/avibactam concentrations may help to suppress bacterial regrowth in the presence of ceftazidime/avibactam. The optimal treatment approach for ceftazidime/avibactam-resistant KPC-Kp likely depends on the mechanism of resistance. Additional studies are warranted to confirm these findings.

Therapeutic Options for Metallo-ß-Lactamase-Producing Enterobacterales.

The spread of metallo-ß-lactamase (MBL)-producing Enterobacterales worldwide without the simultaneous increase in active antibiotics makes these organisms an urgent public health threat. This review summarizes recent advancements in diagnostic and treatment strategies for infections caused by MBL-producing Enterobacterales. Adequate treatment of patients infected with MBL-producing Enterobacterales relies on detection of the ß-lactamase in the clinic. There are several molecular platforms that are currently available to identify clinically relevant MBLs as well as other important serine-ß-lactamases. Once detected, there are several antibiotics that have historically been used for the treatment of MBL-producing Enterobacterales. Antimicrobials such as aminoglycosides, tetracyclines, fosfomycin, and polymyxins often show promising in vitro activity though clinical data are currently lacking to support their widespread use. Ceftazidime-avibactam combined with aztreonam is promising for treatment of infections caused by MBL-producing Enterobacterales and currently has the most clinical data of any available antibiotic to support its use. While cefiderocol has displayed promising activity against MBL-producing Enterobacterales in vitro and in preliminary clinical studies, further clinical studies will better shed light on its place in treatment. Lastly, there are several promising MBL inhibitors in the pipeline, which may further improve the treatment of MBL-producing Enterobacterales.

Optimizing aminoglycoside selection for KPC-producing Klebsiella pneumoniae with the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib.

OBJECTIVES: KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates commonly co-harbour the aminoglycoside-modifying enzyme (AME) gene aac(6')-Ib, which encodes an AME that can confer resistance to some of the commercially available aminoglycosides. We sought to determine the influence of AAC(6')-Ib in KPC-Kp on the pharmacodynamic activity of aminoglycosides. METHODS: Six KPC-Kp clinical isolates, three with and three without aac(6')-Ib, were analysed. Using these isolates, the bacterial killing of amikacin, gentamicin and tobramycin was assessed in static time-kill experiments. The pharmacodynamic activity of the aminoglycosides was then assessed in a dynamic one-compartment infection model over 72 h using simulated human pharmacokinetics of once-daily dosing with amikacin (15 mg/kg), gentamicin (5 mg/kg) and tobramycin (5 mg/kg). RESULTS: At clinically relevant aminoglycoside concentrations in time-kill experiments and the dynamic one-compartment model, gentamicin was more active than amikacin or tobramycin against the isolates harbouring aac(6')-Ib. Amikacin, gentamicin and tobramycin all showed progressively reduced bacterial killing with exposure to repeated doses against most isolates in the dynamic one-compartment model. MIC values were generally not a good predictor of gentamicin pharmacodynamic activity against KPC-Kp, but were more reliable for amikacin and tobramycin. CONCLUSIONS: Gentamicin may be preferred over amikacin or tobramycin for treatment of KPC-Kp infections. However, gentamicin MICs are not a consistent predictor of its pharmacodynamic activity and unexpected treatment failures are possible.

A coup d'état by NDM-producing Klebsiella pneumoniae overthrows the major bacterial population during KPC-directed therapy.

The objective of this study was to utilize a co-culture hollow-fiber infection model (HFIM) to characterize the interplay between a small, difficult-to-detect, New Delhi metallo-ß-lactamase-producing Klebsiella pneumoniae (NDM-Kp) minor population and a larger K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae population in the presence of KPC-directed antibacterial therapy. Selective plating onto agar with ceftazidime-avibactam was used to track the density of the NDM-Kp population. Susceptibility testing and the Verigene System failed to identify the small initial NDM-Kp population. However, a ceftazidime-avibactam Etest detected resistant colonies that were confirmed to be NDM-Kp. In the HFIM, all of the investigated drug regimens caused regrowth within 24 h and resulted in >109 CFU/mL of NDM-Kp. Our study demonstrates that the HFIM is a powerful tool for studying the population dynamics of multiple pathogens during antimicrobial exposure and also highlights that difficult-to-detect minor populations of drug-resistant bacteria may cause treatment failure without appropriate antibacterial therapy.

Unraveling the Gentamicin Drug Product Complexity Reveals Variation in Microbiological Activities and Nephrotoxicity.

The gentamicin drug product is a complex mixture of numerous components, many of which have not individually undergone safety and efficacy assessments. This is in contrast to the majority of medicines that require rigorous characterizations of trace impurities and are dosed as single components. In gentamicin, four components, known as gentamicin congeners C1, C1a, C2, and C2a, comprise the majority of the mixture. A liquid chromatography-mass spectroscopy analysis revealed that the relative abundances of each gentamicin congener in commercial formulations can vary up to 1.9-fold depending on the commercial source of the gentamicin. To determine if the gentamicin used for antibiotic susceptibility testing (AST) would be predictive of the microbiological activity of the product used to dose patients, the relative abundances of the four congeners contained on commercial AST disks were measured. It was found that the congener abundances on the commercial AST disks varied up to 4.1-fold. After purification of the four gentamicin congeners, similar potencies against bacterial strains lacking aminoglycoside-modifying enzymes (AMEs) were observed. However, the potency of the congeners against strains harboring a common AME differed up to 128-fold. Nephrotoxicity of the individual gentamicin congeners also differed significantly in cell-based and repeat-dose rat nephrotoxicity studies. Variations in the composition of commercial gentamicin products combined with toxicity differences between gentamicin congeners suggest that some gentamicin formulations may be more nephrotoxic. Our results also raise the concern that gentamicin susceptibility test results may not be predictive of patient outcomes and could lead to unexpected clinical treatment failures.