Impact of nutritional factors on the in vitro PK/PD modelling of polymyxin B against various strains of Acinetobacter baumannii.

The main objective of this study was to assess the effect of rich artificial cation adjusted Mueller-Hinton broth (CAMHB), on the growth of three Acinetobacter baumannii strains, ATCC 19606 and two clinical A. baumannii strains, either susceptible or resistant to polymyxin B (PMB), and on the PMB bactericidal activity. A pharmacokinetic (PK) / pharmacodynamic (PD) modelling approach was then used to characterize the effect of PMB in various conditions. Time-kill experiments were performed using undiluted CAMHB or diluted at 50%, 25% and 10%, with or without Ca2+ and Mg2+ compensation (known to affect PMB activity), and with PMB concentrations ranging from 0.25 mg/L to 256 mg/L based on the strain's MIC. For each strain, time-kill replicates were modelled using NONMEM®. Unexpectedly, CAMHB dilution by up to 10-fold did not affect the growth rate of any of the three strains in the absence of PMB. Yet, PMB bactericidal activity increased with medium dilution resulting in particular in a reduction of the apparent bacterial regrowth of the various strains observed typically after few hours of experiment. The data of each strain were well characterised by a PK/PD model with two bacterial sub-populations with different susceptibility to PMB (a more susceptible (S+) and a less susceptible (S-)). Relatively large, unexplained strain-to-strain variability was observed regarding the impact of medium dilution as well as cation compensation. Complementary experiments are needed to characterise the mechanism underlying this medium dilution effect.

A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Translational PK/PD for the Development of Novel Antibiotics-A Drug Developer's Perspective.

Antibiotic development traditionally involved large Phase 3 programs, preceded by Phase 2 studies. Recognizing the high unmet medical need for new antibiotics and, in some cases, challenges to conducting large clinical trials, regulators created a streamlined clinical development pathway in which a lean clinical efficacy dataset is complemented by nonclinical data as supportive evidence of efficacy. In this context, translational Pharmacokinetic/Pharmacodynamic (PK/PD) plays a key role and is a major contributor to a "robust" nonclinical package. The classical PK/PD index approach, proven successful for established classes of antibiotics, is at the core of recent antibiotic approvals and the current antibacterial PK/PD guidelines by regulators. Nevertheless, in the case of novel antibiotics with a novel Mechanism of Action (MoA), there is no prior experience with the PK/PD index approach as the basis for translating nonclinical efficacy to clinical outcome, and additional nonclinical studies and PK/PD analyses might be considered to increase confidence. In this review, we discuss the value and limitations of the classical PK/PD approach and present potential risk mitigation activities, including the introduction of a semi-mechanism-based PK/PD modeling approach. We propose a general nonclinical PK/PD package from which drug developers might choose the studies most relevant for each individual candidate in order to build up a "robust" nonclinical PK/PD understanding.

A previously uncharacterized gene, PA2146, contributes to biofilm formation and drug tolerance across the ɣ-Proteobacteria.

Transcriptomic studies have revealed a large number of uncharacterized genes that are differentially expressed in biofilms, which may be important in regulating biofilm phenotypes such as resistance to antimicrobial agents. To identify biofilm genes of unknown function in P. aeruginosa, we made use of RNA-seq and selected 27 uncharacterized genes that were induced upon biofilm growth. Biofilms by respective mutants were subsequently analyzed for two biofilm characteristics, the biofilm architecture and drug susceptibility. The screen revealed 12 out of 27 genes to contribute to biofilm formation and 13 drug susceptibility, with 8 genes affecting both biofilm phenotypes. Amongst the genes affecting both biofilm phenotypes was PA2146, encoding a small hypothetical protein that exhibited some of the most substantial increases in transcript abundance during biofilm growth by P. aeruginosa PAO1 and clinical isolates. PA2146 is highly conserved in É£-proteobacteria. Inactivation of PA2146 affected both biofilm phenotypes in P. aeruginosa PAO1, with inactivation of homologs in Klebsiella pneumoniae and Escherichia coli having similar effects. Heterologous expression of PA2146 homologs complemented the P. aeruginosa ∆PA2146, suggesting that PA2146 homologs substitute for and play a similar role as PA2146 in P. aeruginosa.

Escape mutations circumvent a tradeoff between resistance to a beta-lactam and resistance to a beta-lactamase inhibitor.

Beta-lactamase inhibitors are increasingly used to counteract antibiotic resistance mediated by beta-lactamase enzymes. These inhibitors compete with the beta-lactam antibiotic for the same binding site on the beta-lactamase, thus generating an evolutionary tradeoff: mutations that increase the enzyme's beta-lactamase activity tend to increase also its susceptibility to the inhibitor. Here, we investigate how common and accessible are mutants that escape this adaptive tradeoff. Screening a deep mutant library of the blaampC beta-lactamase gene of Escherichia coli, we identified mutations that allow growth at beta-lactam concentrations far exceeding those inhibiting growth of the wildtype strain, even in the presence of the enzyme inhibitor (avibactam). These escape mutations are rare and drug-specific, and some combinations of avibactam with beta-lactam drugs appear to prevent such escape phenotypes. Our results, showing differential adaptive potential of blaampC to combinations of avibactam and different beta-lactam antibiotics, suggest that it may be possible to identify treatments that are more resilient to evolution of resistance.

A novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model describing ceftazidime/avibactam efficacy against ß-lactamase-producing Gram-negative bacteria.

BACKGROUND: Diazabicyclooctanes (DBOs) are an increasingly important group of non ß-lactam ß-lactamase inhibitors, employed clinically in combinations such as ceftazidime/avibactam. The dose finding of such combinations is complicated using the traditional pharmacokinetic/pharmacodynamic (PK/PD) index approach, especially if the ß-lactamase inhibitor has an antibiotic effect of its own. OBJECTIVES: To develop a novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model for ceftazidime/avibactam against Gram-negative pathogens, with the potential for combination dosage simulation. METHODS: Four ß-lactamase-producing Enterobacteriaceae, covering Ambler classes A, B and D, were exposed to ceftazidime and avibactam, alone and in combination, in static time-kill experiments. A PKPD model was developed and evaluated using internal and external evaluation, and combined with a population PK model and applied in dosage simulations. RESULTS: The developed PKPD model included the effects of ceftazidime alone, avibactam alone and an 'enhancer' effect of avibactam on ceftazidime in addition to the ß-lactamase inhibitory effect of avibactam. The model could describe an extensive external Pseudomonas aeruginosa data set with minor modifications to the enhancer effect, and the utility of the model for clinical dosage simulation was demonstrated by investigating the influence of the addition of avibactam. CONCLUSIONS: A novel mechanism-based PKPD model for the DBO/ß-lactam combination ceftazidime/avibactam was developed that enables future comparison of the effect of avibactam with other DBO/ß-lactam inhibitors in simulations, and may be an aid in translating PKPD results from in vitro to animals and humans.

Meropenem-nacubactam activity against AmpC-overproducing and KPC-expressing Pseudomonas aeruginosa in a neutropenic murine lung infection model.

Nacubactam is a novel non-ß-lactam diazabicyclooctane ß-lactamase inhibitor under development for the treatment of serious Gram-negative infections. This study assessed the efficacy of human-simulated epithelial lining fluid (ELF) exposure of nacubactam in combination with meropenem against AmpC-overproducing (n=4) and Klebsiella pneumoniae carbapenemase (KPC)-expressing (n=3) Pseudomonas aeruginosa isolates in the neutropenic murine lung infection model. Meropenem, nacubactam and meropenem-nacubactam (1:1 concentration ratio) minimum inhibitory concentrations (MICs) were determined in triplicate using broth microdilution. Regimens that provided ELF profiles mimicking those observed in humans given nacubactam 2 g q8h (1.5-h infusion) alone and in combination with a subtherapeutic ELF exposure of meropenem were administered 2 h after inoculation. Efficacy was assessed as the change in log10 colony-forming units (CFU)/lung at 24 h compared with 24-h meropenem monotherapy. Meropenem, nacubactam and meropenem-nacubactam MICs were 8->64, 128->256 and 2-16 mg/L, respectively. Meropenem and nacubactam monotherapy groups demonstrated bacterial growth over 24 h for each isolate. Against AmpC-overproducing and KPC-expressing P. aeruginosa isolates, meropenem-nacubactam resulted in -2.73±0.93 and -4.35±1.90 log10CFU/lung reduction, respectively, relative to meropenem monotherapy. Meropenem-nacubactam showed promising in-vivo activity against meropenem-resistant P. aeruginosa, indicative of a potential role for the treatment of infections caused by these challenging pathogens.

Efficacy of Human-Simulated Epithelial Lining Fluid Exposure of Meropenem-Nacubactam Combination against Class A Serine ß-Lactamase-Producing Enterobacteriaceae in the Neutropenic Murine Lung Infection Model.

Nacubactam is a novel, broad-spectrum, ß-lactamase inhibitor that is currently under development as combination therapy with meropenem. This study evaluated the efficacy of human-simulated epithelial lining fluid (ELF) exposures of meropenem, nacubactam, and the combination of meropenem and nacubactam against class A serine carbapenemase-producing Enterobacteriaceae isolates in the neutropenic murine lung infection model. Twelve clinical meropenem-resistant Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae isolates, all harboring KPC or IMI-type ß-lactamases, were utilized in the study. Meropenem, nacubactam, and meropenem-nacubactam (1:1) combination MICs were determined in triplicate via broth microdilution. At 2 h after intranasal inoculation, neutropenic mice were dosed with regimens that provided ELF profiles mimicking those observed in humans given meropenem at 2 g every 8 h and/or nacubactam at 2 g every 8 h (1.5-h infusions), alone or in combination. Efficacy was assessed as the change in bacterial growth at 24 h, compared with 0-h controls. Meropenem, nacubactam, and meropenem-nacubactam MICs were 8 to >64 µg/ml, 2 to >256 µg/ml, and 0.5 to 4 µg/ml, respectively. The average bacterial density at 0 h across all isolates was 6.31 ± 0.26 log10 CFU/lung. Relative to the 0-h control, the mean values of bacterial growth at 24 h in the untreated control, meropenem human-simulated regimen treatment, and nacubactam human-simulated regimen treatment groups were 2.91 ± 0.27, 2.68 ± 0.42, and 1.73 ± 0.75 log10 CFU/lung, respectively. The meropenem-nacubactam combination human-simulated regimen resulted in reductions of -1.50 ± 0.59 log10 CFU/lung. Meropenem-nacubactam human-simulated ELF exposure produced enhanced efficacy against all class A serine carbapenemase-producing Enterobacteriaceae isolates tested in the neutropenic murine lung infection model.

In Vivo Efficacy of Meropenem with a Novel Non-ß-Lactam-ß-Lactamase Inhibitor, Nacubactam, against Gram-Negative Organisms Exhibiting Various Resistance Mechanisms in a Murine Complicated Urinary Tract Infection Model.

Urinary tract infections (UTIs) are a tremendous burden on the health care system due to the vast number of infections resulting in antibiotic therapy and/or hospitalization. Additionally, these infections are frequently caused by multidrug-resistant (MDR) organisms, limiting the availability of effective antimicrobials. Nacubactam is a novel non-ß-lactam-ß-lactamase inhibitor with in vitro activity against class A and class C ß-lactamases. Nacubactam is being developed in combination with meropenem, providing broad-spectrum activity in addition to improved stability against common ß-lactamases. Here, we utilized a neutropenic murine complicated UTI (cUTI) model to determine the potential clinical utility of meropenem-nacubactam compared with meropenem or nacubactam alone against 10 Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae isolates with diverse genotypic and phenotypic profiles, including NDM, KPC, OXA, CTX-M, SHV, and TEM enzyme-producing isolates. Selected isolates had meropenem-nacubactam MICs between 1 and 8 µg/ml. Meropenem-nacubactam demonstrated the greatest in vivo efficacy against 9 of 10 isolates, achieving a ≥3 log reduction from the 48-h control in all isolates tested, including isolates prepared as high inoculums. Nacubactam alone confirmed antibacterial properties, achieving a >1 log reduction against the majority of isolates. The combination of meropenem-nacubactam further enhanced the activity of either agent alone, notably against meropenem-resistant isolates. Against ceftazidime-avibactam-resistant isolates, meropenem-nacubactam demonstrated increased antibacterial kill upwards of 6 log10 CFU in comparison to the 48-h control. Our data support the potential clinical utility of meropenem-nacubactam for cUTI in humans against MDR Enterobacteriaceae, although further clinical data supporting meropenem-nacubactam efficacy are needed.

Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes.

Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested.

Biofilm induced tolerance towards antimicrobial peptides.

Increased tolerance to antimicrobial agents is thought to be an important feature of microbes growing in biofilms. We address the question of how biofilm organization affects antibiotic susceptibility. We established Escherichia coli biofilms with differential structural organization due to the presence of IncF plasmids expressing altered forms of the transfer pili in two different biofilm model systems. The mature biofilms were subsequently treated with two antibiotics with different molecular targets, the peptide antibiotic colistin and the fluoroquinolone ciprofloxacin. The dynamics of microbial killing were monitored by viable count determination, and confocal laser microscopy. Strains forming structurally organized biofilms show an increased bacterial survival when challenged with colistin, compared to strains forming unstructured biofilms. The increased survival is due to genetically regulated tolerant subpopulation formation and not caused by a general biofilm property. No significant difference in survival was detected when the strains were challenged with ciprofloxacin. Our data show that biofilm formation confers increased colistin tolerance to cells within the biofilm structure, but the protection is conditional being dependent on the structural organization of the biofilm, and the induction of specific tolerance mechanisms.

Activity of ceftibuten, cefaclor, azithromycin, clarithromycin, erythromycin and telithromycin against Streptococcus pyogenes clinical isolates with different genotypes and phenotypes.

BACKGROUND: The growing number of macrolide-resistant strains of Streptococcus pyogenes represents an increasing worldwide problem. Macrolide resistance in S. pyogenes is mediated by several different genes, which determine different levels of resistance to macrolides, lincosamides and streptogramin B (MLS). METHODS: This study compared the in vitro antimicrobial activity of azithromycin, clarithromycin, erythromycin, ceftibuten, cefaclor, and telithromycin against 287 strains of S. pyogenes by the broth microdilution method. All strains were characterized both phenotypically and genotypically for erythromycin resistance and most of them have been M-typed by means of PCR. RESULTS: Ceftibuten and cefaclor showed the best antimicrobial activity, while MIC values for telithromycin were higher against constitutively MLS (cMLS)-resistant strains rather than against the other phenotypes. CONCLUSION: Oral cephalosporins retain the best activity against S. pyogenes; showing good activity except for cMLS-resistant strains, telithromycin is a valid alternative to these antimicrobials.

Genetic diversity of cell-invasive erythromycin-resistant and -susceptible group A streptococci determined by analysis of the RD2 region of the prtF1 gene.

The RD2 region of the internalization-associated gene prtF1, which encodes the fibronectin-binding repeat domain type 2 of protein F1, plays a crucial role in the entry of group A streptococci (GAS) into epithelial cells. A molecular study of the variability of the RD2 region was carried out with 77 independent Italian GAS, 66 erythromycin resistant (ER) and 11 erythromycin susceptible (ES), which had previously been investigated for the association between erythromycin resistance and ability to enter human respiratory cells. The amplicons obtained from PCR analysis of the RD2 region were consistent with a number of RD2 repeats ranging from one to five, more frequently four (n = 30), three (n = 27), and one (n = 18). A new method to type cell-invasive GAS (RD2 typing) was developed by combining PCR analysis of the RD2 region and restriction analysis of PCR products with endonucleases HaeIII, DdeI, and HinfI. Overall, 10 RD2 types (a to j) were distinguished (all detected among the 66 ER isolates, four detected among the 11 ES isolates). Comparison and correlation of RD2 typing data with the genotype and phenotype of macrolide resistance and with data from PCR M typing and SmaI macrorestriction analysis allowed us to identify 41 different clones (31 among the 66 ER isolates and 10 among the 11 ES isolates). Three major clones accounted for 40% of the isolates (47% of ER strains). Some ES isolates appeared to be related to ER isolates with identical combinations of RD2 type and emm type. While simultaneous use of different typing methods is essential for a thorough investigation of GAS epidemiology, RD2 typing may be especially helpful in typing cell-invasive GAS.

emm Gene distribution among erythromycin-resistant and -susceptible Italian isolates of Streptococcus pyogenes.

The phenotypes and genetic determinants for macrolide resistance were determined for 167 erythromycin-resistant Streptococcus pyogenes strains. A cMLS phenotype was shown in 18% of the erythromycin-resistant strains, while inducible resistance was apparent in 31% and the M phenotype was apparent in 50%. The emm gene type of this set of resistant isolates and that of 48 erythromycin-sensitive isolates were determined. emm2 and emm48 were recorded only in the resistant strains of the M phenotype, while approximately all of the strains harboring the emm22 gene had the cMLS phenotype. More than 80% of the emm89-positive strains had the iMLS phenotype, and the same portion of emm4 strains presented the M phenotype. emm3 is recorded only among sensitive strains. The distribution of frequencies of the genetic determinant for the virulence factor M protein was significantly different both among organisms of different types of resistance and between resistant and sensitive populations of S. pyogenes under study.

Erythromycin resistance in italian isolates of Streptococcus pyogenes and correlations with pulsed-field gel electrophoresis analysis.

Erythromycin resistance among Streptococcus pyogenes strains has been reported in Italy at high rates during the last few years. A total of 152 erythromycin-resistant isolates of this species from southern Italian regions were characterized for the macrolide-resistance phenotype and screened by PCR for the corresponding genetic determinant. A close correlation was found between these phenotypic/genotypic data concerning macrolide resistance and results of Sma I macrorestriction fragment patterns (PFGE) analysis. In fact, the vast majority of the isolates assigned to individual PFGE classes mostly belonged to a single phenotype of macrolide resistance. All untypeable isolates belonged to the M phenotype. Twenty-two distinct PFGE types were recognized, of which 11 were recorded in only one isolate (one-strain type); about 50% of typeable isolates fell into five type clusters and 70% in seven. The increased erythromycin resistance among Italian isolates of S. pyogenes does not appear to be due to the spread of a single clone, but results indicate that the majority of group A streptococci examined are probably spread from a limited number of clones.

PCR m typing: a new method for rapid typing of group a streptococci.

A new approach for the M-typing of Streptococcus pyogenes is reported. Oligonucleotide primers were used in a PCR to amplify the N-terminal region of the emm gene. The presence of the PCR amplification product is associated with the corresponding M serotype. This technique offers potential advantages over other molecular typing methods.