In Vitro Activities of Ceftazidime–Avibactam and Aztreonam–Avibactam at Different Inoculum Sizes of Extended-Spectrum β-Lactam-Resistant Enterobacterales Blood Isolates

β-lactam–avibactam combinations have been proposed as carbapenem-sparing therapies, but little data exist on their in vitro activities in infections with high bacterial inocula. We investigated the in vitro efficacies and the inoculum effects of ceftazidime–avibactam and aztreonam–avibactam against extended-spectrum β-lactam-resistant Enterobacterales blood isolates. A total of 228 non-repetitive extended-spectrum β-lactam-resistant Escherichia coli and Klebsiella pneumoniae blood isolates were prospectively collected in a tertiary center. In vitro susceptibilities to ceftazidime, aztreonam, meropenem, ceftazidime–avibactam, and aztreonam–avibactam were evaluated by broth microdilution method using standard and high inocula. An inoculum effect was defined as an eightfold or greater increase in MIC when tested with the high inoculum. Of the 228 isolates, 99% were susceptible to ceftazidime–avibactam and 99% had low aztreonam–avibactam MICs (≤8 mg/L). Ceftazidime–avibactam and aztreonam–avibactam exhibited good in vitro activities; MIC50/MIC90 values were 0.5/2 mg/L, 0.125/0.5 mg/L, and ≤0.03/0.25 mg/L, respectively, and aztreonam–avibactam was more active than ceftazidime–avibactam. The frequencies of the inoculum effect with ceftazidime–avibactam and aztreonam–avibactam were lower than with meropenem (14% vs. 38%, p < 0.001 and 30% vs. 38%, p = 0.03, respectively). The β-lactam-avibactam combinations could be useful as carbapenem-sparing strategies, and aztreonam–avibactam has the better in vitro activity but is more subject to the inoculum effect than ceftazidime–avibactam.

A new PKPD model to characterize the inoculum effect of Acinetobacter baumannii on polymyxin B in vitro

The inoculum effect (i.e., reduction in antimicrobial activity at large starting inoculum) is a phenomenon described for various pathogens. Since limited data exist regarding inoculum effect of Acinetobacter baumannii , we evaluated killing of A. baumannii by polymyxin B, a last-resort antibiotic, at several starting inocula and developed a PKPD model to capture this phenomenon. In vitro static time-kill experiments were performed using polymyxin B at concentrations ranging from 0.125 to 128 mg/L against a clinical A. baumannii isolate at four starting inocula from 10 5 to 10 8 CFU/mL. Samples were collected up to 30 h to quantify the viable bacterial burden and were simultaneously modeled in the NONMEM software program. The expression of polymyxin B resistance genes ( lpxACD , pmrCAB and wzc ), and genetic modifications were studied by RT-qPCR and DNA sequencing experiments, respectively. The PKPD model included a single homogeneous bacterial population with adaptive resistance. Polymyxin B effect was modelled as a sigmoidal E max model and the inoculum effect as an increase of polymyxin B EC 50 with increasing starting inoculum using a power function. Polymyxin B displayed a reduced activity as the starting inoculum increased: a 20-fold increase of polymyxin B EC 50 was observed between the lowest and the highest inoculum. No effects of polymyxin B and inoculum size were observed on the studied genes. The proposed PKPD model successfully described and predicted the pronounced in vitro inoculum effect of A. baumannii on polymyxin B activity. These results should be further validated using other bacteria/antibiotic combinations and in vivo models.

199. Polymorphisms in Key Regulatory Regions of the bla operon Correlate with the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus (MSSA)

Background The cefazolin inoculum effect (CzIE), defined as Cz minimum inhibitory concentration ≥ 16 µg/ml at high inoculum (HI-MIC), has been associated with poor clinical outcomes in patients with MSSA bacteremia or osteomyelitis. The CzIE is correlated with the presence of the blaZ gene, one of the components of the bla operon encoding the BlaZ β-lactamase (type A, B, C or D). Other portions of the bla operon include blaR and blaI (encoding the antibiotic sensor and transcriptional repressor, respectively) and the intergenic region with operator and promoter sequences (Figure 1). In BlaR, residue 293 mediates signal transduction, and the Z and R dyads in the intergenic region are the DNA-binding sites for BlaI (Figure 2). Previous experiments have shown that the regulatory portions of the bla operon play a key role in the CzIE. Here, we investigated the association between the CzIE and specific variations in the regulatory sequences of the bla operon. Figure 1. Functioning of the bla operon and the production of the staphylococcal β-lactamase BlaZ. Figure 2. Structure and key regions of the intergenic region of the bla operon, incluiding the promoter and the BlaI DNA-binding regions (Z and R dyads). Methods A total of 437 MSSA containing blaZ were evaluated for the CzIE using broth microdilution at high inoculum. Using whole genome sequencing, the sequences of the bla operons were classified into cassettes based on unique changes in predicted amino acid sequences of BlaZ, BlaR and BlaI paired with specific nucleotide alterations in the intergenic region. The bla operon sequence of S. aureus ATCC29213 was used as reference (cassette 0). Results Among 437 MSSA isolates, 46% exhibited the CzIE. We identified 55 unique bla cassettes. The bla cassettes were phylogenetically grouped in 7 clusters (Figure 3) which grouped cassettes with different BlaZ types and variations in the Z dyad, the -35 box, residue 293 of BlaR, and the blaI ribosomal binding site. Each cluster had an association to the CzIE and distinct Cz HI-MICs. The combination of: a BlaZ type A, C or D, an adenine in the position -66 of blaZ (-35 box of blaZ), a cytosine in the position -22 of blaZ(Z dyad), and either an arginine or a serine in position 293 of BlaR was a very strong predictor of the CzIE (Figure 4). Figure 3. Phylogenetical organization of bla operon cassettes into clusters, their association with polymorphisms in key regulatory regions and the CzIE. GM Cz-MIC: Geometric mean of the Cefazolin MIC at high Inoculum. Figure 4. Variations of the bla operon, their association with the CzIE, their GM (Geometric Mean) of the Cefazolin MIC and the MIC distribution of the strains with each specific combination of polymorphisms. Conclusion Specific variations in regulatory portions of the bla operon, which are likely to influence BlaZ expression, are highly associated with the CzIE, supporting the notion that regulation of blaZ is the key factor responsible for the CzIE in MSSA. Disclosures Lorena Diaz, PhD , Nothing to disclose William R. Miller, MD , Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support) William R. Miller, MD , Entasis (Individual(s) Involved: Self): Scientific Research Study Investigator; Merck (Individual(s) Involved: Self): Grant/Research Support Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support)

1248. A Machine-Learning Approach to Predict the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

Background The cefazolin (Cz) inoculum effect (CzIE), defined as an increase in the Cz MIC to ≥16 µg/mL at high inoculum (107 CFU/mL), has been associated with poor outcomes in MSSA bacteremia and osteomyelitis. The CzIE is associated with the BlaZ β-lactamase, encoded by blaZ and regulated by BlaR (antibiotic sensor) and BlaI (transcriptional repressor). Here, we aimed to obtain a machine-learning (ML) model to predict the presence of the CzIE based on the nucleotide sequence of the entire bla operon and its regulatory components. Methods Using whole genome sequencing, we analyzed the nucleotide sequences of the entire bla operon in 436 MSSA isolates recovered from blood, soft-tissue infections or pneumonia in adults (training-testing cohort, prevalence of the CzIE: 46%). Also, 32 MSSA recovered from pediatric patients with osteomyelitis with the CzIE were included as validation cohort. The CzIE was determined by broth microdilution at high inoculum. K-mer counts were obtained from the bla operon sequences of the isolates from the testing-training cohort, and then used in a ML pipeline which i) discards uninformative K-mers, ii) identifies optimal hyper-parameters and, iii) performs training of the model using 70% of the sequences as training set and 30% as testing set. The pipeline tested 11 different K-mer sizes and 2 models: Logistic Regression (LR) and Support Vector Machine (SVM). Finally, the model with best predictive ability was applied to the sequences of the MSSA osteomyelitis isolates (validation cohort). Results The ML approach had high specificity ( &gt;90%), accuracy ( &gt;80%) and ROC-AUC values ( &gt;0.7) for detecting the CzIE in the testing set of isolates (Figure 1), independently of the type of model or the K-mer size used. The best predictive ability was with LR using K-mers of 17 nucleotides, with an accuracy of 84%, specificity of 96%, and sensitivity of 70% in the testing set (Figure 2). In the validation cohort, the model was capable to correctly identify all the strains exhibiting the CzIE (100% sensitivity). Figure 1. Prediction metrics of the ML pipeline for the detection of the CzIE in MSSA isolates from the training-test cohort. Predictions are shown accordingly to the model and K-mer sizes tested. Figure 2. ROC of best predictive model (Logistic Regression, K-mer size 17) for the detection of the CzIE in MSSA isolates. Conclusion The ML approach is a promising genomic application to detect the CzIE in MSSA isolates of a variety of sources, bypassing phenotypic testing. Further validation is needed to evaluate its possible utility in clinical settings. Disclosures Jonathon C. McNeil, MD, Agency for Healthcare Research and Quality (Research Grant or Support)Allergan (Grant/Research Support)Nabriva (Grant/Research Support, Other Financial or Material Support, Site PI for a multicenter trial) Anthony R. Flores, MD, MPH, PhD, Nothing to disclose Sheldon L. Kaplan, MD, Pfizer (Research Grant or Support) Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

Effects of Incubation Time and Inoculation Level on the Stabilities of Bacteriostatic and Bactericidal Antibiotics against Salmonella Typhimurium

This study was designed to evaluate the stability of chloramphenicol, erythromycin, tetracycline, cephalothin, ciprofloxacin, and tobramycin against antibiotic-sensitive Salmonella Typhimurium (ASST) and antibiotic-resistant S. Typhimurium (ARST) during the broth microdilution assay. The antimicrobial activity in association with antibiotic stability was measured by using antibiotic susceptibility, time-delayed inoculation, time-extended incubation, and inoculum effect assays. The loss of the antimicrobial activity of cephalothin against ASST exposed to 1 MIC was observed for the 10 h delayed inoculation. The antimicrobial activities of tetracycline and ciprofloxacin against ASST and ARST exposed to ½ MIC were significantly decreased after the 10 h delayed inoculation. All antibiotics used in this study, except for ciprofloxacin, showed the considerable losses of antimicrobial activities against ASST and ARST after 40 h of incubation at 37 °C when compared to the 20 h of incubation during AST. Compared to the standard inoculum level (6 log CFU/mL), the MIC0.1 values of bactericidal antibiotics, ciprofloxacin and tobramycin against ASST were increased by more than 4-fold at the high inoculum level of 9 log CFU/mL. This would provide practical information for better understanding the clinical efficacy of the currently used antibiotics by considering the antibiotic stability during incubation time at different inoculum levels.

Inoculum effect of antimicrobial peptides

The activity of many antibiotics depends on the initial density of cells used in bacterial growth inhibition assays. This phenomenon, termed the inoculum effect, can have important consequences for the therapeutic efficacy of the drugs, because bacterial loads vary by several orders of magnitude in clinically relevant infections. Antimicrobial peptides are a promising class of molecules in the fight against drug-resistant bacteria because they act mainly by perturbing the cell membranes rather than by inhibiting intracellular targets. Here, we report a systematic characterization of the inoculum effect for this class of antibacterial compounds. Minimum inhibitory concentration values were measured for 13 peptides (including all-D enantiomers) and peptidomimetics, covering more than seven orders of magnitude in inoculated cell density. In most cases, the inoculum effect was significant for cell densities above the standard inoculum of 5 × 105 cells/mL, while for lower densities the active concentrations remained essentially constant, with values in the micromolar range. In the case of membrane-active peptides, these data can be rationalized by considering a simple model, taking into account peptide–cell association, and hypothesizing that a threshold number of cell-bound peptide molecules is required in order to cause bacterial killing. The observed effect questions the clinical utility of activity and selectivity determinations performed at a fixed, standardized cell density. A routine evaluation of the dependence of the activity of antimicrobial peptides and peptidomimetics on the inoculum should be considered.

Microscopic Analysis of Bacterial Inoculum Effect Using Micropatterned Biochip

Antimicrobial resistance has become a major problem in public health and clinical environments. Against this background, antibiotic susceptibility testing (AST) has become necessary to cure diseases in an appropriate and timely manner as it indicates the necessary concentration of antibiotics. Recently, microfluidic based rapid AST methods using microscopic analysis have been shown to reduce the time needed for the determination of the proper antibiotics. However, owing to the inoculum effect, the accurate measurement of the minimal inhibitory concentration (MIC) is difficult. We tested four standard bacteria: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, against five different antibiotics: piperacillin, cefotaxime, amikacin, levofloxacin, and ampicillin. The results showed that overall, the microfluidic system has a similar inoculum effect compared to the conventional AST method. However, due to the different testing conditions and determination protocols of the growth of the microfluidic based rapid AST, a few results are not identical to the conventional methods using optical density. This result suggests that microfluidic based rapid AST methods require further research on the inoculum effect for practical use in hospitals and can then be used for effective antibiotic prescriptions.

A Test For The Rapid Detection of the Cefazolin Inoculum Effect in Methicillin-Susceptible Staphylococcus aureus

The cefazolin inoculum effect (CzIE) has been associated with therapeutic failures and mortality in invasive methicillin-susceptible Staphylococcus aureus (MSSA) infections. A diagnostic test to detect the CzIE is not currently available. We developed a rapid (∼3 h) CzIE colorimetric test to detect staphylococcal-β-lactamase (BlaZ) activity in supernatants after ampicillin induction. The test was validated using 689 bloodstream MSSA isolates recovered from Latin-America and US. Cefazolin MIC determination at high inoculum (107 CFU/mL) was used as reference standard (cut-off of ≥16 μg/mL). All isolates underwent genome sequencing. A total of 257 (37.3%) MSSA exhibited the CzIE by the reference standard method. The overall sensitivity and specificity of the colorimetric test was 82.5% and 88.9%, respectively. Sensitivity in MSSA isolates harboring type A BlaZ (most efficient enzyme against cefazolin) was 92.7% with a specificity of 87.8%. The performance of the test was lower against type B and C enzymes (sensitivities of 53.3% and 72.3%, respectively). When the reference value was set to ≥32 μg/mL the sensitivity for isolates carrying type A enzymes was 98.2%. Specificity was 100% for MSSA lacking blaZ. The overall negative predictive value ranged from 81.4% to 95.6% in Latin-American countries using published prevalence rates of the CzIE. MSSA from US were genetically diverse, with no distinguishing genomic differences from Latin-American MSSA, distributed among 18 sequence types. A novel test can readily identify most MSSA isolates exhibiting the CzIE, particularly those carrying type A BlaZ. In contrast to the MIC determination using high-inoculum, the rapid test is inexpensive, feasible and easy to perform. After minor validation steps, it could be incorporated into the routine clinical laboratory workflow.