Effect Of Inoculation Research Articles

Challenging T > MIC Using Meropenem vs. Escherichia coli and Pseudomonas aeruginosa.

Objective: For meropenem 40%T > MIC is associated with optimal killing of P. aeruginosa and E. coli. However, it is unknown how the distribution of %T > MIC through a treatment day impacts the antimicrobial effect in vitro. Therefore, we investigated the in vitro antibiotic activity of meropenem, precisely if 40%T > MIC is achieved in one single long period (single dose), 2 × 20% periods (dosing-bid), or 3 × 13.3% (dosing t.i.d.) thereby keeping the overall period of T > MIC constant. Material/Methods: Time kill curves (TKC) with P. aeruginosa-ATCC-27853 and E. coli-ATCC-25922 and five clinical isolates each were implemented over 24 h in CAMHB with concentrations from 0.25×MIC-32×MIC. Periods over and under MIC were simulated by centrifugation steps (discarding supernatant and refilling with fresh CAMHB). Double and triple dosing involved further addition and removal of antibiotic. Complementary growth controls (GC) with and without centrifugation steps were done and the emergence of phenotypical resistance was evaluated (repeated MIC-testing after antibiotic administration). Results: No impact of centrifugation on bacterial growth was seen. TKC with P. aeruginosa showed the best killing in the triple dosage, followed by the double and single dose. In multiple regimens at least a concentration of 4×MIC was needed to achieve a recommended 2-3 log10 killing. Likewise, a reduction of E. coli was best within the three short periods. Contrary to the TKCs with P. aeruginosa we could observe that after the inoculum reached a certain CFU/mL (≥10^8), no further addition of antibiotic could achieve bacterial killing (identified as the inoculum effect). For P. aeruginosa isolates resistance appeared within all regimens, the most pronounced was found in the 40%T > MIC experiments indicating that a single long period might accelerate the emergence of resistance. Contrary, for E. coli no emergence of resistance was found. Conclusion/Outlook: We could show that not solely the %T > MIC is decisive for an efficient bacterial eradication in vitro, but also the distribution of the selected %T > MIC. Thus, dividing the 40%T > MIC in three short periods requested lowers antibiotic concentrations to achieve efficient bacterial killing and reduces the emergence of resistance in P. aeruginosa isolates. The distribution of the %T > MIC did impact the bacterial eradication of susceptible pathogens in vitro and might play an even bigger role in infections with intermediate or resistant pathogens.

English

This study was to explore the beneficial interactive effects of fungal inoculum alone or in conjunction with phosphatic fertilizer on maize (Zea mays L.). We conducted a greenhouse pot experiment in soil which included four treatments such as control, Penecillium oxalicum, Aspergiilus niger, P. oxalicum + A. niger at three different levels of phosphatic fertilizers (0, 100, and 200 mg kg-1). The plant height, fresh weight of root/shoot biomass and dry weight of root/shoot biomass and soil chemical properties were recorded. We evaluated the growth parameters of maize and phosphorus (P) uptake in maize were highest in treatment I3 × F2 (Penecillium+Aspergillus + 200 mg P kg-1) followed by I2 × F2 (Aspergillus + 200 mg P kg-1) over rest of the treatments. The beneficial effect was decreased where alone fertilizer dose was added as compared to those where it was added with the co-inoculation of phosphorus solubilizing fungi (PSF). It was observed that soil pH, total organic carbon and available P significantly changed because of various treatments. Based on these evidences, it could be inferred that increasing the maize growth by fungal culture improves soil P fertility, which is favorable to succeeding crops. Further research is needed to understand the chemistry of co-inoculation of fungal inoculum with phosphatic fertilizer. © 2022 Friends Science Publishers

Inoculum effect of Enterobacterales co-expressing OXA-48 and CTX-M on the susceptibility to ceftazidime/avibactam and meropenem.

The treatment of infections caused by OXA-48/CTX-M-coproducing Enterobacterales may be based on new beta-lactam/beta-lactamase inhibitors, such as ceftazidime/avibactam (CZA), or on high dose of meropenem (MER). However, bacterial density at the infection site may vary widely, and the inoculum effect of such antimicrobial strategies has never been specifically investigated. To determine if CZA or MER susceptibilities are impacted by high inocula of Enterobacterales co-expressing both enzymes: OXA-48 like and CTX-M. Determination of an inoculum effect was performed with a standard inoculum of 108CFU/mL (0.5 McFarland) as recommended by EUCAST guidelines and compared to a twofold increase as well as a tenfold increase (1 McFarland and 5 McFarland respectively). Thirty-nine isolates of ceftazidime-resistant Enterobacterales were included of which 27 (70%) co-expressed OXA-48 + CTX-M-15, 6 (15%) OXA-48 + CTX-M-14, and 6 (15%) OXA-181 + CTX-M-15. The susceptibility to the CZA combination was preserved whatever the inoculum used. Regarding MER, 24 (61.5%) of the isolates were susceptible to MER with the standard inoculum, 19 (48.7%) with a twofold increase, and only 15 (38.5%) with a tenfold increase. We showed that in vitro inoculum effect was observed with meropenem but not with CZA for OXA-48- combined with CTX-M-producing Enterobacterales.

PKPD Modeling of the Inoculum Effect of Acinetobacter baumannii on Polymyxin B in vivo.

The reduction in antimicrobial activity at high bacterial counts is a microbiological phenomenon known as the inoculum effect (IE). In a previous in vitro study, a significant IE was observed for polymyxin B (PMB) against a clinical isolate of Acinetobacter baumannii, and well described by a new pharmacokinetic-pharmacodynamic model. Few in vivo studies have investigated the impact of inoculum size on survival or antibiotic efficacy. Therefore, our objective was to confirm the influence of inoculum size of this A. baumannii clinical isolate on PMB in vivo effect over time. Pharmacokinetics and pharmacodynamics of PMB after a single subcutaneous administration (1, 15 and 40 mg/kg) were studied in a neutropenic murine thigh infection model. The impact of A. baumannii inoculum size (105, 106 and 107 CFU/thigh) on PMB efficacy was also evaluated. In vivo PMB PK was well described by a two-compartment model including saturable absorption from the subcutaneous injection site and linear elimination. The previous in vitro PD model was modified to adequately describe the decrease of PMB efficacy with increased inoculum size in infected mice. The IE was modeled as a decrease of 32% in the in vivo PMB bactericidal effect when the starting inoculum increases from 105 to 107 CFU/thigh. Although not as important as previously characterized in vitro an IE was confirmed in vivo.

Staphylococcus aureus in Non-Cystic Fibrosis Bronchiectasis: Prevalence and Genomic Basis of High Inoculum β-Lactam Resistance.

Rationale: The pathobiology of Staphylococcus aureus in non-cystic fibrosis bronchiectasis (nCFB) is poorly defined. When present at high density or "inoculum," some methicillin-sensitive S. aureus (MSSA) can inefficiently degrade antistaphylococcal β-lactam antibiotics via BlaZ penicillinases (termed the "inoculum effect" [IE]). Given the high burden of organisms in bronchiectatic airways, this is particularly relevant. Objectives: Drawing from a prospectively collected biobank, we sought to understand the prevalence, natural history, potential for transmission, and antibiotic resistance profiles among nCFB-derived MSSA isolates. Methods: All individuals attending a regional consultancy nCFB clinic with sputum collected between 1981 and 2017 were considered, and those with one or more S. aureus-positive cultures composed the cohort. Each individual's most recent biobank isolate was subjected to whole-genome sequencing (including the blaZ gene), antibacterial susceptibility testing, and comparative β-lactam testing at standard (5 × 105 colony-forming unit [cfu]/ml) and high (5 × 107 cfu/ml) inocula to assess for the IE and pronounced IE. Results: Seventy-four (35.4%) of 209 individuals had one or more sputum samples with S. aureus (68 MSSA, 6 methicillin-resistant S. aureus). Those with S. aureus infection were more likely to be female. Among 60 of 74 MSSA isolates subjected to whole-genome sequencing, no evidence of transmission was identified, although specific multilocus sequence typing types were prevalent, including ST-1, ST-15, ST-30, and ST-45. Antibiotic resistance was uncommon, except for macrolides (∼20%). Among the 60 MSSA samples, the prevalence of IE and pronounced IE was observed to be drug specific: meropenem (0% and 0%, respectively), cefepime (3% and 5%, respectively), ceftazidime (8% and 0%, respectively), cloxacillin (12% and 0%, respectively), cefazolin (23% and 0%, respectively), and piperacillin-tazobactam (37% and 17%, respectively). The cefazolin IE was associated with blaZ type A (P < 0.01) and ST-30 (P < 0.01), whereas the piperacillin-tazobactam IE was associated with type C blaZ (P < 0.001) and ST-15 (P < 0.05). Conclusions:S. aureus infection was common, although no evidence of transmission was apparent in our nCFB cohort. Although routine susceptibility testing did not identify significant resistance, inoculum-related resistance was found to be relevant for commonly used nCFB antibiotics, including cefazolin and piperacillin-tazobactam. Given previous associations between IEs and negative patient outcomes, further work is warranted to understand how this phenotype impacts nCFB disease progression.

Droplet Microfluidics for High-Throughput Analysis of Antibiotic Susceptibility in Bacterial Cells and Populations.

ConspectusAntibiotic-resistant bacteria are an increasing concern both in everyday life and specialized environments such as healthcare. As the rate of antibiotic-resistant infections rises, so do complications to health and the risk of disability and death. Urgent action is required regarding the discovery of new antibiotics and rapid diagnosis of the resistance profile of an infectious pathogen as well as a better understanding of population and single-cell distribution of the resistance level. High-throughput screening is the major affordance of droplet microfluidics. Droplet screens can be exploited both to look for combinations of drugs that could stop an infection of multidrug-resistant bacteria and to search for the source of resistance via directed-evolution experiments or the analysis of various responses to a drug by genetically identical bacteria. In droplet techniques that have been used in this way for over a decade, aqueous droplets containing antibiotics and bacteria are manipulated both within and outside of the microfluidic devices. The diagnostics problem was approached by producing a series of microfluidic systems with integrated dilution modules for automated preparation of antibiotic concentration gradients, achieving the speed that allowed for high-throughput combinatorial assays. We developed a method for automated emulsification of a series of samples that facilitated measuring the resistance levels of thousands of individual cells encapsulated in droplets and quantifying the inoculum effect, the dependence of resistance level on bacterial cell count. Screening of single cells encapsulated in droplets with varying antibiotic contents has revealed a distribution of resistance levels within populations of clonally identical cells. To be able to screen bacteria from clinical samples, a study of fluorescent dyes in droplets determined that a derivative of a popular viability marker is more suitable for droplet assays. We have developed a detection system that analyzes the growth or death state of bacteria with antibiotics for thousands of droplets per second by measuring the scattering of light hitting the droplets without labeling the cells or droplets. The droplet-based microchemostats enabled long-term evolution of resistance experiments, which will be integrated with high-throughput single-cell assays to better understand the mechanism of resistance acquisition and loss. These techniques underlie automated combinatorial screens of antibiotic resistance in single cells from clinical samples. We hope that this Account will inspire new droplet-based research on the antibiotic susceptibility of bacteria.

Anaerobic co-digestion of cheese whey and septage: Effect of substrate and inoculum on biogas production

Cheese whey is an industrial waste generated from the cheese processing unit of the dairy industry and requires treatment before its disposal. The present study investigated the possibilities of improving the digestibility from anaerobic digestion of lipid rich dairy by-product, cheese whey using septage as the co-substrate with different inoculum. Biochemical methane potential assays were conducted under mesophilic temperature conditions and results were validated using Modified Gompertz Model. Two sets of BMP tests were done; to assess the individual and combined digestion abilities of septage in anaerobic co-digestion of whey and to assess the ability of 3 inoculum sources (cattle manure, sewage sludge, and acclimatized anaerobic sludge) in the co-digestion process. The results indicated that septage is an excellent co-substrate that has better adaptability with cheese whey and the optimum mix ratio was found as 40:60 (SCW: SSP). BMP tests were also conducted with inoculum at S/I ratio of 1 and statistical analysis was performed to study the synergistic effect of both co-digestion and inoculum. The tests revealed that the cattle manure resulted in the highest biogas production (342.22mL/gVS) at 60% whey fraction. Modified Gompertz model fitted the experimental data well and identified an increase in lag phase times when whey fraction is increased. Comparatively higher lag phase times ranging from 1.98 to 4.35 days were obtained for sewage sludge inoculated samples. The maximum methane production (Pmax) was obtained at 60% whey fraction (369.63 ± 4.05mL/gVS) at a very short lag time of 0.76 ± 0.17days for cattle manure inoculated mixture.

The effect of pH, temperature, and inoculum on the fermentation of pulp and paper biosludge: increasing the nutrient availability for rearing of black soldier fly larvae

Pulp and paper production is one of the largest global industries producing annually 400 million metric tons of pulp and paper products and 6 million tons of pulp and paper biosludge (PPBS). From a resource efficiency and sustainability perspective, there is a need for improving PPBS management. This study assessed fermentation of PPBS as pretreatment to improve PPBS feasibility as feed for black soldier fly larvae. The impact of temperature, pH, and inoculum on the concentration of soluble chemical oxygen demand (sCOD) and volatile fatty acids (VFA) was assessed. An initial pH of 10 and the addition of inoculum from an anaerobic digester substantially increased the concentration of sCOD. The obtained concentration of VFA was low compared to the VFA concentration needed to improve the growth of Black Soldier Fly Larvae (BSFL). The PPBS is recalcitrant to fermentation because of the high content of lignocellulose. Fermentation as done in this study does not convert PPBS to a feasible feed for black soldier fly larvae; thus, further research on improved fermentation is needed. However, fermentation at alkaline pH and addition of inoculum do increase the final pH of PPBS which improves its feasibility as feed for BSFL. Future studies should explore pH > 10 and temperatures > 55 °C to increase sCOD and improving generation of VFA by removal of inhibiting substances, testing other types of inoculum (rumen microorganisms) and co-fermentation.

Application of Semi-Mechanistic Pharmacokinetic and Pharmacodynamic Model in Antimicrobial Resistance.

Antimicrobial resistance is a major public health issue. The pharmacokinetic/pharmacodynamic (PK/PD) model is an essential tool to optimize dosage regimens and alleviate the emergence of resistance. The semi-mechanistic PK/PD model is a mathematical quantitative tool to capture the relationship between dose, exposure, and response, in terms of the mechanism. Understanding the different resistant mechanisms of bacteria to various antibacterials and presenting this as mathematical equations, the semi-mechanistic PK/PD model can capture and simulate the progress of bacterial growth and the variation in susceptibility. In this review, we outline the bacterial growth model and antibacterial effect model, including different resistant mechanisms, such as persisting resistance, adaptive resistance, and pre-existing resistance, of antibacterials against bacteria. The application of the semi-mechanistic PK/PD model, such as the determination of PK/PD breakpoints, combination therapy, and dosage optimization, are also summarized. Additionally, it is important to integrate the PD effect, such as the inoculum effect and host response, in order to develop a comprehensive mechanism model. In conclusion, with the semi-mechanistic PK/PD model, the dosage regimen can be reasonably determined, which can suppress bacterial growth and resistance development.

Effects of Microbial Inoculum on Microbial Community and Enzyme Activity Involved in Nitrogen-Sulfur Metabolism During Sewage Sludge Composting

Effects of Microbial Inoculum on Microbial Community and Enzyme Activity Involved in Nitrogen-Sulfur Metabolism During Sewage Sludge Composting

Effect of Homemade Compound Microbial Inoculum on the Reduction of Terramycin and Antibiotic Resistance Genes in Terramycin Mycelial Dreg Aerobic Composting and its Mechanism

Effect of Homemade Compound Microbial Inoculum on the Reduction of Terramycin and Antibiotic Resistance Genes in Terramycin Mycelial Dreg Aerobic Composting and its Mechanism

The Effect of Tempe Yeast Inoculum Amount on the Quantity and Quality of Cocos nucifera viridis Virgin Coconut Oil (VCO)

This study aims to determine the effect of tempeh yeast inoculum on the color, odor, and yield in the manufacture of Virgin Coconut Oil (VCO) from Cocos nucifera viridis. This research was conducted in November 2020 in Brebes, Magelang, and Lampung. This research employed the descriptive quantitative method. The population of this experiment was coconut fruit found in Lampung, Brebes, and Magelang. The sample used was green coconut (Cocos nucifera viridis) found in Lampung, Brebes, and Magelang. The working procedure of this research consisted of fermenting the sample and observing the oil produced by Virgin Coconut Oil. This research's data collection techniques were observation, documentation, and products calculation. Based on the observations, the researchers found the highest yield in the experiment of 80 ml cream with an inoculum of 8 grams (30.4%), followed by a volume of 80 ml of cream with a 6-gram inoculum (28.4%). The smallest yield was formed at a volume of 80 ml cream with inoculum 4 grams (14.6%). The VCO obtained in this research had a distinctive aroma of coconut oil and was clear in color. Therefore, the amount of tempeh yeast inoculum affects the color, odor, and yield in the manufacture of Virgin Coconut Oil (VCO) from Cocos nucifera viridis.

Kinetics of In Vitro Gas Production and Fitting Mathematical Models of Corn Silage

This study aimed at examining the effects of rumen inoculum of steers receiving different combinations of ionophore and probiotics in their diets on in vitro gas production of corn silage. The fitting of gas production was performed with five mathematical models and its kinetics was evaluated. Four crossbred steers (403.0 ± 75.5 kg body weight) with ruminal cannula were assigned to a 4 × 4 Latin square design. The additives used were Monensin sodium (Rumensin® 100, 3 g/day), Bacillus toyonensis (Micro-Cell Platinum® 109, 1 g/day) and Saccharomyces cerevisiae boulardii (ProTernative®20, 0.5 g/day). Additives were arranged into the following treatments, supplied daily into total mixed diet: (1) Monensin; (2) Monensin + B. toyonensis; (3) Monensin + S. boulardii; and (4) B. toyonensis + S. boulardii. The gas production data were fitted into the models of Gompertz, Groot, Ørskov, Brody, Richards, and Dual-pool Logistic. A perfect agreement between observed and predicted values in curves of accumulated in vitro gas production was observed in the Groot and Richards models, with higher coefficient of determination (R2 = 0.770 and 0.771, respectively), concordance correlation coefficient (CCC = 0.871 and 0.870, respectively), and root mean square error of prediction (RMSEP = 1.14 and 1.15, respectively). Evaluating the feed additives throughout the Groot model, the B. toyonensis + S. boulardii treatment presented higher VF (12.08 mL/100 mg of DM; p = 0.0022) than Monensin and Monensin + S. boulardii (9.16 and 9.22 mL/100 mg of DM, respectively). In addition, the fractional rate of gas production (k) was higher (p = 0.0193) in B. toyonensis + S. boulardii than in Monensin, not presenting a statistical difference (p &gt; 0.05) from the other two treatments. Additionally, with the time of beginning to gas production, the lag time (λ), was greater (p &lt; 0.001) with Monensin and Monensin + B. toyonensis than with Monensin + S. boulardii and B. toyonensis + S. boulardii. The combination of Monensin and probiotics (B. toyonensis + S. boulardii) resulted in better kinetics of degradation of corn silage, being that the Groot and Richards models had the best fit for estimates of the in vitro gas production data of corn silage tested with different feed additive combinations.

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.

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

Abstract 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 ( &amp;gt;90%), accuracy ( &amp;gt;80%) and ROC-AUC values ( &amp;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

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

BackgroundThe 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). MethodsA 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).ResultsAmong 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. ConclusionSpecific 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)

A New Pharmacokinetic-Pharmacodynamic 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. Given that 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 pharmacokinetic-pharmacodynamic (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 105 to 108 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 modeled as a sigmoidal Emax model and the inoculum effect as an increase of polymyxin B EC50 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 EC50 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.

The effect of inoculum size on the activity of different ratios of Ceftriaxone – Sulbactam [(1:1) vs. (2:1)] against ESBL producing organisms

Background and Objective: Antibiotic resistance has become the most importunate health issue and remains a major public health concern worldwide. Production of extended spectrum β-lactamases (ESBL) is one of the major causes of resistance to β-lactam antibiotics. Several in-vitro studies have demonstrated the significant impact of inoculum size on the effectiveness of β-lactam-β-lactamase inhibitor combination. Higher concentration of β-lactamase inhibitor (Sulbactam) will be needed to combat the increased production of ESBLs in infections with high inoculum. The primary objective of the present study is to evaluate Minimum Inhibitory Concentration (MICs) of ESBL producing Enterobacteriaceae, against Ceftriaxone alone and Ceftriaxone with Sulbactam combination (1:1 and 2:1) in presence of normal and higher inoculum of these bacteria. The secondary objective of the study is to demonstrate the in-vitro efficacy of Ceftriaxone-Sulbactam (CS) in presence of higher inoculum of bacteria. Method: ESBL producing strains of Enterobacteriaceae (E. coli, K pneumoniae 30 each) were isolated from patient samples. MIC of Ceftriaxone and Sulbactam alone and in combination (1:1 and 2:1) was determined in presence of both normal (105CFU/ml) and higher inoculum (107CFU/ml) of these bacteria using micro broth dilution method of antimicrobial susceptibility testing. Resultsand Conclusion: The study finds an inoculum effect with Ceftriaxone. After addition of Sulbactam, MIC value for Ceftriaxone was reduced by 4-128 folds and 2-32 folds in ESBL producing strains of E. coli and K. pneumoniae respectively. The findings of the present study demonstrated that CS at 1:1 ratio has better in-vitro activity against ESBL producing strains of E. coli and K. pneumoniae than at a 2:1 ratio in both normal bacterial inoculum and higher bacterial inoculum. This is suggestive of the need for the 1:1 ratio combination of Ceftriaxone-Sulbactam in management of severe infections caused by ESBL producing organisms.

Comparing the in vitro digestibility of leaves from tropical trees when using the rumen liquor from cattle, sheep or goats

This study compared the effect of ruminal inoculum from different donor species (goat vs. cattle and sheep vs. cattle) on the in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD) of leaves from tropical plant species. Comparisons were performed with and without the addition of N and polyethylene glycol (PEG). Leaves from six tropical trees with different condensed tannin content were used (Caesalpinia gaumeri, Cordia alliodora, Gymnopodium floribundum, Neomillspaughia emarginata, Piscidia piscipula and Senegalia gaumeri). Donors of ruminal liquor included a ruminal cannulated adult cow (500 kg live weight [LW]), two growing Criollo goats (22 ± 3 kg LW, study one) and two growing Pelibuey lambs (24.8 ± 0.28 LW, study two). Donors were maintained in individual pens and received a basal diet with concentrate feed and Pennisetum purpureum grass. The IVDMD and IVOMD using sheep or goat vs. cow rumen liquor were determined from dry samples of a commercial concentrate feed, P. purpureum grass, and the leaves from experimental plants with a 2 × 2 factorial arrangement: with or without N-enriched media (+N and -N), and with or without polyethylene glycol (+PEG and -PEG). Respective standard linear regressions and orthogonal regressions were used to compare equivalence of IVDMD and IVOMD results between ruminal inoculum from different donor species (cattle vs. goat in study one and cattle vs. sheep in study two). The standard linear regressions suggested strong associations between the ruminal inocula compared, in both IVDMD and IVOMD with the four treatments (R2 > 87 % for study one and R2 > 89 % for study two, P < 0.001). However, orthogonal regressions showed differences between inocula of cattle and goats on IVDMD, when using + PEG media, and on IVDMD and IVOMD when using + N media (P < 0.05). Differences between inocula of cattle and sheep were only evident in the absence of PEG (P < 0.05). The standard linear and orthogonal regressions suggested that the IVDMD and IVOMD of leaves from tropical trees obtained with sheep or goats’ ruminal liquors, within the evaluated range of digestibilities (c. 0.2 to 0.65), can be predicted from the IVDMD and IVOMD obtained with the cattle ruminal liquor. However, when evaluating digestibilities of leaves from tropical trees it is important to evaluate the specific incubation conditions using the donor species of interest to improve accuracy of results.

Malus Antioxidant Metabolism Following Bacterial–Fungal Inoculation in Organic Farming: From Root to Fruit

This study investigated the antioxidant status of roots, leaves, and fruit upon microbial inoculation (AMF+PGPR, arbuscular mycorrhizal fungi, and plant growth-promoting rhizobacteria, respectively) of young organically farmed apple trees over two growing seasons. Three cultivars—‘Topaz’, ‘Chopin’, and ‘Odra’—were selected to test the relationship between inoculation and enzymatic and non-enzymatic antioxidant components. The antioxidant metabolism was highly dependent on tissue type and growing season. The greatest effect on antioxidant status following application of the inoculum was found in roots, then leaves, but it was almost negligible in fruit. Roots were influenced most by application of the inoculum in the first growing season, while leaves were influenced most in the second season. Considerable differences between the inoculated and control plants were found for root glutathione reductase (GR) and catalase (CAT) activity, as well as glutathione and ascorbate contents; root phenolics were not influenced by inoculation. In the case of leaves, effect of microbial inoculation on GR activity was revealed in the first growing season, while for global phenolics in the second season, and only the concentration of glutathione was significantly higher in the leaves of inoculated trees in both growing seasons. Leaf ascorbate content and CAT activity were not influenced by the microbial inoculation. The control and inoculated trees expressed a similar total antioxidant capacity, irrespective of the tissue type tested. Furthermore, the response of the cultivars to inoculation varied and also changed in consecutive growing seasons. Based on this study, it is likely that the effect of microbial inoculum as a tool for enhancing health-promoting properties in the fruit of perennial plants is weaker than that described for vegetables where different plant organs are edible.