High Inoculum Research Articles

1186. Cefazolin inoculum effect predicts reduced susceptibility to other antibiotics and patient outcomes in MSSA endovascular infections

BackgroundMSSA Infective endocarditis (IE) is inherently a high-burden infection with up to a 30% mortality rate. Cefazolin is an appealing treatment option for IE with low toxicity and a favorable dosing scheme. However, cefazolin has been associated with treatment failure in IE, attributed to an inoculum effect. The specific mechanism underlying the cefazolin inoculum effect (CIE) remains undetermined, but CIE has been linked to both blaZ expression and agr dysfunction. This study aims to determine whether CIE is linked to reduced susceptibility to other antibiotics and worse outcomes regardless of therapy in MSSA endovascular infections.MethodsSixty-four MSSA strains were collected from patients with endovascular infections not treated with cefazolin. To determine CIE phenotype, strains were cultured and MICs assayed for cefazolin, nafcillin, and vancomycin at 107 CFU/mL for high-inocula (HI) and 105 CFU/mL for standard-inocula (SI). This study defined CIE as a ≥ 4-fold increase in MIC at HI compared to SI, with at least an MIC of 4 mg/L at HI. Nitrocefin disks identified blaZ expression, and beta lysin disks were used to determine hemolysin type and agr function. Patient outcomes of mortality and bacteremia duration were assessed across cohorts.ResultsTwenty-four strains exhibit a CIE (38%), with 10 strains having an MIC of ≥ 32mg/L at HI. Nafcillin and vancomycin also had an inoculum effect, uncoupled from the CIE and occurring at a lower frequency and amplitude at HI. Presence of CIE had a greater association with blaZ expression (71% vs 25%) than agr dysfunction (38% vs 20%). 50% (9/18) of CIE infections were cleared within 48 hours while 77% (20/26) of CIE-negative infections were cleared within 48 hours (P=0.106). However, presence of CIE was not associated with increased mortality (25% CIE-positive vs 35%; P=0.578)ConclusionPrevious studies for CIE failed to enrich for isolates from endovascular sources, where inocula are known to be high. This study presents one of the largest endovascular source cohorts for CIE evaluation. It identifies that CIE prevalence (38%) is higher than reports from diverse infection sources (10-36%). CIE appears to predict bacteremia duration with other MSSA treatment options, suggesting mechanisms independent of blaZ and agr function for this phenomenon.Disclosures Warren Rose, PharmD, MPH, Merck (Grant/Research Support)Paratek (Grant/Research Support)

28. Regulation of the Staphylococcal β-lactamase Plays a Major Role in the cefazolin Inoculum Effect

BackgroundCefazolin (Cz) is commonly used to treat methicillin-sensitive Staphylococcus aureus (MSSA) bacteremia. Yet, some MSSA isolates producing the staphylococcal β-lactamase (BlaZ) exhibit the Cz inoculum effect (CzIE), defined as an increase in the minimum inhibitory concentration (MIC) to ≥ 16 µg/mL at high inoculum (107 CFU/mL, HI-MIC). Retrospective clinical data linked the CzIE to increased 30-day mortality and Cz treatment failure in patients with MSSA bacteremia, yet the mechanistic bases of this phenomenon are unknown. We aimed to explore the contribution of blaZ regulation, via BlaR (antibiotic sensor) and BlaI (transcriptional repressor) (Fig 1) to the CzIE by i) in trans expression assays and ii) analysis of their sequences in a set of isolatesFigure 1. Structure of the Staphylococcal bla OperonMethodsThe blaZ genes (with putative promoters) of strains exhibiting and lacking the CzIE (TX0117 and ATCC29213, respectively) were expressed in trans in RN4220 (blaZ neg) using the promotor-less vector pWM401 (Figure 2). We subsequently cloned the blaR and blaI genes of each TX0117 and ATCC29213 upstream of each blaZ allele (Figure 3). The presence of the CzIE was assessed in transformants using broth microdilution at standard (105 CFU/mL, SI-MIC) and high inoculum. We also performed whole-genome sequencing (WGS) in 104 MSSA isolates exhibiting and lacking the CzIE to compare the sequences of BlaZ, BlaR, and BlaI and classified them by allotypes (unique amino acid sequences) using ATCC29213 as reference. Figure 2. In trans expression of blaZ genes from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220 Figure 3. In trans expression of the bla Operons from a CzIE+ strain (TX0117) and a CzIE- strain (ATCC29213) in RN4220ResultsExpression of blaZTX0117 and blaZATCC29213 with their native promoters in RN4220 resulted in the CzIE with Cz HI-MICs ≥ 64 µg/mL regardless of the origin of the allele (Table 1). Inclusion of the regulatory elements blaR and blaI from TX0117 (CzIE+) did not change the phenotype. In contrast, addition of blaR and blaI from ATCC29213 (CzIE-) led to a marked decrease in the Cz HI-MIC (Table 1). Sequence analyses of 104 MSSA isolates revealed 10, 17 and 6 BlaZ, BlaR and BlaI allotypes, respectively (Table 2). BlaZ-2 and BlaR-4 were linked to the CzIE in 90% of isolates. Table 1. MIC values of transformans after In trans expression of blaZ genes and bla Operons from a S. aureus CzIE+ strain(TX0117) and a CzIE- strain (ATCC29213) in RN4220 Table 2. BlaZ allotypes of 104 Staphylococcus aureus isolates and their association with the CzIEConclusionOur results suggest that overexpression of blaZ can lead to the CzIE in any MSSA strain. Thus, the regulation of blaZ expression via BlaR and BlaI seem to play a major role in the CzIE. Identification of specific BlaR and BlaI allotypes could predict the presence of the CzIE. Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Scientific Research Study Investigator)MeMed (Scientific Research Study Investigator)Merck (Grant/Research Support)

251. High prevalence of the cefazolin inoculum effect in methicillin-susceptible Staphylococcus aureus causing bloodstream infections in a hospital network in Houston, TX

BackgroundAnti-staphylococcal β-lactams, such as anti-staphylococcal penicillins (AsPen) or cefazolin are the drugs of choice for methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections. However, cefazolin has seen increasing use due to its better tolerance, lower cost, ease of administration and possibly better outcomes when compared to AsPen. Nevertheless, its efficacy may be compromised by the cefazolin inoculum effect (CzIE), defined as an increase in the minimum inhibitory concentration (MIC) of cefazolin to ≥16mg/L when a high inoculum (5x107 CFU/ml) is present. Previous studies have suggested that the prevalence of the CzIE varies geographically, with high prevalence in some Latin American countries. Prospective data evaluating the presence of the CzIE in deep-seated MSSA infections across the United States are lacking.MethodsWe performed a prospective observational study of MSSA bacteremia in a network of 13 hospitals in Houston, TX. Patients ≥ 18 years old, with a positive blood culture with MSSA, with at least one follow-up blood culture confirming clearance of the bacteremia, who received cefazolin or nafcillin as definitive therapy (72 hours or longer after culture results known) and whose original isolate was available for evaluation of the CzIE, were included. Patients with polymicrobial BSI, or those who received another antibiotic with activity against MSSA in the definitive therapy period were excluded. Cefazolin MICs were determined by broth microdilution at standard and high inoculum.ResultsWe report the results of 50 patients enrolled from February 15, 2020-April 30, 2020. The baseline characteristics of each group are outlined in Table 1. A total of 37/50 (74%) received cefazolin as definitive therapy, and complicated bacteremia was seen in 27/50 (54%). A total of 16/50 (32%) of the MSSA isolates exhibited the CzIE. Two patients in our cohort died: both of whose isolates exhibited the CzIE and received cefazolin as definitive therapy. ConclusionWe report a high prevalence of the CzIE in MSSA BSIs in a major US urban hospital network. Further evaluation of the clinical implications of the CzIE is urgently needed.Disclosures William R. Miller, MD, Entasis Therapeutics (Scientific Research Study Investigator)Merck (Grant/Research Support)Shionogi (Advisor or Review Panel member) Cesar A. Arias, MD, MSc, PhD, FIDSA, Entasis Therapeutics (Scientific Research Study Investigator) MeMed (Scientific Research Study Investigator) Merck (Grant/Research Support)

1325. Things that go Bump in the Night: Combating Klebsiella pneumoniae co-producing New Delhi metallo-beta-lactamase (NDM) and Mobile Colistin Resistance (MCR)

BackgroundThe scourge of MBLs among Gram negatives, such New Delhi Metallo-beta-lactamase-producing Klebsiella pneumoniae, has resulted in an overwhelming need for new treatmens. Worryingly, additional acquisition of plasmid-mediated polymyxin resistance through the mcr gene can produce strains resistant to all last line agents. The novel combination of aztreonam (ATM, which is not an MBL substrate) with avibactam (AVI, to inhibit extended spectrum beta-lactamases that inactivate ATM) has been proposed to restore ATM activity.Methods K. pneumoniae SZ04 harboring blaNDM-5, blaCTX-M-55, and mcr-1 (MICATM = 128 mg/L, MICPolymyxin B = 4 mg/L, MICAmikacin = 1 mg/L, MICceftazidime/avibactam > 16/4 mg/L) was studied at two initial inoculum (106 and 108 cfu/mL) over 24h in static time kills (SCTK). Concentration arrays of 2-, 3-, and 4-drug combinations of low- and package insert-dose polymyxin B (PMB) ± low- and package insert-dose amikacin (AMI) ± package insert dosing of ATM/AVI were simulated in > 200 individual arms of SCTK. Data were summarized using the Log Ratio Area (LRA) which is calculated by integrating the area under the bacterial killing curve, normalizing to the growth control, then log-transforming. A Hill-type function was fit to the data in order to determine the maximum effect (Emax) and drug concentration for 50% effect (EC50).ResultsWhen simulating the maximum free concentration of amikacin 15 mg/kg (52 mg/L) in combination with package insert concentrations of ATM/AVI, there was a marked reduction of 3.22 in the LRA compared to the growth control for the 108 cfu/mL starting inocula. Combining ATM with low amikacin (0.813 mg/L) and polymyxin B (0.125 mg/L) resulted in a reduction in LRA of 4.32 at 106 cfu/mL. Model fitting results showed a statistically significant difference in EC50 to amikacin between low and high inocula at 1.24 and 18.1 mg/L, respectively. Combination of AMI with low-concentration PMB (0.125 mg/L) resulted in an increase in the Emax of amikacin to -5.68.ConclusionThe use of ATM/AVI combinations is a promising option against MBL and MCR co-producing K. pneumoniae. Low-dose strategies of polymyxin or amikacin dosing in combination with ATM/AVI is merits further testing for future translation to the clinical setting to improve efficacy and optimize treatment.DisclosuresThomas Lodise, PharmD, PhD, Paratek Pharmaceuticals, Inc. (Consultant) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

1263. Assessment of Cefepime (FEP)-Taniborbactam (TAN) Human Exposures to Suppress the Emergence of Resistance among Serine (SBL)- and Metallo-β-Lactamase (MBL)-Producing Gram-Negative Bacteria (GNB) in a Hollow Fiber Infection Model (HFIM)

BackgroundFEP-TAN (FTB) efficacy and safety are currently being evaluated in a Phase 3 trial (NCT03840148). TAN, a boronic acid-based β-lactamase inhibitor, restores susceptibility to FEP when resistance is driven by SBL or MBL (ie, NDM, VIM). This in vitro study assessed whether clinical FTB exposures suppress treatment-emergent resistance in pathogenic Enterobacterales and Pseudomonas aeruginosa.MethodsBioreactors (C2011, FiberCell) were inoculated with clinical GNB strains (N=6) using highly concentrated log phase cultures (> 108 CFU). Syringe pumps supplied humanized exposures of FEP (2 g), FTB (2 g/0.5 g), ceftazidime-avibactam (CZA, 2 g/0.5 g), each as 2 h infusions q8h, or meropenem-vaborbactam (MEV, 2 g/2 g q8h, 3 h infusion) for 7 days. Exposures were confirmed by UPLC-MS/MS for all agents. Subpopulations with elevated FTB MICs (4x) were monitored with drug-supplemented agar. CZA or MEV served as positive or negative controls for selected strains. Samples, serially removed from bioreactors, were saline-washed prior to quantitative culture to prevent drug carryover.ResultsAll strains grew rapidly in the presence of FEP (Figure 1), consistent with resistance by broth microdilution (BMD, Table 1). With the addition of TAN, there was extensive killing of the total bacterial populations by FTB, and subpopulations with elevated FTB MICs were never recovered. Like FTB against Klebsiella pneumoniae (KP) BAA-1705, CZA initially decreased the inoculum to the lower limit of detection, but unlike FTB, allowed regrowth to 3.7 log10 CFU/mL by day 7. The first dose of FTB was bactericidal against VIM+ and NDM+ KP strains while regrowth occurred prior to 8 h of MEV and CZA challenge, respectively. Notably, early failure of MEV is discordant with susceptibility by BMD (MIC= 4 µg/mL). By day 7, FTB sterilized an OXA-48+ KP strain that when challenged by MEV, grew to 9.8 log10 CFU/ml at 24 h.Figure 1. Bacterial burdens observed in the HFIM when treated with FEP alone or FEP+TAN (FTB) Table 1. Characterization of strains assessed in the HFIM; minimum inhibitory concentration (MIC) values for treatments not assessed are included in parentheses. ConclusionIn a 7-day HFIM with humanized exposures and high initial inoculums, FTB provided sustained bactericidal activity against multidrug-resistant Enterobacterales and P. aeruginosa strains harboring a diversity of β-lactamases and suppressed growth of resistant subpopulations. These data are crucial to inform understanding of the potential role for FTB in GNB infections and future clinical studies.DisclosuresLindsay M. Avery, PharmD, Venatorx Pharmaceuticals (Employee, Shareholder) Salvador Vernacchio, BS, Venatorx Pharmaceuticals (Employee, Shareholder) Lisa McLaughlin, BS, Venatorx Pharmaceuticals (Employee, Shareholder) Luigi Xerri, PhD, Venatorx Pharmaceuticals (Employee, Shareholder) Greg Moeck, PhD, Venatorx Pharmaceuticals (Employee, Shareholder) Daniel Pevear, PhD, Venatorx Pharmaceuticals (Employee, Shareholder)

Disentangling the interactive effects of climate change and Phytophthora cinnamomi on coexisting Mediterranean tree species

The structure and function of Mediterranean mixed oak forests are seriously threatened by climate change and the exotic pathogen Phytophthora cinnamomi. Both factors have been individually studied in some detail, but we have a poor understanding of their interactive effects on plant performance and species coexistence. We conducted a greenhouse experiment where seedlings of three coexisting species with contrasting susceptibility to the pathogen (Quercus suber, Quercus canariensis and Olea europaea var. sylvestris) were planted in pairs under different scenarios of drought (control vs. 30% rainfall reduction), warming (control vs. 3°C increase) and pathogen presence (control vs. soil infested with P. cinnamomi). At the end of the experiment, we assessed the severity of root symptoms and plant morphological traits (shoot and root biomass, root tissue density and specific root length). Results showed that seedlings of the three species, including O. europaea and Q. canariensis (species considered as resistant and tolerant to the pathogen, respectively), showed in general significantly higher root symptoms and lower shoot and root biomass than non-inoculated seedlings, independently of the drought, warming and plant treatments. We found, however, some interactive effects of the climatic treatments and P. cinnamomi on root traits of the two Quercus species. Variations in the specific root length (SRL) and root tissue mass density (RTD) of Quercus spp. suggest a compensation of the negative effects of both stressors on seedling performance. Overall, our results suggest that the warming and drought predicted for Mediterranean forests during spring might not have a relevant impact on the infectivity ability of P. cinnamomi, supporting its great adaptability to these changing climatic conditions. Therefore, in Mediterranean forests infected by P. cinnamomi, it becomes crucial to avoid the spread and multiplication of the pathogen during spring, since its destructive ability at high inoculum levels is largely independent of environmental conditions.

Identification of the gamma irradiation dose applied to ground beef that reduces Shiga toxin producing Escherichia coli but has no impact on consumer acceptance

The aims of the present study were: a) to estimate the minimal dose of gamma irradiation required to reduce 5 log CFU/g of native O157 and non-O157 Shiga toxin-producing Escherichia coli population in ground beef samples inoculated with high inoculum; b) to assess its effectiveness in samples with low inoculum and 3) to evaluate consumer acceptance. Based on the results, 1 kGy was estimated as the minimal dose of gamma irradiation required to reduce 5 log CFU/g of STEC in ground beef. However, when samples with low inoculum level were subjected to 1 kGy, 3.9% of the samples were positive for stx and eae genes after an enrichment step. Consumer acceptance analysis was carried out with samples subjected to 2.5 kGy and no significant differences were found between irradiated and control samples. Therefore, 2.5 kGy was identified as the gama irradiation dose that reduces STEC but has no impact on consumer acceptance of ground beef.

In Vitro Activities and Inoculum Effects of Ceftazidime-Avibactam and Aztreonam-Avibactam against Carbapenem-Resistant Enterobacterales Isolates from South Korea

Ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (AZT-AVI) are novel antibiotic combinations active against multidrug-resistant Gram-negative pathogens. This study aimed to evaluate their in vitro activities and inoculum effects in carbapenem-resistant Enterobacterales (CRE), including carbapenemase-producing (CP)-CRE and non-CP-CRE. A total of 81 independent clinical isolates of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae were collected. CAZ-AVI and AZT-AVI minimal inhibitory concentrations (MICs) were evaluated by broth microdilution using standard and high inocula. The inoculum effect was defined as an ≥8-fold increase in MIC with high inoculum. Phenotypic determination of β-lactam resistance mechanism and PCR for carbapenemase genes were performed. Of the 81 CRE isolates, 35 (43%) were CP-CRE. Overall, 73% of the isolates were susceptible to CAZ-AVI, and 95% had low AZT-AVI MICs (≤8 µg/mL). The MIC50/MIC90s of CAZ-AVI and AZT-AVI were 4/≥512 µg/mL and 0.5/4 µg/mL, respectively. CAZ-AVI was more active against non-CP-CRE than against CP-CRE (susceptibility 80% vs. 63%, p = 0.08; MIC50/MIC90, 2/16 μg/mL vs. 4/≥512 μg/mL), whereas AZT-AVI was more active against CP-CRE (MIC50/MIC90, 0.25/1 μg/mL vs. 0.5/8 μg/mL). All four isolates with high AZT-AVI MIC (≥16 μg/mL) were resistant to CAZ-AVI, but only 18% (4/22) of CAZ-AVI-resistant isolates had high AZT-AVI MIC. The rates of the inoculum effect for CAZ-AVI and AZT-AVI were 18% and 47%, respectively (p < 0.001). Interestingly, the frequency of the AZT-AVI inoculum effect was higher in K. pneumoniae than E. coli (64% vs. 8%, p < 0.001). AZT-AVI is more active against CRE than CAZ-AVI, even in CP-CRE and CAZ-AVI-resistant isolates. The presence of a substantial inoculum effect may contribute to clinical failure in high-inoculum infections treated with AZT-AVI.

Synthesis and antibacterial activities of quaternary ammonium salts with different alkyl chain lengths grafted on polyvinyl alcohol-formaldehyde sponges

Antibacterial polyvinyl alcohol-formaldehyde (PVF) sponges containing quaternary ammonium salts (QA) with different alkyl chain lengths are obtained through the grafting reaction of QA onto the PVF network under alkaline conditions. The grafting percentages of obtained PVF-g-QA sponges are in the range of 1.1–1.6%. As-prepared PVF-g-QA sponges show average pore sizes ranging from 60 μm to 90 μm with high porosity and interconnected pore structure. PVF-g-QA sponges with different alkyl chain lengths display the absorption capacity (Qs) of 13.3–14.2 g·g−1 in deionized water, 13.9–14.8 g·g−1 in saline solution, and 13.9–14.8 g·g−1 in artificial blood and can attain absorption equilibrium within 1 min. Notably, the PVF-g-QA sponges demonstrate excellent antibacterial effects on both S. aureus and E. coli bacteria strains. With high initial inoculum concentration (1 × 108 CFU/mL), with increasing the alkyl chain length of QA, the antibacterial activities of PVF-g-QA sponges against E. coli increase from 20.47% to 99.98% firstly and then decrease to 92.91%, the antibacterial activities against S. aureus increase from 16.67% to 99.96% and then decrease to 93.21%. None of the samples have cytotoxicity for cells. The as-prepared PVF-g-QA sponges with remarkable absorption capacity and excellent antibacterial performance can be considered as a new kind of antibacterial functional material.

Impact of antibiotic timing on mortality from Gram-negative bacteraemia in an English district general hospital: the importance of getting it right every time.

There is limited evidence that empirical antimicrobials affect patient-oriented outcomes in Gram-negative bacteraemia. We aimed to establish the impact of effective antibiotics at four consecutive timepoints on 30 day all-cause mortality and length of stay in hospital. We performed a multivariable survival analysis on 789 patients with Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa bacteraemias. Antibiotic choices at the time of the blood culture (BC), the time of medical clerking and 24 and 48 h post-BC were reviewed. Patients that received ineffective empirical antibiotics at the time of the BC had higher risk of mortality before 30 days (HR = 1.68, 95% CI = 1.19-2.38, P = 0.004). Mortality was higher if an ineffective antimicrobial was continued by the clerking doctor (HR = 2.73, 95% CI = 1.58-4.73, P < 0.001) or at 24 h from the BC (HR = 1.83, 95% CI = 1.05-3.20, P = 0.033) when compared with patients who received effective therapy throughout. Hospital-onset infections, 'high inoculum' infections and elevated C-reactive protein, lactate and Charlson comorbidity index were independent predictors of mortality. Effective initial antibiotics did not statistically significantly reduce length of stay in hospital (-2.98 days, 95% CI = -6.08-0.11, P = 0.058). The primary reasons for incorrect treatment were in vitro antimicrobial resistance (48.6%), initial misdiagnosis of infection source (22.7%) and non-adherence to hospital guidelines (15.7%). Consecutive prescribing decisions affect mortality from Gram-negative bacteraemia.

Propionic acid production by Propionibacterium freudenreichii using sweet sorghum bagasse hydrolysate.

Propionic acid, a widely used food preservative and intermediate in the manufacture of various chemicals, is currently produced from petroleum-based chemicals, raising concerns about its long-term sustainability. A key way to make propionic acid more sustainable is through fermentation of low-cost renewable and inedible sugar sources, such as lignocellulosic biomass. To this end, we utilized the cellulosic hydrolysate of sweet sorghum bagasse (SSB), a residue from a promising biomass source that can be cultivated around the world, for fermentative propionic acid production using Propionibacterium freudenreichii. In serum bottles, SSB hydrolysate supported a higher propionic acid yield than glucose (0.51 vs. 0.44 g/g, respectively), which can be attributed to the presence of additional nutrients in the hydrolysate enhancing propionic acid biosynthesis and the pH buffering capacity of the hydrolysate. Additionally, SSB hydrolysate supported better cell growth kinetics and higher tolerance to product inhibition by P. freudenreichii. The yield was further improved by co-fermenting glycerol, a renewable byproduct of the biodiesel industry, reaching up to 0.59 g/g, whereas volumetric productivity was enhanced by running the fermentation with high cell density inoculum. In the bioreactor, although the yield was slightly lower than in serum bottles (0.45 g/g), higher final concentration and overall productivity of propionic acid were achieved. Compared to glucose (this study) and hydrolysates from other biomass species (literature), use of SSB hydrolysate as a renewable glucose source resulted in comparable or even higher propionic acid yields. KEY POINTS: • Propionic acid yield and cell growth were higher in SSB hydrolysate than glucose. • The yield was enhanced by co-fermenting SSB hydrolysate and glycerol. • The productivity was enhanced under high cell density fermentation conditions. • SSB hydrolysate is equivalent or superior to other reported hydrolysates.

Optimizing pharmacokinetics/pharmacodynamics of β-lactam/β-lactamase inhibitor combinations against high inocula of ESBL-producing bacteria

Reduced in vitro β-lactam activity against a dense bacterial population is well recognized. It is commonly attributed to the presence of β-lactamase(s) and it is unknown whether the inoculum effect could be diminished by a β-lactamase inhibitor. We evaluated different β-lactam/β-lactamase inhibitor combinations in suppressing a high inoculum of ESBL-producing bacteria. Three clinical isolates expressing representative ESBLs (CTX-M-15 and SHV-12) were examined. The impact of escalating β-lactamase inhibitor (tazobactam or avibactam) concentrations on β-lactam (piperacillin or ceftazidime) MIC reduction was characterized by an inhibitory sigmoid Emax model. The effect of various dosing regimens of β-lactam/β-lactamase inhibitor combinations was predicted using %T>MICi and selected exposures were experimentally validated in a hollow-fibre infection model over 120 h. The threshold exposure to suppress bacterial regrowth was identified using recursive partitioning. A concentration-dependent reduction in β-lactam MIC was observed (r2 ≥0.93). Regrowth could be suppressed in all six experiments using %T>MICi ≥73.6%, but only one out of six experiments below the threshold (P = 0.015). The exposures to suppress regrowth might be attained using the clinical dose of avibactam, but a much higher dose than the standard dose would be needed for tazobactam. A dense population of ESBL-producing bacteria could be suppressed by an optimized dosing regimen of selected β-lactam/β-lactamase inhibitor combinations. The reversibility of enzyme inhibition could play an important role in diminishing the inoculum effect. In vivo investigations to validate these findings are warranted.

Highly infested soils undermine the use of resistant olive rootstocks as a control method of verticillium wilt

AbstractVerticillium wilt of olive (VWO) is probably the most devastating fungal disease for olive trees worldwide, and currently the main cultivars are susceptible or moderately susceptible to this disease. The evaluation of resistant cultivars as rootstocks to control the disease has scarcely been explored, and mainly in short‐term studies under controlled conditions, which usually do not correspond with field evaluations. The main objective of this study was to assess the responses to VWO of different scion × rootstock combinations of the olive cultivars Picual, Arbequina, Changlot Real, and Frantoio in a long‐term field experiment with a soil highly infested with the defoliating pathotype of Verticillium dahliae. The results showed that grafting the susceptible cultivar Picual onto resistant rootstocks delayed the onset of the disease symptoms; however, after 4 years, it was observed that all combinations that contain Picual (a) were extensively colonized by V. dahliae; (b) developed severe symptoms of the disease; and (c) had plant mortality similar to Picual growing on its own roots. This result highlights the importance of long‐term field experiments to evaluate VWO and shows that grafting susceptible olive cultivars onto resistant ones does not provide a durable control of VWO under high inoculum potential, as V. dahliae is able to progress through the resistant rootstock and then extensively colonize and kill the susceptible scion. However, the high inoculum potential observed in this study does not allow us to consider the evaluated resistant cultivars as completely ineffective under lower inoculum densities.

The effects of koruk products used as marination liquids against foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Typhimurium) inoculated on poultry meat

In this study, the effects of marination liquids (MLs) prepared using koruk juice (25% and 50%) and dried koruk pomace (1% and 2%) with or without additives [salt (1%) and thyme (0.1%)] on meat safety were determined. In the first stage, the minimum inhibition concentration (MIC) values of MLs were observed in the range of 6.50% - >50% (v/v). In the second stage, Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Typhimurium were inoculated on poultry meat at high and low inoculum doses (≅ 6 log and ≅ 3 log) and marinated at 4 °C for 2, 24 and 48 h. When high inoculum doses were used, E. coli O157:H7, L. monocytogenes and S. Typhimurium counts on meats were decreased by 0.78–2.52, 0.42–1.50 and 0.58–2.00 log CFU/g, respectively. Marination with 50% koruk juice without additives (ML1) for 48 h was determined as the most effective treatment on E. coli O157:H7 and S. Typhimurium, while the highest reduction of L. monocytogenes was achieved by 50% koruk juice with additives (ML2) for 48 h. The counts of pathogens inoculated on samples at low inoculum doses were reduced to undetectable levels for all treatment times. The results of this study revealed that koruk-based MLs are effective products for providing food safety.

β,γ-Diaryl α-methylene-γ-butyrolactones as potent antibacterials against methicillin-resistant Staphylococcus aureus

A selected series of racemic α-methylene-γ-butyrolactones (AMGBL) synthesized via allylboration or allylindation reactions were screened against methicillin-resistant Staphylococcus aureus (MRSA) USA300. Unlike natural AMGBLs, such as parthenolide, synthetic analogs bearing aryl moieties at the β- and γ-positions are potent against MRSA. The most potent molecules were comparable to vancomycin and linezolid, the drugs of the last resort for MRSA infections, in their effectiveness with minimum inhibitory concentrations (MICs) ranging from 3.0 to 5.2 μM. These lactones also exhibited potent antibacterial activity against other clinically important multidrug-resistant Gram-positive bacteria (except enterococci), while also showing high tolerability to mammalian cells. Several of these molecules surpassed vancomycin in their rapid killing of the high MRSA inoculum (2 h vs 12 h) in a standard time-kill kinetics assay, with compounds 1l and 1m significantly reducing the intracellular burden of MRSA by about 98–99%, at low concentrations. Additionally, the compounds surpassed vancomycin in inhibiting staphylococcal protease production, indicating that synthetic methylene lactones warrant further investigations as promising anti-MRSA candidates.

Cloxacillin or fosfomycin plus daptomycin combinations are more active than cloxacillin monotherapy or combined with gentamicin against MSSA in a rabbit model of experimental endocarditis.

In vitro and in vivo activity of daptomycin alone or plus either cloxacillin or fosfomycin compared with cloxacillin alone and cloxacillin plus gentamicin were evaluated in a rabbit model of MSSA experimental endocarditis (EE). Five MSSA strains were used in the in vitro time-kill studies at standard (105-106 cfu/mL) and high (108 cfu/mL) inocula. In the in vivo EE model, the following antibiotic combinations were evaluated: cloxacillin (2 g/4 h) alone or combined with gentamicin (1 mg/kg/8 h) or daptomycin (6 mg/kg once daily); and daptomycin (6 mg/kg/day) alone or combined with fosfomycin (2 g/6 h). At standard and high inocula, daptomycin plus fosfomycin or cloxacillin were bactericidal against 4/5 and 5/5 strains, respectively, while cloxacillin plus gentamicin was bactericidal against 3/5 strains at standard inocula but against none at high inocula. Fosfomycin, cloxacillin, gentamicin and daptomycin MIC/MBCs of the MSSA-678 strain used in the EE model were: 8/64, 0.25/0.5, 0.25/0.5 and 1/8 mg/L, respectively. Adding gentamicin to cloxacillin significantly reduced bacterial density in vegetations compared with cloxacillin monotherapy (P = 0.026). Adding fosfomycin or cloxacillin to daptomycin [10/11 (93%) and 8/11 (73%), respectively] significantly improved the efficacy of daptomycin in sterilizing vegetations [0/11 (0%), P < 0.001 for both combinations] and showed better activity than cloxacillin alone [0/10 (0%), P < 0.001 for both combinations] and cloxacillin plus gentamicin [3/10 (30%), P = 0.086 for cloxacillin plus daptomycin and P = 0.008 for fosfomycin plus daptomycin]. No recovered isolates showed increased daptomycin MIC. The addition of cloxacillin or fosfomycin to daptomycin is synergistic and rapidly bactericidal, showing better activity than cloxacillin plus gentamicin for treating MSSA EE, supporting their clinical use.

Cefazolin Inoculum Effect and Methicillin-Susceptible Staphylococcus aureus Osteoarticular Infections in Children.

Select methicillin-susceptible Staphylococcus aureus (MSSA) strains may produce β-lactamases with affinity for first-generation cephalosporins (1GCs). In the setting of a high inoculum, these β-lactamases may promote the cleavage of 1GCs, a phenomenon known as the cefazolin inoculum effect (CzIE). We evaluated the prevalence and impact of CzIE on clinical outcomes among MSSA acute hematogenous osteomyelitis (AHO) cases. MSSA AHO isolates obtained from two children's hospitals between January 2011 and December 2018 were procured through ongoing surveillance studies. Isolates were tested for CzIE via a broth macrodilution assay using an inoculum of 107 CFU/ml; CzIE was defined as a cefazolin MIC of ≥16 μg/ml. Isolates were characterized by accessory gene regulator group (agr). The progression from acute to chronic osteomyelitis was considered an important outcome. A total of 250 cases with viable isolates were included. Notably, 14.4% of isolates exhibited CzIE with no observed temporal trend; and 4% and 76% of patients received a 1GC as an empirical and definitive therapy, respectively. CzIE isolates were more often resistant to clindamycin, belonged to agrIII, and associated with the development of chronic osteomyelitis. In multivariable analyses, agrIII, multiple surgical debridements, delayed source control, and CzIE were independently associated with progression to chronic osteomyelitis. A higher rate of chronic osteomyelitis was observed with CzIE isolates regardless of definitive antibiotic choice. CzIE is exhibited by 14.4% of MSSA AHO isolates in children. CzIE is independently associated with progression to chronic osteomyelitis in cases of AHO irrespective of final antibiotic choice. These data suggest that negative outcomes reported with CzIE may more accurately reflect strain-dependent virulence factors rather than true antibiotic failure.

Ortiva top® fungicide as an alternative to Bavistin® fungicide against cercospora leaf spot of sugar beet

This study aimed at validating the combination of azoxystrobin + difenoconazole (Ortiva top®) as an alternative to carbendazim (Bavistin®) against Cercospora leaf spot of sugar beet caused by Cercospora beticola and identifying the lowest effective dosage. Experiments plots were designed as completely randomised blocks and subjected to foliar sprays of azoxystrobin + difenoconazole at three doses of 0.75, 1 and 1.25 L/ha and at the recommended dose of 0.5 kg/ha for carbendazim. The lowest effective dosage for azoxystrobin + difenoconazole fungicide was determined 1 L/ha under field condition. In this study, the optimal dosage of azoxystrobin + difenoconazole fungicide significantly reduced the disease severity (DS) compared with carbendazim fungicide and untreated plots and significantly increased the yield per hectare. Inclusion of the optimal dosage of azoxystrobin + difenoconazole in the cercospora leaf spot management can offer an appropriate alternative to carbendazim even under high inoculum pressure of the pathogen.

Early Polylysine Release from Dental Composites and Its Effects on Planktonic Streptococcus mutans Growth.

The study aim was to assess the effect of incorporating polylysine (PLS) filler at different mass fractions (0.5, 1 and 2 wt%) on PLS release and Streptococcus mutans planktonic growth. Composite containing PLS mass and volume change and PLS release upon water immersion were assessed gravimetrically and via high-performance liquid chromatography (HPLC), respectively. Disc effects on bacterial counts in broth initially containing 8 × 105 versus 8 × 106 CFU/mL Streptococcus mutans UA159 were determined after 24 h. Survival of sedimented bacteria after 72 h was determined following LIVE/DEAD staining of composite surfaces using confocal microscopy. Water sorption-induced mass change at two months increased from 0.7 to 1.7% with increasing PLS concentration. Average volume increases were 2.3% at two months whilst polylysine release levelled at 4% at 3 weeks irrespective of composite PLS level. Early percentage PLS release, however, was faster with higher composite content. With 0.5, 1 and 2% polylysine initially in the composite filler phase, 24-h PLS release into 1 mL of water yielded 8, 25 and 93 ppm respectively. With initial bacterial counts of 8 × 105 CFU/mL, this PLS release reduced 24-h bacterial counts from 109 down to 108, 107 and 102 CFU/mL respectively. With a high initial inoculum, 24-h bacterial counts were 109 with 0, 0.5 or 1% PLS and 107 with 2% PLS. As the PLS composite content was raised, the ratio of dead to live sedimented bacteria increased. The antibacterial action of the experimental composites could reduce residual bacteria remaining following minimally invasive tooth restorations.

Evaluation of the GENE-UP® Listeria monocytogenes Method for the Detection of Listeria monocytogenes in a Variety of Foods and Select Environmental Surfaces: Collaborative Study, First Action 2019.11.

The GENE-UP® Listeria monocytogenes 2 (LMO 2) assay (Performance Tested MethodSM 121804) uses real-time PCR technology and a proprietary detection platform, the GENE-UP Thermocycler, to detect Listeria monocytogenes in a variety of foods and environmental surfaces. The purpose of this validation was to evaluate the method's interlaboratory performance and submit the result to AOAC INTERNATIONAL for adoption as First Action Official MethodSM for the detection of Listeria monocytogenes in a variety of foods and select environmental surfaces. The GENE-UP method was evaluated in a multi-laboratory study as part of the AFNOR NF VALIDATION certification process using unpaired test portions for one food matrix, full-cream goat milk cottage cheese (8.4% fat). The candidate method was compared to the ISO 11290-1/Amd.1:2004 reference method. Sixteen participants from 15 laboratories throughout the European Union participated. Three levels of contamination were evaluated: a non-inoculated control level (0 CFU/test portion), a low inoculum level (∼2 CFU/test portion), and a high inoculum level (∼10 CFU/test portion). Data from the study were analyzed according to the Probability of Detection (POD) statistical model as presented in the AOAC validation guidelines. The dLPODC values with 95% confidence interval for each comparison were; -0.02 (-0.07, 0.03), -0.08 (-0.31, 0.16), and 0.00 (-0.03, 0.03) for the non-inoculated, low, and high contamination levels, respectively. The dLPODC results demonstrate no difference in performance between the candidate method and reference method for the matrix evaluated. The GENE-UP LMO method demonstrated accuracy and precision in detecting and discerning L. monocytogenes from other Listeria species.