Evaluating mono and combination therapy of meropenem and amikacin against Pseudomonas aeruginosa bacteremia in the Hollow-Fiber Infection Model

Debate continues as to the role of combination antibiotic therapy for the management of Pseudomonas aeruginosa infections. We studied extent of bacterial killing and resistance emergence of meropenem and amikacin as monotherapy and as a combination therapy against susceptible and resistant P. aeruginosa isolates from bacteremic patients using the dynamic in vitro hollow-fiber infection model. Three P. aeruginosa isolates (meropenem MICs 0.125, 0.25 & 64 mg/L) were used simulating bacteremia with an initial inoculum ~ 1×105 CFU/mL and the expected pharmacokinetics of meropenem and amikacin in critically ill patients. For isolates susceptible to amikacin and meropenem (isolates 1 and 2), the rate of bacterial killing was increased with the combination regimen when compared with monotherapy of either antibiotic. Both the combination and meropenem monotherapy were able to sustain bacterial killing throughout the seven-day treatment course, whereas regrowth of bacteria occurred with amikacin monotherapy after 12 hours. For the meropenem-resistant P. aeruginosa isolate (isolate 3), only the combination regimen demonstrated bacterial killing. Given that tailored antibiotic regimens can maximize potential synergy against some isolates, future studies should explore the benefit of combination therapy against resistant P. aeruginosa.

Similarity between corn hybrids using sanitary and physiological seed parameters

One of the elements capable of explaining the epidemics of diseases in the fields of corn production is the amount of initial inoculum present in seeds. The objective of this paper was to study the health and physiological quality of maize hybrids grown in the 2019 crop. The experiment was carried out using 22 hybrids represented by: 13K21PW Morgan©, 14K185PW Morgan©, 230 PRO 3 DKB©, 290 PRO 3 DKB© , 3040 VIP3 LG©, 3055 VIP3 LG©, 30F35 Pioneer ©, 310 PRO 3 DKB©, 345 PRO 3 DKB©, 8882 Coded DKB, CBS15C385PW Morgan©, CBS15D089PW Morgan©, CBS15SE137PW Morgan©, Coded x40K205 (3898) Pioneer, K9606 VIP3 KWS©, MG580PW Morgan©, MG652PW Morgan©, SH 7930 PRO 2 Sta Helena©, SH 7990 PRO 2 Sta Helena©, SX5885 VIP3 Syngenta©, SX71715 VIP3 Syngenta© and SX8934 VIP3 Syngenta© that were grown in the municipality of Ipameri, GO. Analyzed the percentage of primary root emission (ERP),% of shoot emission (EPA),% of microorganism incidence, % of fungus genera (% GF). Sixteen fungi were detected representing the microflora detected in the seeds of the evaluated hybrids recognized as Penicillium sp., Aspergillus sp., Curvularia sp., Fusarium verticillioides, Gliomastix sp., Cladosporium sp., Gliocladium sp., Bacillus sp., Geotrichum sp. , Trichoderma sp., Mucor sp., Stenocarpela maydis, Pythium sp., Chaetomium sp., Sporotrix sp. and Scopulariopsis sp. The multivariate analysis jointed the cultivars with highest physiological activity represented by SX 8934 Vip3 SYN®, SX 71715 Vip 3®, 3040 Vip3 LG®, SHS 7990 PRO2, SX 5885 Vip3, MG 652 PW®, K9606 Vip3®, DKB 345 PRO3. The lowest incidence of microorganism was in hybrids DKB 345 PRO3 and 8882 cod.

Production of bacterial cellulose films by Gluconoacetobacter xylinus for lipase immobilization

Bacterial cellulose (BC), a microbial polysaccharide, has chemically equivalent structure to plant cellulose with unbranched pellicle structure of only glucose monomers. Due to the unique nanostructure, BC has great potential in enzyme immobilization. In this study, the effects of different cultivation conditions including rotational speed, initial inoculum concentration and medium pH on the film-like cellulosic biomass formation of Gluconacetobacter xylinus JCM 9730 were examined. The resultant BC films were then studied for its feasibility in the immobilization of lipase, a widely used enzyme in biotechnological and industrial processes including food, pharmaceutical, chemical and paper industries. Results showed that increasing in rotational speed from 0 rpm to 200 rpm converted cellulose-producing cells to non-cellulose-producing ones, leading to a significant decline in BC film formation. The increase in initial inoculum size from 0.01 g/L to 0.1 g/L reduced sugar concentration and surface area of the medium, and therefore inhibiting the formation of film-like cellulosic biomass. In addition, the optimum pH range of Acetobacter species from 5.4 – 6.3 was found not optimal for BC film formation. The highest amount of film-like cellulosic biomass of 19.01 g/L was obtained under static condition (0 rpm) with initial cell concentration of 0.04 g/L and initial pH of 4.0. The BC film samples were then acetylated with acetic anhydride/iodine system to convert the hydroxyl groups to less hydrophilic acetyl groups and were used for lipase immobilization. Results showed that lipase immobilized on acetylated BC still maintained its lipid hydrolytic activity. It can be hence concluded that BC films produced by G. xylinus JCM 9730 were potential for lipase immobilization.

In vitro Bactericidal Activities of Combination Antibiotic Therapies Against Carbapenem-Resistant Klebsiella pneumoniae With Different Carbapenemases and Sequence Types

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is becoming increasingly problematic due to the limited effectiveness of new antimicrobials or other factors such as treatment cost. Thus, combination therapy remains a suitable treatment option. We aimed to evaluate the in vitro bactericidal activity of various antibiotic combinations against CRKP with different carbapenemase genotypes and sequence types (STs). Thirty-seven CRKP with various STs and carbapenemases were exposed to 11 antibiotic combinations (polymyxin B or tigecycline in combination with β-lactams including aztreonam, cefepime, piperacillin/tazobactam, doripenem, meropenem, and polymyxin B with tigecycline) in static time-kill studies (TKS) using clinically achievable concentrations. Out of the 407 isolate-combination pairs, only 146 (35.8%) were bactericidal (≥3 log10CFU/mL decrease from initial inoculum). Polymyxin B in combination with doripenem, meropenem, or cefepime was the most active, each demonstrating bactericidal activity in 27, 24, and 24 out of 37 isolates, respectively. Tigecycline in combination with β-lactams was rarely bactericidal. Aside from the lower frequency of bactericidal activity in the dual-carbapenemase producers, there was no apparent difference in combination activity among the strains with other carbapenemase types. In addition, bactericidal combinations were varied even in strains with similar STs, carbapenemases, and other genomic characteristics. Our findings demonstrate that the bactericidal activity of antibiotic combinations is highly strain-specific likely owing to the complex interplay of carbapenem-resistance mechanisms, i.e., carbapenemase genotype alone cannot predict in vitro bactericidal activity. The availability of WGS information can help rationalize the activity of certain combinations. Further studies should explore the use of genomic markers with phenotypic information to predict combination activity.

The microbial composition of the initial insult can predict the prognosis of experimental sepsis

We hypothesized that the composition of sepsis-inducing bacterial flora influences the course of fecal peritonitis in rodents. Saline or fecal suspensions with a standardized dose range of bacterial colony-forming units (CFUs) were injected intraperitoneally into Sprague–Dawley rats. The qualitative composition of the initial inoculum and the ascites was analyzed separately by MALDI-TOF mass spectrometry. Invasive monitoring was conducted in separate anesthetized groups (n = 12–13/group) after 12, 24, 48 and 72 h to determine rat-specific organ failure assessment (ROFA) scores. Death and ROFA scores peaked at 24 h. At this time, 20% mortality occurred in animals receiving a monomicrobial E. coli suspension, and ROFA scores were significantly higher in the monomicrobial subgroup than in the polymicrobial one (median 6.5; 5.0–7.0 and 5.0; 4.75–5.0, respectively). ROFA scores dropped after 48 h, accompanied by a steady decrease in ascites CFUs and a shift towards intra-abdominal monomicrobial E. coli cultures. Furthermore, we found a relationship between ascites CFUs and the evolving change in ROFA scores throughout the study. Hence, quantitatively identical bacterial loads with mono- or polymicrobial dominance lead to a different degree of sepsis severity and divergent outcomes. Initial and intraperitoneal microbiological testing should be used to improve translational research success.

In vivo Serial Passaging of Human–Simian Immunodeficiency Virus Clones Identifies Characteristics for Persistent Viral Replication

We previously reported that a human immunodeficiency virus type 1 with a simian immunodeficiency virus vif substitution (HSIV-vifNL4-3) could replicate in pigtailed macaques (PTMs), demonstrating that Vif is a species-specific tropism factor of primate lentiviruses. However, infections did not result in high-peak viremia or setpoint plasma viral loads, as observed during simian immunodeficiency virus (SIV) infection of PTMs. Here, we characterized variants isolated from one of the original infected animals with CD4 depletion after nearly 4years of infection to identify determinants of increased replication fitness. In our studies, we found that the HSIV-vif clones did not express the HIV-1 Vpr protein due to interference from the vpx open reading frame (ORF) in singly spliced vpr mRNA. To examine whether these viral genes contribute to persistent viral replication, we generated infectious HSIV-vif clones expressing either the HIV-1 Vpr or SIV Vpx protein. And then to determine viral fitness determinants of HSIV-vif, we conducted three rounds of serial in vivo passaging in PTMs, starting with an initial inoculum containing a mixture of CXCR4-tropic [Vpr-HSIV-vifNL4-3 isolated at 196 (C/196) and 200 (C/200) weeks post-infection from a PTM with depressed CD4 counts] and CCR5-tropic HSIV (Vpr+ HSIV-vif derivatives based NL-AD8 and Bru-Yu2 and a Vpx expressing HSIV-vifYu2). Interestingly, all infected PTMs showed peak plasma viremia close to or above 105 copies/ml and persistent viral replication for more than 20weeks. Infectious molecular clones (IMCs) recovered from the passage 3 PTM (HSIV-P3 IMCs) included mutations required for HIV-1 Vpr expression and those mutations encoded by the CXCR4-tropic HSIV-vifNL4-3 isolate C/196. The data indicate that the viruses selected during long-term infection acquired HIV-1 Vpr expression, suggesting the importance of Vpr for in vivo pathogenesis. Further passaging of HSIV-P3 IMCs in vivo may generate pathogenic variants with higher replication capacity, which will be a valuable resource as challenge virus in vaccine and cure studies.

Predicting Experimental Sepsis Survival with a Mathematical Model of Acute Inflammation

Sepsis is characterized by an overactive, dysregulated inflammatory response that drives organ dysfunction and often results in death. Mathematical modeling has emerged as an essential tool for understanding the underlying complex biological processes. A system of four ordinary differential equations (ODEs) was developed to simulate the dynamics of bacteria, the pro- and anti-inflammatory responses, and tissue damage (whose molecular correlate is damage-associated molecular pattern [DAMP] molecules and which integrates inputs from the other variables, feeds back to drive further inflammation, and serves as a proxy for whole-organism health status). The ODE model was calibrated to experimental data from E. coli infection in genetically identical rats and was validated with mortality data for these animals. The model demonstrated recovery, aseptic death, or septic death outcomes for a simulated infection while varying the initial inoculum, pathogen growth rate, strength of the local immune response, and activation of the pro-inflammatory response in the system. In general, more septic outcomes were encountered when the initial inoculum of bacteria was increased, the pathogen growth rate was increased, or the host immune response was decreased. The model demonstrated that small changes in parameter values, such as those governing the pathogen or the immune response, could explain the experimentally observed variability in mortality rates among septic rats. A local sensitivity analysis was conducted to understand the magnitude of such parameter effects on system dynamics. Despite successful predictions of mortality, simulated trajectories of bacteria, inflammatory responses, and damage were closely clustered during the initial stages of infection, suggesting that uncertainty in initial conditions could lead to difficulty in predicting outcomes of sepsis by using inflammation biomarker levels.

Behavior of Native Food Isolates of Listeria monocytogenes and Listeria innocua under the Influence of Selected Cultural Attributes and Heat and Cold Treatments

The present investigation assesses the influence of cultural attributes and heat and cold treatments on the behaviour of native toxigenic L. monocytogenes CFR 1302 and non-toxigenic L. innocua CFR 1304 in selected medium. The growth responses of L. monocytogenes and L. innocua under the influencing factors of storage temperature (10-40°C), pH level (5.5-7.5), and storage period (6-48 h) in brain heart infusion and nutrient broths revealed closeness between observed and predicted populations. Response surface plots were generated for the growth behavior of the two test cultures as a function of pH level. The toxigenic L. monocytogenes CFR 1302 could reach a higher viable population. The effect of heat treatment on Listeria spp. in selected five heating menstra showed the lowest D-value of 3.7 min at 60°C for L. innocua CFR 1304 in Milli-Q water to the highest of 8.4 min at 56°C for L. monocytogenes CFR 1302 in skim milk. The average z-value across the heating menstra for L. monocytogenes was 27.3°C as against that of 22°C for L. innocua. In the case of cold treatment, storage of 4 and 8°C resulted in appreciable increase in counts of L. monocytogenes CFR 1302 from the initial inoculum introduced in selected media. At -20°C, there was a slight decrease in the viable population. The research data helps to predict the viable populations of L. monocytogenes as a part of risk assessment in the food chain. This is of significance in providing safe and healthy food to human population.

PSX-A-4 Late-Breaking: Assessing the antifungal efficacy of EverWildTM D01 against Aspergillus flavus in semi-moist pet treats

Semi-moist pet treats contain moisture levels ranging from 20–30% making them ideal for mold growth and mycotoxin production. To control mold, synthetic mold inhibitors such as potassium sorbate or calcium propionate are used. Synthetic additives are effective, but there is a shift in the pet owner preferences for ‘natural’ products. Fermented and (or) cultured whey has been shown to contain antifungal metabolites and has been used to control fungal growth in bread. EverWildTM D01 is a commercially available cultured whey product. The objective of this study was to assess the efficacy of EverWildTM D01 against Aspergillus flavus in a semi-moist pet treat. A model semi-moist pet treat fortified to be nutritionally complete was produced with three levels of powdered whey fermentate, [1.0%, 3.0% and 5% (w/w)], a negative control with no antifungal and a “positive control” which contained potassium sorbate as a standard industrial mold inhibitor. Treats were produced by baking in 3cm x 3cm squares. Individual squares were inoculated with 0.1 mL of Aspergillus flavus (ATCC 204304) to achieve a final concentration of ~ 5.0 Log CFU/square. The inoculated squares were individually incubated at 250 C. Fungal analysis was performed at day 3, 6, 9, 12, 15, 18, 21, 24 and 28 by surface plating on Potato Dextrose Agar (PDA) followed by incubation at 250 C for 72h. The total log reductions were calculated by subtracting the initial inoculum with the colony counts on day 28. EverWildTM D01 at 1.0%, 3.0%, and 5% (w/w) had a 1.90, 3.89 and 4.58 Log CFU/square log reduction, respectively. The positive control had a 1.19 Log CFU/square log reduction. All treatments differed from the negative control (P < 0.05). In conclusion, EverWildTM D01 may be effective as a natural alternative to synthetic mold inhibitors in semi-moist pet treats.

Seed-transmission of Cowpea mild mottle virus on several varieties of soybean in Indonesia

Sutrawati M, Hidayat SH, Suastika G, Sukarno BPW, Nurmansyah A. 2021. Seed-transmission of Cowpea mild mottle virus on several varieties of soybean in Indonesia. Biodiversitas 22: 4182-4185. Seeds and infected plants play important role as source of disease in the field for seed-transmitted virus, such as Cowpea mild mottle virus (CPMMV). Research was conducted to determine seed transmission nature of CPMMV on 10 soybean varieties based on growing on test method and dot immunobinding assay to confirm CPMMV infection. Field experiment was conducted to evaluate the efficiency of seed-transmitted CPMMV as the source of initial inoculum in the field. Soybean var. ‘Anjasmoro’ from 3 cultivation areas (Cianjur, Bogor, and Cirebon) was used for field experiment. Seed transmission of CPMMV was confirmed on soybean var. ‘Detam 2’, ‘Detam 3’, ‘Malika’, ‘Anjasmoro’, and ‘Argomulyo’; but was not found on ‘Detam 1’, ‘Detam 4’, ‘Wilis’, ‘Grobogan’, and ‘Dena 1’. The infection of CPMMV did not show symptom, either on the seedcoat and the unifoliolate leaves. Infection rate of CPMMV on seeds were relatively high, ranged between 27 to 86%. Disease incidence on var. ‘Anjasmoro’ from Cianjur, Cirebon, and Bogor varied from 32.9 to 75% and 57.9 to 81.3% in screenhouse and field experiment, respectivelly.