Standard Inoculum Research Articles

Effect of delayed vial entry on the recovery and time to positivity of microorganisms from automated blood culture vials in a tertiary care hospital, South India

Recommendations by manufacturers depict that blood culture bottles are to be loaded into the automated machines as soon as possible after the blood collection. But it has been noticed that in peripheral set-ups where round the clock laboratory facility is not available or which are located far from the reference labs, or even in the tertiary care centres, delay occurs prior to the bottle loading into the instruments. In our study, we have compared the effect of pre-incubating the blood culture bottles at various temperatures like 4°C, room temperature and 37°C for various holding times, such as 2, 6, 12 and 24 hours simulating the delay in transport time in BACTEC plus Aerobic and BACTALERT FA plus systems in a tertiary care set up. We included five recent clinical isolates of different microorganisms in our study, such as Staphylococcus aurous, Streptococcus, Escherichia coli, Pseudomonas aeruginosa and Candida albicans. Standard inoculum was prepared for each organism and 3 ml of it was added with 10 ml of citrated human blood and then inoculated into blood culture vial (BACTALERT FA plus and BACTEC Plus). Organism recovery rate, time to detection and rates of false-negativity from both the instruments were evaluated by using seeded blood culture vial and controls with delayed entry. Performance wise analysis showed that the overall isolation rate of the organisms from BACTEC bottles was higher compared to BACTALERT. Also BACTEC showed less average TTD compared to BACTALERT for Pseudomonas aeruginosa, Streptococcus and candida albicans but for E. coli and S. aureus BACTALERT showed earlier detection. To conclude, storage of the inoculated bottles at room temperature gave optimal recovery of the organisms for both BACTEC and BACTALERT systems, if delayed entry is inevitable. Keyword: Delayed vial entry effect on recovery of microorganisms.

The relevance of environment vs. composition on dissolved organic matter degradation in freshwaters

AbstractDissolved organic matter (DOM) composition exerts a direct control on its degradation and subsequent persistence in aquatic ecosystems. Yet, under certain conditions, the degradation patterns of DOM cannot be solely explained by its composition, highlighting the relevance of environmental conditions for DOM degradation. Here, we experimentally assessed the relative influence of composition vs. environment on DOM degradation by performing degradation bioassays using three contrasting DOM sources inoculated with a standardized bacterial inoculum under five distinct environments. The DOM degradation kinetics modeled using reactivity continuum models showed that composition was more important than environment in determining the bulk DOM decay patterns. Changes in DOM composition resulted from the interaction between DOM source and environment. The role of environment was stronger on shaping the bacterial community composition, but the intrinsic nature of the DOM source exerted stronger control on the DOM degradation function.

A new ophthalmic formulation containing antiseptics and dexpanthenol: In vitro antimicrobial activity and effects on corneal and conjunctival epithelial cells

Antibiotic resistance is increasing even in ocular pathogens, therefore the interest towards antiseptics in Ophthalmology is growing. The aim of this study was to analyze the in vitro antimicrobial efficacy and the in vitro effects of an ophthalmic formulation containing hexamidine diisethionate 0.05%, polyhexamethylene biguanide (PHMB) 0.0001% disodium edetate (EDTA) 0.01%, dexpanthenol 5% and polyvinyl alcohol 1.25% (Keratosept, Bruschettini, Genova, Italy) on cultured human corneal and conjunctival cells. The in vitro antimicrobial activity was tested on Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mitis. For each microbial strain 10 μL of a 0.5 MacFarland standardized bacterial inoculum were incubated at 25 °C with 100 μL of ophthalmic solution for up to 6 h. After different periods of time, samples were inoculated on blood agar with 5% sheep blood. Moreover, a 0.5 MacFarland bacterial inoculum was seeded in triplicate on Mueller-Hinton Agar or on Mueller-Hinton Fastidious Agar; then a cellulose disc soaked with 50 μL of ophthalmic solution was applied on the surface of agar and plates were incubated for 18 h at 37 °C, in order to evaluate the inhibition of bacterial growth around the disc. Human corneal and conjunctival epithelial cells in vitro were incubated for 5, 10 and 15 min with Keratosept or its components. The cytotoxicity was assessed through the release of cytoplasmic enzyme lactate dehydrogenase (LDH) into the medium immediately after exposure to the drugs; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the metabolic cell activity. Our results show that Keratosept ophthalmic solution gave an average logarithmic (log) reduction of bacterial load of 2.14 ± 0.35 within 6 h of exposure (p-value < 0.05 versus control saline solution). On agar plates, all microbial strains, excluding P. Aeruginosa, showed an inhibition zone of growth around the Keratosept-soaked discs. Keratosept and its components after 5 and 10 min did not show any cytotoxic effect on cultured corneal and conjunctival cells, and only after 15 min a significant reduction of cell viability and an increase of cytotoxicity compared to control (vehicle) was seen; dexpanthenol 5% and polyvinyl alcohol accelerated the wounding of corneal cells in vitro. In conclusion, Keratosept showed good antimicrobial activity on the tested strains; the ophthalmic solution and its components were safe and non-toxic for the corneal and conjunctival epithelial cells for 5 and 10 min at the concentrations analyzed, and dexpanthenol 5% and polyvinyl alcohol promoted the wounding of corneal cells.

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.

Hippocampal damage and seizure severity during acute viral encephalitis are determined by the intensity of the infiltrating inflammatory monocyte response

Abstract The immunological response to CNS viral infection must balance the need for effective viral clearance with the need to preserve irreplaceable neurons. Failure to achieve this balance may result in death or severe cognitive impairment. We have observed hippocampal injury, compromised cognitive function, and seizures in C57BL/6 mice following acute infection with TMEV. We established that an initial innate immune response controlled by inflammatory monocyte infiltration into the brain is responsible for this neural injury. To better understand the pathogenic mechanisms involved in monocyte recruitment to the CNS we asked whether reducing the initial viral inoculum impacted the evolution of neural injury. We found that mice infected with standard (2×105 PFU) or low inoculum (1.25×104 PFU) TMEV exhibited a graded inflammatory response. Notably, a 16-fold reduction in viral inoculum resulted in 80% attenuation of the inflammatory monocyte response, concomitant with a 90% attenuation in acute production of CCL2 and complete abrogation of acute increases in TNFα and IL-6 in the hippocampus. Cognitive performance was preserved in low inoculum mice and seizure burden was highly attenuated. Despite the introduction of 16-times less virus, viral load in the brain was equivalent at 3 dpi between groups, indicating that the amount of replicating virus was not associated with reduced neuropathology. Our findings support a model in which the earliest host response to pathogen in the CNS shapes the size of the inflammatory response and constrains the downstream pathological sequelae.

Minimum inhibitory concentration of vancomycin to methicillin resistant &lt;em&gt;Staphylococcus aureus&lt;/em&gt; at a tertiary care hospital in Sri Lanka

Background: Methicillin Resistant Staphylococcus aureus (MRSA) has evolved as a serious threat to public health. Objectives: The objectives of the present study were to determine the antibiotics susceptibility patterns of S. aureus and the minimum inhibitory concentration (MIC) of vancomycin to MRSA isolated from different clinical samples at the Colombo South Teaching Hospital (CSTH) in Sri Lanka. Methodology: A total 72 isolates of S. aureus, obtained from different clinical samples at the CSTH, from January to May 2017 were included in the study. S. aureus isolates were identified by Gram stain, colony morphology, catalase, slide/tube coagulase tests. The antibiotic susceptibility tests were carried as per Clinical Laboratory Standards Institute (CLSI) guidelines. MRSA isolates were detected using the cefoxitin (30 μg) disk diffusion test. Inducible clindamycin resistance (MLSB i) was detected by the disk approximation (D test) test. The vancomycin MICs were determined by the E-test method with a 0.5 McFarland standard inoculum. The MIC clinical breakpoints were defined according to the CLSI guidelines (susceptible, ≤2 μg/ml; intermediate, 4–8 μg/ml; and resistant, ≥16 μg/ml). Results: Of the 72 S. aureus clinical isolates, 29 (40.2%) were MRSA. Inducible clindamycin resistance was detected in 16% of the MRSA isolates. Minimum inhibitory concentrations of vancomycin to the isolates of MRSA ranged from 0.125 μg/ml to 2 μg/ml. Conclusions: The rate of isolation of MRSA was high and it has emerged as a serious public health threat to Sri Lanka. The Minimum Inhibitory Concentration (MIC) of all the MRSA isolates were ≤2 μg/ml. None of the MRSA isolates were found to be intermediate-sensitive or vancomycin resistant. Therefore, vancomycin can be used as the drug of choice for treatment of infections caused by MRSA.

Using of Some Agro-industrial Wastes for Improving Carotenoids Production from Yeast Rhodotorula glutinis 32 and Bacteria Erwinia uredovora DSMZ 30080

Some agro-industrial wastes such as clarified cane molasses, high test molasses, sweet whey, potato starch and corn steep liquor were tested as carbon sources or nitrogen source for growth and carotenoid accumulation using bacteria Erwinia uredovora DSMZ 30080 and yeast Rhodotorula glutinis number 32. Erlenmeyer flasks containing 100 ml of production media, the flasks were inoculated with 1 ml of standard inoculum and incubated at 150 rpm for 4 days at 30°C. Samples were collected periodical every 24h, cell dry weight and carotenoids concentration were determined. Sweet whey and highest molasses gave the highest growth being 2.85 and 7.34 gl-1, respectively and scored the same layout on carotenoids conc. which reach the peak during stationary phase (72 h of fermentation). Using of high test molasses and sweet whey as carotenoid production media were incremented carotenoid conc. about 1.7 and 2 fold (with respect to reference media). Increasing high test molasses conc. to give 5% initial sugar led to up great growth, carotenoids conc., productivity, yield and Yc/x from Rhodo. glutinis 32 to be 7.31 gl-1, 2.67 mgl-1, 0.037 mgl-1h-1, 0.067% and 0.365, respectively. Furthermore, using corn steep liquor (30%) as nitrogen sources augmented carotenoids concentration about 3.8 and 4fold for incomplete and complete production media using Rhodo. glutinis32. Also, a negligible effect on growth was observed with dark incubation with both strains which dropped about 75 and 48% with regard to control for E. uredovora DSMZ 30080 and Rhod. glutinis 32, respectively, whereas, carotenoids conc. was increased about 21% for E. uredovora DSMZ 30080 in dark condition.

Activity of pulmonary vancomycin exposures versus planktonic and biofilm isolates of methicillin-resistant Staphylococcus aureus from cystic fibrosis sputum

Vancomycin is commonly used to treat methicillin-resistant Staphylococcus aureus (MRSA) infections in patients with cystic fibrosis (CF) lung disease. However, there are limited data to support the in vitro activity of this agent against MRSA isolated from CF sputum. The primary objective of this study was to evaluate the activity of vancomycin at pulmonary concentrations (intravenous and inhaled) against four clinical MRSA CF sputum isolates in planktonic and biofilm time-kill (TK) experiments. Vancomycin minimum inhibitory concentrations (MICs) were determined for these isolates at standard inoculum (SI) (~106 CFU/mL) and high inoculum (HI) (~108 CFU/mL) as well as in biofilms cultivated using physiological medium representing the microenvironment of the CF lung. Vancomycin concentrations of 10, 25, 100 and 275 µg/mL were evaluated in TK experiments against planktonic MRSA at varying inocula and versus biofilm MRSA. Vancomycin MICs increased from 0.5 µg/mL when tested at SI to 8-16 µg/mL at HI. Vancomycin MICs were further increased to 16-32 µg/mL in biofilm studies. In TK experiments, vancomycin displayed bactericidal activity (≥3 log10 killing at 24 h) against 1/4 and 0/4 planktonic MRSA isolates at SI and HI, respectively, whereas vancomycin was bactericidal against 0/4 isolates against MRSA biofilms. Based on these findings, vancomycin monotherapy appears unlikely to eradicate MRSA from the respiratory tract of patients with CF, even at high concentrations similar to those observed with inhaled therapy. Novel vancomycin formulations with enhanced biofilm penetration or combination therapy with other potentially synergistic agents should be explored.

The Antibacterial Efficacy and In Vivo Toxicity of Sodium Hypochlorite and Electrolyzed Oxidizing (EO) Water-Based Endodontic Irrigating Solutions.

The objective of this study was to evaluate the antibacterial efficacy against Enterococcus faecalis and Streptococcus mutans and in vivo toxicity using embryonic zebrafish assays of sodium hypochlorite (NaOCl) and electrolyzed oxidizing (EO) water (containing hypochlorous acid (HOCl))-based root canal irrigating solutions. Methodology: Using 100 μL microbial count of 1 × 108 cfu/mL Enterococcus faecalis to mix with each 10 mL specimen of NaOCl or HOCl for designed time periods. The above protocol was also repeated for Streptococcus mutans. The concentration of viable microorganisms was estimated based on each standardized inoculum using a plate-count method. Zebrafish embryo assays were used to evaluate acute toxicity. Results: All the HOCl or NaOCl treatment groups showed > 99.9% antibacterial efficacy against Enterococcus faecalis and Streptococcus mutans. Zebrafish embryos showed almost complete dissolution in 1.5% NaOCl within 5 min. Both survival rates after being treated with 0.0125% and 0.0250% HOCl for 0.5 min or 1.0 min were similar to that of E3 medium. Conclusions: Both NaOCl and HOCl revealed similar antibacterial efficacy (> 99.9%) against Enterococcus faecalis and Streptococcus mutans. While 1.5% NaOCl fully dissolved the Zebrafish embryos, both 0.0125% and 0.0250% HOCl showed little in vivo toxicity, affirming its potential as an alternative irrigation solution for vital pulp therapy.

Evaluation of Antimicrobial Activity and Synergistic Effect of Spices against Few Selected Pathogens

Objectives: The main objective of this study was to evaluate antimicrobial activity of ethanolic extract of spices along with determination of its synergistic effect against few selected pathogens.&#x0D; Methods: In this study, ethanolic extract of 5 different spices; Zingiber officinale (Ginger), Allium sativum (Garlic), Curcuma longa (Turmeric), Capsicum annum (Chili) and Allium cepa (Onion) were obtained by using Soxhlet apparatus. The ethanolic extract was concentrated by evaporation and different concentrations of extract were prepared in Dimethy Sulphoxide (DMSO) solvent. Test organisms included mainly pathogens i.e. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae. The antimicrobial activities of the extracts were determined by well diffusion technique both individually and in combination. On the other hand, Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) was determined by serial dilution technique. The result were interpreted on the basis of the fact that the growth occurs in positive control and other tubes with inadequate amount of extract whereas the lowest concentration of agent that inhibits growth of organism, detected by lack of visible turbidity by inhibition of 99% is designed as the MIC. The MBC is identified by determining the lowest concentration of extract solution that reduces the viability of the initial bacterial inoculum by a predetermined reduction such as ≥99.9%. Likewise, for determination of Fractional Inhibitory Concentration Index (FICI), two extracts were combined along with standardized inoculum of bacterial strain. Tubes without visible turbidity were streaked on agar plate and observed for 99.9% killing. &#x0D; Results: All the tested extract of spices were found effective against S. aureus and K. pneumoniae only. The highest zone of inhibition (ZOI) was found in chili extract (ZOI=26 mm) against S. aureus whereas lowest zone of inhibition was found in garlic extract against K. pneumoniae (ZOI=12mm). Similarly, highest ZOI was produced by combined extract of both Turmeric and Ginger (ZOI= 26 mm). Turmeric extract was found to be effective against S. aureus (MIC value = 62.5 mg /ml and MBC value = 31.25 mg/ml) and K. pneumoniae (MIC value 125 mg/ml and MBC value = 62.5 mg/ml). The Fractional Inhibitory Concentration (FIC) values of combined extract suggested synergistic and additive effect (0.5&lt;FIC&lt;1). Chili and ginger were effective with FIC value of 0.25.&#x0D; Conclusion: To recapitulate, the extract of spices can be used to prevent the pathogenic organism.

713. Preventive Administration of MEDI6389, a Combination of Monoclonal Antibodies (mAbs) Targeting Alpha-Toxin (AT), Panton-Valentine Leukocidin (PVL), Leukocidin ED (LukED), Gamma-Hemolysin and Clumping Factor A (ClfA), in a Rabbit Model of USA300 MRSA Prosthetic Joint Infection (PJI)

BackgroundmAbs targeting staphylococcal virulence factors could represent an interesting preventive strategy in PJI. We evaluate here MEDI6389 compared with isotype-matched control IgG (c-IgG) in a rabbit model of USA300 MRSA PJI.MethodsRabbits were randomized for prophylaxis with either c-IgG (n = 13; 30 mg/kg; controls) or MEDI6389 (n = 13; 30 mg/kg of each mAb) administered intravenously 12h before infection. A cemented screw and ultrahigh-molecular-weight polyethylene washer were placed intraarticularly in the external femoral condyle. After suturing the joint capsule and musculocutaneous layers, 300 µL of a standardized bacterial inoculum containing 5 × 105 CFU of a USA300 MRSA clinical isolate were injected intraarticularly. Animals were euthanized on day 8 and the knee joint was harvested for bacteriological analysis (synovial bacterial counts, and enumeration of screw-adherent bacteria after sonication) and histology (conventional pathology, and transmission electron microscopy [TEM] for neutrophils analysis). In vivo observations made on neutrophils were confirmed by TEM analysis of human neutrophils incubated in vitro with purified PVL, LukED, and gamma-hemolysin with or without the corresponding mAb.ResultsIn comparison with the control group, the average amount of pus (1.7 ± 1.8 vs. 3.1 ± 1.2 g, P = 0.026) and the number of bacteria in the synovial pus (5.9±1.5 vs. 7.2±1.4 log10 CFU, P = 0.031) and on the screw (2.7 ± 1.5 vs. 4.1 ± 1.6 log10 CFU, P = 0.035) were decreased in animals pretreated by MEDI6389. Conventional pathological examination showed a marked reduction in synovitis of MEDI6389-pretreated animals. TEM of synovitis harvested from infected knee joints of control animals showed significant greater number of abnormal neutrophils that appeared rounded, with condensed nucleus and no granules, compared with those pretreated with MEDI6389 (P = 0.002). This classical leukocidin-induced neutrophilic killing phenotype could be neutralized with anti-leukocidin mAbs using ex vivo human neutrophils incubated with PVL, LukED, HlgAB, or HlgCB.ConclusionThe preventive administration of MEDI6389 allows a reduction of local inflammation and bacterial burden in this USA300 MRSA rabbit PJI model. Disclosures All authors: No reported disclosures.

2176. A New Rapid Test for Detection of The Cefazolin Inoculum Effect (CIE) in Methicillin-Susceptible Staphylococcus aureus (MSSA)

BackgroundMost MSSA harbor one of the four different variants of β-lactamase (BlaZ) (A, B, C and D). The CIE is defined as an MIC >16 μg/mL when a high inoculum (107 CFU/mL) is used and depends on the presence of BlaZ. The presence of the CIE has been associated with therapeutic failure in invasive MSSA infections. In some countries of South America, the prevalence of CIE is high, ranging from 36% to 51% (Colombia and Argentina, respectively). Type A BlaZ is most often associated with the CIE due to its high affinity for cefazolin. Here, we developed a rapid test based on the premise that the extracellular form of BlaZ is responsible for the CIE. We aimed to identify invasive MSSA that exhibit the CIE and validate the test in two cohorts of isolates from patients in Colombia and ArgentinaMethods152 MSSA clinical isolates were collected from Colombia (n = 71) and Argentina (n = 81). We determined MIC at standard and high inoculum. We developed a test using induction of BlaZ with ampicillin (150 μg/mL) for 20 minutes and, using the supernatant for incubation with nitrocefin for 30 min. A change in color from yellow to red was considered positive. MSSA TX0117 (BlaZ +, with the CIE), ATCC 29213 (BlaZ-negative) and ATCC 25923 (BlaZ + lacking the CIE) were used as controls. BlaZ typing of all Argentinian isolates was available by sequencingResultsA high proportion (43%) of MSSA exhibited the CIE (34% and 52% of Colombian and Argentinian isolates, respectively) by MIC. The rapid test identified 76% of isolates exhibiting the CIE and correctly ruled out all isolates lacking the CIE (sensitivity 80%, specificity 100%). Furthermore, the rapid test detected all isolates with the CIE that harbored Type A BlaZ from Argentina. Conversely, the test failed to identify the CIE in Argentinian isolates that produce type B and C BlaZ. The sensitivity and specificity of the rapid test for the Colombian isolates whose BlaZ type was unknown were 89% and 100%, respectively.ConclusionA rapid test of less than 2 h can readily identify MSSA isolates exhibiting the CIE. For isolates carrying type A BlaZ, which is highly associated with the CIE, the test had a sensitivity and specificity of 100%. Rapid identification of MSSA with the CIE may have important therapeutic consequences in deep-seated infectionsDisclosures All authors: No reported disclosures.

207. High Severity and Mortality Due to Methicillin-Susceptible Staphylococcus aureus Infections in a Colombian Hospital

BackgroundMethicillin-susceptible Staphylococcus aureus (MSSA) infections are considered less severe than those caused by methicillin-resistant S. aureus. However, we have observed an important increase in severe cases of invasive MSSA infections in a hospital in Pereira, Colombia. Here, we characterize the clinical outcomes and epidemiology of these infections.MethodsWe included adult and pediatric patients hospitalized between February 2018 and April 2019 presenting with invasive infections caused by MSSA. All isolates were sent to a central laboratory to confirm identification. We determined cefazolin MICs at standard (105 CFU/mL) and high inoculum (107 CFU/mL) by broth microdilution and a rapid test to detect cefazolin inoculum effect (CIE). The CIE was defined as an increase of MIC to ≥16 µg/mL when tested at high inoculum. Clinical data (demographics, intensive care unit (ICU) admission, therapy and mortality) were obtained from medical records.ResultsA total of 60 patients were included in the study and 41.6% were women. Most (63.3%) infections were hospital-associated. Bacteremia was the most frequent type of infection (71.6%). The mean duration of hospital stay was 24.5 days (IQR, 14–44). 61.6% of patients were admitted to the ICU with a mean length of stay of 14 days (IQR, 7–30). Mortality at 30 days was 28.3% (17 out of 60 patients) and was slightly higher (30.2%) in patients with bacteremia. Early mortality (48 h) was 10% (n = 6). Most patients (75%) received β-lactams (28.8% cefazolin and 84% oxacillin). 18 patients (33%) had isolates that exhibited the CIE but most (n = 11) received oxacillin. Among 17 patients who died, 35% had received antibiotics other than β-lactams (5 vancomycin, 1 ampicillin-sulbactam) and two did not received any therapy due to rapid death. The mean duration of antibiotic therapy was 11 days. Source control was deemed appropriate in 65% of the cases.ConclusionAn increase in severe invasive infections caused by MSSA was observed in our hospital with a high proportion of patients requiring ICU care. A significant proportion of patients received inappropriate treatment. Due to the aggressive nature of invasive MSSA infections, efforts to optimize appropriate therapy for these infections are urgently needed in Colombia.Disclosures All authors: No reported disclosures.

2202. Validation of a Rabbit Model of Pseudomonas aeruginosa Acute Pneumonia

BackgroundSeverity and antimicrobial resistance of P. aeruginosa (PA) hospital-acquired pneumonia led the FDA to encourage the development of animal models for preclinical evaluation of new therapeutic strategies. We present here the validation of a rabbit model of PA acute pneumonia.MethodsRabbits were infected by endotracheal instillation of 1.8 mL of a standardized inoculum containing 9 × 107 CFU of PA clinical strain 6206 (predetermined 100% lethal dose). The natural history of the disease was described by the following parameters evaluated at 3, 4, 5, 6, 10 hours post-infection (hpi) and at the time of spontaneous death (6 rabbits/group): lung-to-body weight ratio (LW/BW), pulmonary, splenic and renal bacterial counts, pulmonary histology and blood markers (blood cell counts, blood gas and IL-8). Three groups of 12 rabbits were then treated with saline (controls), tobramycin or meropenem at doses determined by PK/PD analysis to confirm the efficacy of a human-equivalent dosing regimen.ResultsPA strain 6,206 caused fatal pneumonia in 13–23 hours by acute respiratory distress syndrome (pulmonary edema and necrosis with LW/BW > 10, pO2 <40 mmHg) and/or sepsis (hyperlactatemia, hypoglycemia, cytopenias). LW/BW and pulmonary bacterial counts increased significantly over time. The splenic and renal bacterial spread was constant after 6 hpi. Hypoxemia <60 mmHg appeared at 5 hpi for 4/6 rabbits, associated with elevated plasma IL-8 concentration, massive neutrophilic influx into the airspace, lung necrosis, hemorrhage, and pulmonary edema formation. Consequently, 5 hpi appeared as the most appropriate time to trigger a therapeutic intervention. Meropenem (80 mg/kg/q2h) or tobramycin (1 injection of 2.5 mg/kg, then saline/q2h) showed superiority over saline, with a mortality rate of 33% and 17% vs. 100%, and an LW/BW ratio of 8.53 and 8.54 vs. 13.9, respectively. Tobramycin was less effective than meropenem in clearing bacteria, with, respectively, 1 and 9 out of 12 rabbits having sterile samples.ConclusionThis rabbit model of PA acute pneumonia is a reliable evaluation tool for new therapeutic strategies. Our study also provides guidance for the development of animal models by describing the natural history of disease and therapeutic validation. Disclosures All authors: No reported disclosures.

Alternative method in Galleria mellonella larvae to study biofilm infection and treatment

In vivo studies are crucial decision-maker step in order to translate in vitro data to an applied therapy. Considering this we describe a simple method that analyzes and quantifies biofilm formation inside the Galleria mellonella larvae. Toothbrush bristles were employed as an abiotic surface to mimic a medical device. A standardized inoculum of Staphylococcus aureus was systemically injected in the larvae together with the insertion of a bristle in the last proleg pair. After incubation adhered cells were detached from bristles and quantified by colony-forming units (CFU) counting using staphylococci-selective medium. About 3 × 106 CFU of S. aureus were recovered from bristles and scanning electron microscopy (SEM) images confirmed biofilm formation. Control group did not show adherent bacteria, as demonstrated by absence of CFU counting and SEM images, indicating that the insertion procedure is free of bacterial contamination. We present a feasible method to evaluate bacterial biofilm formation in vivo that in the near future can be used to evaluate antibiofilm compounds.

Comparative evaluation of plazomicin MICs obtained using agar dilution versus broth microdilution methods and impact of inoculum size against ESBL-producing Escherichia coli, carbapenemase-producing Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus clinical isolates

MICs of plazomicin were determined by agar dilution and broth microdilution in 187 ESBL-producing Escherichia coli (n = 73), carbapenemase-producing Klebsiella pneumoniae (n = 55) methicillin-resistant Staphylococcus aureus (n = 59) clinical isolates. Inoculum effect was determined by broth microdilution assay using two different inocula; 1–5 × 105 (standard inoculum) and 1–5 × 108 CFU/mL. For all isolates tested >98% categorical agreement and ≥95% of essential agreement (±1elog2) was found. At high inocula, MICs of plazomicin increased ≥ eight-fold for 25% of E. coli, 24% of K. pneumoniae and 7% of S. aureus isolates tested. The results indicate that agar dilution and broth microdilution methods were equally suitable for determining plazomicin MICs. Inoculum effect was observed for plazomicin in Escherichia coli and Klebsiella pneumoniae isolates. Further studies that establish the genetic background of the isolates are required to better understand the reasons behind the inoculum effect.

Efficacy of Antiseptic Solutions in Treatment of Staphylococcus Aureus Infected Surgical Wounds with Patches of Vascular Graft: An Experimental Study in Rats.

Background and objectives: Treatment of a prosthetic vascular graft infection (PVGI) remains a challenging problem in vascular surgery. The aim of this study was to design a novel rat model for treatment of peripheral vascular prosthesis infection caused by Staphylococcus aureus (S. aureus) and to determine the efficacy of different antiseptic solutions in suppressing or eradicating infection from the wound and the graft material itself. Materials and methods: A piece of Dacron vascular prosthesis was surgically implanted at the dorsum of 48 Wistar rats and the wounds were infected with 5 McFarland standard inoculum of S. aureus. Suppurating wounds were daily irrigated with different antiseptic solutions: octenidine dihydrochloride, povidone-iodine, chlorhexidine digluconate, and sterile saline. The antimicrobial action of antiseptics was defined according to their capability to eradicate bacteria from the graft surroundings and bacteriological examination of the graft itself. Extended studies on wound microbiology, cytology, and histopathology were performed with an additional group of 10 rats, treated with the most effective antiseptic-octenidine dihydrochloride. Results: Four-day treatment course with octenidine, povidone-iodine, and chlorhexidine resulted in 99.98% (p = 0.0005), 90.73% (p = 0.002), and 65.97% (p = 0.004) decrease in S. aureus colonies in wound washouts, respectively. The number of S. aureus colonies increased insignificantly by 19.72% (p = 0.765) in control group. Seven-day treatment course with octenidine eradicated viable bacteria from nine out of 10 wound washouts and sterilized one vascular graft. Conclusions: A reproducible rat model of PVGI with a thriving S. aureus infection was designed. It is a first PVGI animal model where different antiseptic solutions were applied as daily irrigations to treat peripheral PVGI. Seven-day treatment with octenidine eradicated bacteria from the wound washouts for 90% of rats and one vascular graft. Further studies are needed to investigate if irrigations with octenidine could properly cure vascular bed from infection to assure a successful implantation of a new synthetic vascular substitute.

Antifungal activity of acidic electrolyzed water against strawberry postharvest molds (Fragaria x ananassa Duch cv. Camarosa)

The aim of this word was to assess the in vitro antifungal activity of acidic electrolyzed water (AEW) and the required contact time to inhibit the development of post-harvest molds common in strawberry fruit. Ninety-four strawberry samples were used. A five-fold assay in a randomized design was carried out using standard inoculum of fungal conidia, which were subjected to an array of contact times and AEW concentrations, using a controlled agitation (120 r.p.m) at 23°C. The conidia treated with AEW were grown on potato dextrose agar and their germination was evaluated. Mycelial growth of the treated samples was measured and compared against conidia control samples treated with distilled water instead of AEW. Botrytis cinerea and Rhizopus stolonifer were the most commonly isolated species at 54.25% and 28.72%, respectively. AEW inhibited conidia germination after 7 minutes of exposure. The AEW concentration required to inhibit conidia germination was 10 ppm and 50 ppm for R. stolonifer and B. cinerea respectively, a variance possibly due to the constitution of their cellular walls. A short exposure of AEW is able to inhibit post-harvest fungi growth, and is thus recommended as an efficient, economical, and environmentally sustainable sanitation agent, with the ability of extending the strawberry post-harvest consumption period.

Effect of inoculum type and organic loading on biogas production of sunflower meal and wheat straw

Inoculum plays a vital role in providing initial microbial population in anaerobic process. There is unavailability of standard inoculum in the Pakistan and the available inoculum requires pretreatment and preparation before use. Thus, in this study the effect of inoculum type and organic loading (OL) on the biogas yield of sunflower meal and wheat straw was evaluated. For this purpose, lab scale batch experiments were conducted at OL of 2 g VS L− 1 in 225 mL glass bottle using digested manure, acclimatized sludge and septic tank sludge as inoculum. The highest biogas yield and volatile solids reduction of 768 NmL g− 1 VS and 78%, respectively, were observed from sunflower meal with digested manure. Reactor inoculated with digested manure also showed better buffering capacity in terms of pH. The inoculum selected from first experiment was used to study the effect of OL (2, 6, 10 and 14 g VS L− 1) on biogas yield. Results showed that biogas yield decreased with the increased in OL. Stability parameters revealed that reactors can be operated safely up to OL of 10 g VS L− 1. Biogas production data were modelled by modified Gompertz function.

OPTIMIZATION THE ALGINATE PRODUCTION CONDITIONS IN SWEET WHEY MEDIUM BY AZOTOBACTER CHROCOCCUM

Sweet whey was used as medium for microbial alginate production by Azotobacter chrococcum NCBI MH249629. To optimize alginate production and achieve high yield of alginate, sweet whey medium was prepared using different lactose concentrations (20, 25, 30, 35, 40, 45, 50 g/L), added organic and inorganic nitrogen sources (yeast extract, Malt extract, ammonium sulphate and ammonium nitrate), kept at different temperatures (25, 30, 32, 35 & 37oC), different pH values (4, 5, 6, 7, 8, 9), different agitation speed (100, 120, 150, 170 & 200 rpm), different inoculum size (1,2,3,4,5,6,7,8,9,10%) and cultivated for different fermentation time (0, 12, 24, 36, 48, 60, 72, 84, 96, 108 & 120h). After fermentation time, produced microbial alginate was measured. The results showed that sweet whey could be used as a fermentation medium by Azotobacter chrococcum to produce microbial alginate. The highest yield of alginate (12.56%) as well as productivity of alginate (0.08 g/L/h) was obtained by fermentation of Azotobacter chrococcum in sweet whey medium optimized to 45 g/L lactose concentration as a carbon source and by providing the following conditions which yeast extract 1.5 g/L and ammonium nitrate 0.1 g/L as a nitrogen source and growth promoter, Na2HPO4 1 g/L as mineral salts, Initial pH at 7, size of standard inoculums was 9 % of fermentation medium and the incubation period was 72 hrs.