Antimicrobial Efficacy Testing Research Articles

Variables Affecting the Recovery of Acanthamoeba Trophozoites.

While the results of Acanthamoeba testing have been extensively published, laboratories conducting such testing are left to develop their own methods in the absence of a standardized methodology. The wide disparity of methods has resulted in equally inconsistent reported results for contact lens care (CLC) products. This study’s objective was to determine the source of these discrepancies by evaluating basic Acanthamoeba biology and their impact on antimicrobial efficacy testing, including the ability of a recovery method to stimulate a single trophozoite to proliferate. Antimicrobial efficacy testing was conducted using well-published Acanthamoeba strains, storage conditions, and growth-based recovery methods. To identify variables that influence results, test solutions with low Acanthamoeba disinfection rates were utilized to prevent differences from being masked by high log reductions. In addition, single-cell proliferation assays were executed to understand the growth requirements to stimulate trophozoite propagation in two recovery methods. These studies indicated that both nutrient density (>106 CFU) and the length of plate incubation (at least 14 days) could significantly influence the accurate recovery of trophozoites. Together, this study emphasizes the need to understand how Acanthamoeba trophozoites biology can impact test methods to create divergent results.

Antimicrobial preservation efficacy of liquid glucose and liquid maltitol syrups with and without 0.1% sorbic acid

Abstract Objectives Amongst paediatric pharmaceutical forms, syrups offer advantages such as ease of administration and good palatability. They also exhibited microbial self-preservation properties that may be useful to enhance shelf life of liquid formulation. The objective of our works is to test the self-preservation efficacy of maltitol and glucose syrup without or with sorbic acid as described in the European pharmacopoeia. Methods The European Pharmacopoeia test of antimicrobial preservation efficacy was performed on liquid glucose syrup and liquid maltitol syrup with and without 0.1% sorbic acid. Results Unpreserved glucose and maltitol syrups did not meet the European Pharmacopoeia acceptance criteria for antimicrobial preservative efficacy due to the regrowth of Aspergillus brasiliensis on day 28 whereas glucose and maltitol syrups with 0.1% sorbic acid pass the test. Conclusions The addition of a preservative (sorbic acid) in glucose and maltitol syrups allows the validation of the antimicrobial preservative efficacy test of the European Pharmacopoeia. Further tests are needed to see if preservative efficacy is maintained despite dilutions or in the presence of active pharmaceutical ingredients.

Evaluating Alternate Methods of Determining the Antimicrobial Efficacy of Contact Lens Care Products against Acanthamoeba Trophozoites

Acanthamoeba keratitis (AK) is a serious ocular infection caused by a ubiquitous free-living amoeba, Acanthamoeba. This infection often results in extensive corneal damage and blindness, and is notoriously difficult to cure. While Acanthamoeba is an abundant organism, AK is most associated with contact lens hygiene noncompliance and inadequate contact lens care (CLC) disinfection regimens. Thus, accurate and timely antimicrobial efficacy testing of CLC solutions is paramount. Published methods for antimicrobial efficacy testing of Acanthamoeba trophozoites requires 14 days for results. Presently, alternate and/or rapid methods for evaluating CLC products rarely demonstrate equivalent results compared to commonly-reported methods. Propidium iodide is a cellular stain that can only bind to cells with damaged outer membranes. We evaluated propidium iodide staining as an alternative method for determining the relative antimicrobial efficacy of 11 different CLC products against Acanthamoeba trophozoites. Following exposure to a CLC product, the fluorescence intensity of propidium iodide in an Acanthamoeba population demonstrated a strong correlation to the log reduction determined by established, growth-based Acanthamoeba testing used to evaluate the antimicrobial efficacy of CLC products. Thus, propidium iodide was found to be an effective rapid tool for determining cell death in Acanthamoeba trophozoites following exposure to CLC solutions.

Method development for efficacy testing of veterinary disinfectants using bacteriophage MS2

Method development for efficacy testing of veterinary disinfectants using bacteriophage MS2

Hydrogen Peroxide Disinfecting Solution for Gas Permeable Contact Lenses: A Review of the Antimicrobial Efficacy, Compatibility, and Safety Performance of a One-Step Lens Care System.

CLEAR CARE PLUS (CCP), also known as AOSEPT PLUS with HydraGlyde, is approved for use with gas permeable (GP) lenses, and the indication is supported by the scientific evidence that is reviewed in this article. Antimicrobial efficacy testing of CCP both as a stand-alone disinfectant and as part of a regimen shows that CCP exceeds the ISO 14729 criteria against bacteria, yeast, and mold. In real-world conditions, it is effective against clinically relevant bacterial strains isolated from adverse events and against the two forms, trophozoites and cysts, of resilient Acanthamoeba species. Compatibility tests of CCP with two types of GP lenses indicate that the physical and/or optical parameters of lenses are unaffected through 30 cycles of simulated use with CCP, and a clinical trial shows substantial equivalence of clinical performance with a commonly used GP multipurpose solution. These results indicate that CCP is well suited for cleaning and disinfection of GP contact lenses.

Virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus and avian influenza virus.

This study evaluated the virucidal efficacy of acidic electrolyzed water (AEW) against African swine fever virus (ASFV) and avian influenza virus (AIV), according to the Animal and Plant Quarantine Agency (APQA) guidelines for efficacy testing of veterinary disinfectants. AEW (pH 5.0–6.5) was prepared using a commercially available “Electrolyzed Water Generator” with a free chlorine concentration (FCC) of 5–140 ppm, and its efficiency in reducing the titer of ASFV and AIV was tested in a suspension under low- and high-level organic soiling. Under low-level organic soiling conditions, AEW with FCC ≥40 ppm was effective against ASFV; under high-level organic soiling conditions, AEW with FCC ≥80 ppm was effective against ASFV. Under low-level organic soiling conditions, AEW with FCC ≥60 ppm was effective against AIV; under high-level organic soiling conditions, AEW with FCC ≥100 ppm was effective against AIV. The virucidal effect of AEW seemed dependent on the FCC and the presence of organic soiling. Based on these data, we recommend the following minimum FCCs in AEW treatment for routine disinfection in veterinary field under low- and high-level organic soiling conditions: for ASFV, 50 ppm and 100 ppm; and for AIV, 75 ppm and 125 ppm, respectively. In conclusion, the virucidal effects of AEW against ASFV and AIV emphasize its potential utility as a disinfectant, and we suggest considering organic soiling conditions while using AEW for implementing effective control measures for field applications.

Recovery and chemical disinfection of foot-and-mouth disease and African swine fever viruses from porous concrete surfaces.

AimsDevelop an effective laboratory method to consistently recover viral loads from porous concrete coupons sufficient for disinfectant efficacy testing. Investigate the role of concrete matrix pH on the recovery of foot‐and‐mouth disease virus (FMDV) and African Swine Fever virus (ASFV) from porous concrete. Compare parameters off FMDV and ASFV inactivation on porous and nonporous surfaces in quantitative carrier tests of a liquid chemical disinfectant.Methods and resultsConcrete test coupons were fabricated from commercial and industrial sources and carbonated by exposure to 5% CO2 in a humidified incubator, lowering the matrix pH. Neither dried FMDV nor ASFV were recovered from high‐pH concrete control coupons. Recovery of infectious virus from lower pH carbonated concrete was similar to stainless steel coupon controls. Exposure to the liquid disinfectant Virkon™ S inactivated FMDV and ASFV on porous concrete.ConclusionsConcrete matrix pH had a greater impact than surface porosity on the ability to recover viable virus from unsealed concrete.Significance and Impact of the StudyConcrete is commonly found in environments where virus decontamination is required. This study demonstrates a reproducible method to recover sufficient viral loads from porous concrete coupons to facilitate quantitative carrier testing. This method provides a basis for evidence‐based validation testing of chemical disinfectants to inactivate pH‐sensitive viruses on unsealed concrete.

Impact of pH on efficacy of peroxy acetic acid against Salmonella, Campylobacter, and Escherichia coli on chicken wings

Peroxy acetic acid (PAA) is widely used as an antimicrobial in poultry processing, specifically in the chiller. While the natural pH of PAA at the concentrations used is between 4.5 and 6.0, poultry processors adjust the pH to ≥8.0 to maintain product yield. The objective of this study was to evaluate 1) efficacy of PAA at different concentrations, pH, and contact times against Salmonella, Campylobacter, and Escherichia coli and 2) use of E. coli as a surrogate for Salmonella and Campylobacter to conduct validations studies for poultry processing. Fresh chicken wings (0.45 Kg) were inoculated with a cocktail of nalidixic acid–resistant Salmonella Typhimurium, rifampicin-resistant E. coli (5-strain cocktail), and gentamicin-resistant Campylobacter coli. Inoculated chicken wings were immersed in PAA solutions of 50, 250, and 500 ppm adjusted to pH 8.2 and 10.0 as well as nonadjusted PAA solutions for 10 s and 60 min. Treated chicken wings were rinsed in chilled buffered peptone water, serially diluted, and plated on Petrifilm APC for enumerating Salmonella and E. coli populations and spread plated on Campy Cefex Agar containing gentamicin (200 ppm) to enumerate Campylobacter. Immersion of chicken wings in 500 ppm of PAA (non–pH-adjusted) for 60 min resulted in greater microbial reductions (P ≤ 0.05) of Salmonella, Campylobacter, and E. coli populations of 2.56, 1.90, and 2.53 log CFU/mL, respectively. Higher concentrations and longer exposure times resulted in greater reductions (P ≤ 0.05) of Salmonella, E. coli, and Campylobacter populations, and increasing pH of PAA solution did not affect (P > 0.05) its efficacy. A high correlation (r = 0.93) was observed between E. coli (surrogate) and Salmonella populations suggesting that E. coli can be used as a surrogate for Salmonella for conducting validation studies for antimicrobial efficacy testing in poultry processing.

A rapid model for developing dry surface biofilms of Staphylococcus aureus and Pseudomonas aeruginosa for in vitro disinfectant efficacy testing

BackgroundBacterial biofilms persistent on dry environmental surfaces in healthcare facilities play an important role in the occurrence of healthcare associated infections (HAI). Compared to wet surface biofilms and planktonic bacteria, dry surface biofilms (DSB) are more tolerant to disinfection. However, there is no official method for developing DSB for in vitro disinfectant efficacy testing. The objectives of this study were to (i) develop an in vitro model of DSB of S. aureus and P. aeruginosa for disinfectant efficacy testing and (ii) investigate the effect of drying times and temperatures on DSB development. We hypothesized that a minimum six log10 density of DSB could be achieved on glass coupons by desiccating wet surface biofilms near room temperatures. We also hypothesized that a DSB produced by the model in this study will be encased in extracellular polymeric substances (EPS).MethodsS. aureus ATCC-6538 and P. aeruginosa ATCC-15442 wet surface biofilms were grown on glass coupons following EPA MLB SOP MB-19. A DSB model was developed by drying coupons in an incubator and viable bacteria were recovered following a modified version of EPA MLB SOP MB-20. Scanning electron microscopy was used to confirm the EPS presence on DSB.ResultsOverall, a minimum of six mean log10 densities of DSB for disinfectant efficacy were recovered per coupon after drying at different temperatures and drying times. Regardless of strain, temperature and dry time, 86% of coupons with DSB were confirmed to have EPS.ConclusionA rapid model for developing DSB with characteristic EPS was developed for disinfectant efficacy testing against DSB.

Borderline resistance to oxacillin in Staphylococcus aureus after treatment with sub-lethal sodium hypochlorite concentrations

Surface disinfectants are regularly used in prophylactic and infection control measures. Concern has been raised whether residues of sub-inhibitory disinfectant concentrations may constitute a selective pressure and could contribute to the development of strains which are tolerant and/or resistant to biocides including antibiotics. The current study investigated whether Staphylococcus (S.) aureus ATCC® 29213™ and ATCC® 6538™ would change their growth characteristics and antimicrobial susceptibility profiles after prolonged treatment with sub-inhibitory concentrations of sodium hypochlorite (NaOCl). NaOCl is a fast-acting disinfectant with a broad-spectrum activity, inexpensive and widely used in healthcare and the food production industry. Minimum inhibitory concentration (MIC) for NaOCl was determined by broth macrodilution according to the guidelines for disinfectant efficacy testing provided by the German Veterinary Medical Society. Serial passages after 24 h and 72 h, respectively, in defined sub-inhibitory concentrations of NaOCl resulted in a number of phenotypic variants. Two of these variants, derived from S. aureus ATCC® 29213™, showed elevated MICs of oxacillin and were considered as in vitro-generated borderline oxacillin-resistant S. aureus (BORSA). Transmission electron microscopy revealed a significantly thickened cell wall in these isolates, a phenomenon that has also been described for Listeria monocytogenes after low-level exposure to NaOCl. Whole genome sequencing revealed an early stop codon in the gene coding for the GdpP protein and thereby abolishing the function of this gene. GdpP represents a phosphodiesterase that regulates gene expression, and loss of function of the GdpP protein has been described in association with borderline oxacillin resistance. Our findings suggest that a mutation in the GdpP protein gene and morphological changes of the cell wall were induced by repeated exposure to sub-lethal NaOCl concentrations, and most likely accounted for a BORSA phenotype in two variants derived from S. aureus ATCC® 29213™.

Antimicrobial photodynamic therapy efficacy against specific pathogenic periodontitis bacterial species.

To determine the safety and efficacy of antimicrobial photodynamic therapy (aPDT) combination of 0.33 mM Toluidine Blue O (TBO) with 60 mW/cm2 LED irradiation for 5 min that we had established, this study investigated the cytotoxic effect of aPDT combination on mammalian oral cells (gingival fibroblast and periodontal ligament cells) and compared the antimicrobial efficacy of antibiotics (the combination of amoxicillin (AMX) and metronidazole (MTZ)) against representative periodontitis pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans) versus our aPDT combination. aPDT combination did not show any detectable effect on the viability of Streptococcus sanguinis or Streptococcus mitis, the most common resident species in the oral flora. However, it significantly reduced CFU values of P. gingivalis, F. nucleatum, and A. actinomycetemcomitans. The cytotoxicity of the present aPDT combination to mammalian oral cells was comparable to that of standard antiseptics used in oral cavity. In antimicrobial efficacy test, the present aPDT combination showed equivalent bactericidal rate compared to the combination of AMX + MTZ, the most widely used antibiotics in the periodontitis treatment. The bactericidal ability of the AMX + MTZ combination was effective against all five bacteria tested regardless of the bacterial species, whereas the bactericidal ability of the aPDT combination was effective only against P. gingivalis, F. nucleatum, and A. actinomycetemcomitans, the representative periodontitis pathogenic bacterial species. The present study demonstrated the safety and efficacy of the present aPDT combination in periodontitis treatment. TBO-mediated aPDT with LED irradiation has the potential to serve as a safe single or adjunctive antimicrobial procedure for nonsurgical periodontal treatment without damaging adjacent normal oral tissue or resident flora.

Customized antimicrobial efficacy tests offer superior evaluation of growth inhibitor efficacy for liquid microbial products

Antimicrobial effectiveness tests are common methods used to assess the risk of microbial contamination in pharmaceuticals and cosmetics. These assays may be inappropriate for endospore-based microbial products which often carry a similar – if not greater – risk of microbial contamination. In the present study, we compared the antimicrobial efficacy assessment provided by United States Pharmacopeia Chapter <51> Antimicrobial Effectiveness Testing with a modified test which utilized a customized bacterial challenge. The customized challenge inoculum comprised an assemblage of 12 bacterial strains (both pathogens and spoilage organisms) isolated from the product's end-use geography. Results suggest that some microbial inhibitor systems which pass industry standard antimicrobial effectiveness tests may fail when challenged with a customized bacterial assemblage. In order to provide the best possible assessment of microbial inhibitor systems for liquid Bacillus products, we suggest that new antimicrobial effectiveness tests be developed for this product class which include the addition of field-relevant contaminants in addition to the industry standard pathogen challenge.

Production of Garbage Enzyme from Different Fruit and Vegetable Wastes and Evaluation of its Enzymatic and Antimicrobial Efficacy

Objectives: To evaluate the enzymatic and antimicrobial efficacy of enzyme from garbage produced from different fruits and vegetable wastes.&#x0D; Methods: This study was conducted from October-2018 to February-2019 in the laboratory of Padma Kanya Multiple College, Bagbazar, Kathmandu, Nepal. This study was carried for production, analysis of enzymatic and antimicrobial efficacy by using yeast (Saccharomyces cerevisae) and bacteria (Bacillus species) in 5 fruits peels, Mosambi (Citrus limetta), Pomegranate (Punica granatum), Pineapple (Ananas comosus), Papaya (Carica papaya) and mixed fruits collected from fresh fruit stall and vegetable peels collected from college’s hostel. The fermentation mixture was made in the ratio 1:3:10 (1 part brown sugar, 3 parts fruits/vegetable peels and 10 parts water) and left for 3 months for fermentation.&#x0D; Results: After fermentation, enzyme activity (amylase, protease, caseinase, cellulase and lipase) and antimicrobial efficacy (S. aureus, S. aureus (ATCC 25923), Bacillus spp, Salmonella Typhi, E. coli, E. coli (ATCC 25922), Shigella spp, Pseudomonas aeruginosa) were analyzed. All the samples showed amylase and caseinase enzyme activity, only Pineapple (Ananascomosus), Papaya (Carica papaya) and Mixed fruit showed protease enzyme activity while only Pomegranate (Punicagranatum) showed lipase enzyme activity. In antimicrobial efficacy test, garbage enzyme produced from vegetable sample didn’t show antimicrobial activity with bacteria used except E. coli (ATCC 25922)and S. aureus (ATCC 25923). Similarly, garbage enzyme produced from Mixed fruit and Papaya (Carica papaya) didn’t show antimicrobial activity with Salmonella Typhi and S. aureus (ATCC 25923) respectively but garbage enzyme from other wastes showed antimicrobial activity with bacteria used in test.&#x0D; Conclusion: Different fruits and vegetables wastes showed different enzyme activity and antimicrobial activity.

Temporal efficacy of antimicrobials against aerobic bacteria isolated from equine endometritis: an Italian retrospective analysis (2010–2017)

This study aimed to describe bacteria isolated from the reproductive tract of mares and to identify changes in antimicrobial susceptibility patterns to those antibiotics commonly used for the treatment of...

Conditions for the disinfectant efficacy test under subzero temperatures

Conditions for the disinfectant efficacy test under subzero temperatures

Novel Methods for Efficacy Testing of Disinfectants – Part II

AbstractThe control of infections and maintenance of hygienic conditions are of central importance and the insights gained through several investigations have practical significance today. In contrast, the maintenance of environment and surface disinfection is still controversial and demands novel disinfectants to meet the required criteria. The healthcare centers are fraught with various microorganisms and serve as a point source for multidrug resistance in patients which is more critical to treat. Therefore, it has begun a comprehensive plan in hospitals to focus on disinfection and maintenance of hygiene which is not always appreciated. The urgency to determine the effectiveness of disinfectants is often questioned. The review article shows how the existing problems could be solved by a systematic approach and reports on effective evaluation studies that meet the requirements.

A Comprehensive In Vitro Evaluation of Medihoney as an Anti-Biofilm Agent in Preventing Ventricular Assist Device Driveline Infections

Purpose Medihoney is routinely applied to the exit site of Ventricular Assist Device (VAD) drivelines in patients in many Australian hospitals to prevent driveline infections. Its effectiveness as a prophylactic measure remains unclear. We performed a comprehensive in vitro study using microbiological assays that closely mimic the in vivo environment of the driveline exit-site to assess the effectiveness of medihoney in preventing biofilm-related driveline infections. Methods Antimicrobial efficacy testing of medihoney was performed against 24 clinically-relevant bacterial and fungal strains grown as planktonic and biofilm cells. The minimum inhibitory concentration (MIC), minimum biofilm inhibitory concentration, minimum eradication concentration of medihoney, and the activity of individual components of medihoney were assessed. A colony biofilm assay and a drip-flow biofilm reactor assay that mimic chronic wounds and the driveline exit site were used to evaluate medihoney activity in inhibiting early adherent organism monolayers and mature biofilms. Results Medihoney demonstrated activity against planktonic organism cells in 5/6 S. aureus strains, 6/6 of S. epidermidis strains, 1/6 of P. aeruginosa strains and 4/6 of Candida strains using a qualitative disk diffusion susceptibility assay and a quantitative broth microdilution assay [MICs, 10% weight/volume (W/V) medihoney concentrations]. Higher concentrations of medihoney (30-50%, W/V) were required to inhibit the growth of biofilm cells. Eradication of biofilms could not be achieved by medihoney, even at the highest concentration studied. The antibiofilm properties of medihoney were multi-faceted, including sugar content, pH, osmolality and the proposed active ingredient, methylglyoxal (MGO). MGO actually played a less important role than other medihoney characteristics. The colony biofilm assay and the drip flow biofilm reactor showed that medihoney was unable to prevent the maturation of biofilms after early organism adherence, and only had a weak activity against established biofilms. Conclusion Our work suggests a suboptimal effectiveness of medihoney in preventing driveline exit site infections due to biofilm development, warranting further clinical trials before its widespread use can be justified.

Novel Methods for Efficacy Testing of Disinfectants – Part I

Abstract The pathogens which are the effective transmitters of various infections pose a serious problem in restraining their interference in maintaining a sterile environment. The practical applicability of traditional methods of disinfection is restricted due to their cumbersomeness, toxic product generation, and cost-effectiveness. Therefore, the objective of the current review is to elaborate the efficacies and limitations of various novel disinfectants that can show their activity in a few minutes of treatment. The expected outcome would be feasibility for selection of a favorable disinfectant through various technologies that can generate uniform results and form a basis for the true estimation required parameters. Hence, the current paper ends with the consideration of unique new techniques that distinguishes their simplicity, safety, and efficacy in generating a sterile environment.

Environmental and Experimental Factors Affecting Efficacy Testing of Nonporous Plastic Antimicrobial Surfaces.

Test methods for efficacy assessment of antimicrobial coatings are not modelled on a hospital environment, and instead use high humidity (>90%) high temperature (37 °C), and no airflow. Therefore, an inoculum will not dry, resulting in an antimicrobial surface exhibiting prolonged antimicrobial activity, as moisture is critical to activity. Liquids will dry quicker in a hospital ward, resulting in a reduced antimicrobial efficacy compared to the existing test, rendering the test results artificially favourable to the antimicrobial claim of the product. This study aimed to assess how hospital room environmental conditions can affect the drying time of an inoculum, and to use this data to inform test parameters for antimicrobial efficacy testing based on the hospital ward. The drying time of different droplet sizes, in a range of environmental conditions likely found in a hospital ward, were recorded (n = 630), and used to create a model to inform users of the experimental conditions required to provide a drying time similar to what can be expected in the hospital ward. Drying time data demonstrated significant (p < 0.05) variance when humidity, temperature, and airflow were assessed. A mathematical model was created to select environmental conditions for in vitro antimicrobial efficacy testing. Drying time in different environmental conditions demonstrates that experimental set-ups affect the amount of time an inoculum stays wet, which in turn may affect the efficacy of an antimicrobial surface. This should be an important consideration for hospitals and other potential users, whilst future tests predict efficacy in the intended end-use environment.

Bioluminescent murine models of bacterial sepsis and scald wound infections for antimicrobial efficacy testing.

There are very few articles in the literature describing continuous models of bacterial infections that mimic disease pathogenesis in humans and animals without using separate cohorts of animals at each stage of disease. In this work, we developed bioluminescent mouse models of partial-thickness scald wound infection and sepsis that mimic disease pathogenesis in humans and animals using a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). Two days post-scald wound infection, mice were treated twice daily with a 2% topical mupirocin ointment for 7 days. For sepsis experiments, mice were treated intraperitoneally with 6 mg/kg daptomycin 2 h and 6 h post-infection and time to moribund monitored for 72 h. Consistent bacterial burden data were obtained from individual mice by regular photon intensity quantification on a Xenogen IVIS Lumina XRMS Series III biophotonic imaging system, with concomitant significant reduction in photon intensities in drug-treated mice. Post-mortem histopathological examination of wounds and bacterial counts in blood correlated closely with disease severity and total flux obtained from Xen29. The bioluminescent murine models provide a refinement to existing techniques of multiple bacterial enumeration during disease pathogenesis and promote animal usage reduction. The models also provide an efficient and information-rich platform for preclinical efficacy evaluation of new drug classes for treating acute and chronic human and animal bacterial infections.