Quantitative Suspension Test Research Articles

Antimicrobial efficacy of amine fluoride based tooth gels compared to a toothpaste in a phase 2/step 2 in-vitro test model.

Introduction: The aim of the present study was to determine the antimicrobial effect of various gel formulations on plaque formation; different tooth gels were compared to a toothpaste containing comparable antimicrobial ingredients with regard to its microbiocidal activity. The study was conducted under the assumption, that a chief requirement for the prevention of plaque formation is the combination of mechanical removal and antimicrobial activity, and not the sole capability of mechanical plaque removal.Methods: Ledermix® fluoride gel as commercially available with preservative, and without preservative and perfume oils, Elmex® gelée, and Meridol® toothpaste were tested in a standardized in-vitro test modification of the quantitative suspension test EN 1040. Instead of testing in a suspension, the respective product was directly placed on a standardized contaminated sterile stainless steel disk without adding any bio-burden. 50% egg yolk in Aqua dest. was used as a neutralizer.Results: Within 1 min, Elmex® gelée showed a RF >5 log10 against S. pyogenes and S. sanguinis. Against S. mutans, a log10 RF of ≥5 was achieved after 2 min, against C. albicans after 5 min, and against P. aeruginosa after 10 min S. aureus was the most difficult organisms to be reduced. After an application time of 10 min, only a log10 RF of 2.4 was achieved. Ledermix exceeded the antimicrobial efficacy of Elmex® gelée against S. mutans and C. albicans and was already effective against these organisms after 1 min, but did not show the same antimicrobial efficacy as Elmex® gelée against P. aeruginosa. Similar to Elmex® gelée, a required reduction of >5 log10 for antimicrobials under no organic challenge was not achieved against S. aureus. Ledermix® fluoride gel without preservatives and Ledermix® fluoride gel without preservatives and perfume oil did not show the antimicrobial efficacy of the standard Ledermix® fluoride gel formulation, indicating that the observed antimicrobial efficacy is chiefly based on the preservative, and possibly the perfume oil. Compared to the tested gels, Meridol® toothpaste was less effective and reached any antimicrobial effect >5 log10 only against S. sanguinis after 10 min.Conclusion: All unmodified tested gels showed an antimicrobial effect. Because no relevant antimicrobial efficacy against plaque forming bacteria was achieved within 2 min, in practice, an anti-plaque forming effect based on the antimicrobial action of gels cannot be assumed when used in the oral cavity. However, the results of the present study indicate that the antimicrobial efficacy of gels is determined by their formulation and that for the prevention of plaque formation the combination of mechanical removal and antimicrobial activity is not the chief requirement only, but a sustained antimicrobial effect may be of greater importance.

Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) of polihexanide and triclosan against antibiotic sensitive and resistant Staphylococcus aureus and Escherichia coli strains.

Background: An in-vitro study was conducted investigating the antimicrobial efficacy of polihexanide and triclosan against clinical isolates and reference laboratory strains of Staphylococcus aureus and Escherichia coli.Methods: The minimal inhibitory concentration (MIC) and the minimal microbicidal concentration (MMC) were determined following DIN 58940-81 using a micro-dilution assay and a quantitative suspension test following EN 1040. Polihexanide was tested in polyethylene glycol 4000, triclosan in aqueous solutions.Results: Against all tested strains the MIC of polihexanide ranged between 1–2 µg/mL. For triclosan the MICs varied depending on strains ranging between 0.5 µg/mL for the reference strains and 64 µg/mL for two clinical isolates. A logRF >5 without and logRF >3 with 0.2% albumin burden was achieved at 0.6 µg/mL triclosan. One exception was S. aureus strain H-5-24, where a triclosan concentration of 0.6 µg/mL required 1 minute without and 10 minutes with albumin burden to achieve the same logRFs. Polihexanide achieved a logRF >5 without and logRF >3 with albumin burden at a concentration of 0.6 µg/mL within 30 sec. The exception was the North-German epidemic MRSA strain, were an application time of 5 minutes was required. Conclusion: The clinical isolates of E. coli generally showed higher MICs against triclosan, both in the micro-dilution assay as well in the quantitative suspension test than comparable reference laboratory strains. For polihexanide and triclosan strain dependant susceptibility was shown. However, both antimicrobial compounds are effective when used in concentrations common in practice.

Antimicrobial Efficacy of the Silver Wound Dressing Biatain Ag in a Disc Carrier Test Simulating Wound Secretion

Aim: The efficacy of antimicrobial compounds included in wound dressings has been determined using the quantitative suspension test according to EN 13727 before. However, as suspension tests are not an accurate reflection of the conditions under which wound antiseptics are used, it was investigated if a disc carrier test would yield results simulating practical conditions on wound surfaces. A silver-leaching foam wound dressing was used for evaluation of the disc carrier test method. Method: The disc carriers consisted of circular stainless-steel discs measuring 2 cm in diameter and 1.5 mm in thickness, complying with the requirements of EN 10088-2. Carriers were contaminated with Staphylococcus aureus, methicillin-resistant S. aureus or Pseudomonas aeruginosa, respectively, together with an artificial wound secretion and left to dry at room temperature for 30 min. The wound dressings being tested were placed on the discs for the length of the exposure time, and after neutralization by thioglycolate in phosphate-buffered saline the number of surviving test organisms was then counted. The logarithmic reduction factor was calculated from the difference between the initial inoculum and the number of recovered test organisms. Results: The disc carrier test allowed determination of an antimicrobial efficacy in a realistic setting. It also imposed more stringent requirements on efficacy over time than the quantitative suspension test. The silver foam wound dressing showed a time-dependent antimicrobial efficacy. After 24-hour application time, the reduction factors against S. aureus, P. aeruginosa and the methicillin-resistant S. aureus were 1.9 ± 0.15, 2.1 ± 0.14 and 3.1 ± 0.18, respectively. Conclusion: The disc carrier test was a useful method for testing the antimicrobial efficacy of a foam silver dressing. The antimicrobial dressing exhibited an antimicrobial effect after 3 h and achieved a reduction >2 log against the tested bacterial strains in the presence of a simulated wound secretion after 24 h.

Investigative study on the antimicrobial activity and effects of virucidal hand antiseptic on skin

Methods The efficacy of several commercially available virucidal hand antiseptics against FCV was compared by quantitative suspension test. The efficacy of an acidic formulation VH (Alsoft V, Saraya Co., Ltd.) against various types of microorganisms was further investigated according to international standards such as ASTM or EN. The influence on mouse skin after repeated use of VH was also examined. After 5 days of use simulated in clinical practical condition, physiological properties were measured. In addition, a test on skin irritation was performed electrophysiologically.

Microbicidal efficacy of thiocyanate hydrogen peroxide after adding lactoperoxidase under saliva loading in the quantitative suspension test

ObjectiveAs shown in the quantitative suspension test adding lactoperoxidase to a thiocyanate (SCN−) hydrogen peroxide (H2O2) combination over the physiological saliva level has significant positive antimicrobial effects to a level of totally killing Streptococcus mutans, Streptococcus sanguinis, and Candida albicans. The aim of this study was to evaluate this positive effect under human saliva loading. MethodsThe bactericidal and fungicidal effect of lactoperoxidase was evaluated in a quantitative suspension test by using two test mixtures of a 2.0% thiocyanate and 1.2% hydrogen peroxide solution, one without (Group A) and one with (Group B) lactoperoxidase under saliva loading. Following the quantitative suspension tests (EN-13727/EN-13624), the growth of surviving bacteria and fungi in a nutrient broth was measured. The exposure times were restricted to 1, 3, 5, and 15min. All statistical analyses were carried out with SPSS 11.5. ResultsIn the quantitative suspension test, the combination of thiocyanate and hydrogen peroxide showed relatively low antimicrobial effectiveness on S. mutans, S. sanguinis, and C. albicans in the presence of human saliva at measured time points in comparison to the mixture with lactoperoxidase, which showed a high bactericidal activity within 15min (S. mutans and S. sanguinis) and fungicidal activity within 3min (C. albicans). ConclusionThe antimicrobial effectiveness of the tested thiocyanate hydrogen peroxide combination was increased significantly by adding lactoperoxidase in the quantitative suspension test under human saliva loading.

Importance of Porins for Biocide Efficacy against Mycobacterium smegmatis

Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of porins for the susceptibility of M. smegmatis to biocides.

EFFICIENCY OF ALCOHOLIC SOLUTION «VITASEPT-SKZ» FOR EXTERNAL APPLICATION

The purpose of this work was to study physical-chemical, antimicrobial and toxicological -hygienic properties of the developed antiseptic for external application «Vitasept-SKZ». It has been established that developed spirit solution «Vitasept-SKZ» for external application possesses physical and chemical properties satisfactory to sanitary regulations for antiseptics. In working concentration the drug action results in absence of growth of all museum test cultures of microorganisms within 1 and 2 minutes. In the quantitative suspension test the reduction factor of all test cultures of microorganisms within 1 minutes «Vitasept-SKZ» exceeded 6 lg. For hygienic hand antiseptics the given handwash causes the reduction factor of E.coli test-culture more than 4 lg. In test of microbiological cleanliness microorganisms Enterobacteriaceae bacteria, Р aeruginosa and S. аureus were determined. The developed antiseptic solution pertains to practically nontoxical and low-dangerous substances with low irritant, cumulative properties, does not demonstrate irritating and sensitizing action, being hygienic safe. Stability tests revealed no considerable changes in appearance and physical and chemical properties of the drug. Developed spirit solution «Vitasept-SKZ» for external application can be recommended for use in public medical health care units.

Correlation between reduced susceptibility to disinfectants and multidrug resistance among clinical isolates of Acinetobacter species

The aim of this study was to investigate the susceptibility profiles to disinfectants and antimicrobial agents of 283 non-repetitive Acinetobacter clinical isolates obtained in 97 Japanese hospitals in March 2002. Susceptibility profiles of the above isolates to four disinfectants, six antimicrobial agents and two dyes were investigated. MICs were measured by the agar dilution method recommended by the CLSI (formerly NCCLS). MBC measurements and time-kill assays were performed using a slightly modified quantitative suspension test based on the European Standard EN 1040. No evident resistance to disinfectants was seen among the 283 strains of Acinetobacter spp. isolated in 2002, but the MIC(90)s of chlorhexidine gluconate, benzalkonium chloride and alkyldiaminoethylglycine hydrochloride were 50, 50 and 400 mg/L, respectively. Interestingly, the MICs of alkyldiaminoethylglycine hydrochloride and benzethonium chloride for four and three clinical isolates, respectively, reached 800 mg/L (approximately half the in-use concentration). The MBCs for the 28 disinfectant reduced susceptibility (DRS) isolates, for which the MICs of at least one of the four disinfectants tested were higher than the MIC(90), were comparable to those for susceptible isolates, in general; however, significant differences (P < 0.01) were observed between disinfectant-susceptible and DRS isolates in the time-kill assays of chlorhexidine gluconate, benzalkonium chloride and benzethonium chloride. Furthermore, DRS isolates tended to demonstrate multiresistance profiles to ceftazidime, ciprofloxacin and amikacin (P < 0.05). Since several Acinetobacter clinical isolates have developed augmented resistance to multiple antimicrobials and disinfectants, it is worth checking the susceptibility to disinfectants if multidrug-resistant Acinetobacter spp. are recurrently isolated clinically.

Can vaccinia virus be replaced by MVA virus for testing virucidal activity of chemical disinfectants?

BackgroundVaccinia virus strain Lister Elstree (VACV) is a test virus in the DVV/RKI guidelines as representative of the stable enveloped viruses. Since the potential risk of laboratory-acquired infections with VACV persists and since the adverse effects of vaccination with VACV are described, the replacement of VACV by the modified vaccinia Ankara strain (MVA) was studied by testing the activity of different chemical biocides in three German laboratories.MethodsThe inactivating properties of different chemical biocides (peracetic acid, aldehydes and alcohols) were tested in a quantitative suspension test according to the DVV/RKI guideline. All tests were performed with a protein load of 10% fetal calf serum with both viruses in parallel using different concentrations and contact times. Residual virus was determined by endpoint dilution method.ResultsThe chemical biocides exhibited similar virucidal activity against VACV and MVA. In three cases intra-laboratory differences were determined between VACV and MVA - 40% (v/v) ethanol and 30% (v/v) isopropanol are more active against MVA, whereas MVA seems more stable than VACV when testing with 0.05% glutardialdehyde. Test accuracy across the three participating laboratories was high. Remarkably inter-laboratory differences in the reduction factor were only observed in two cases.ConclusionsOur data provide valuable information for the replacement of VACV by MVA for testing chemical biocides and disinfectants. Because MVA does not replicate in humans this would eliminate the potential risk of inadvertent inoculation with vaccinia virus and disease in non-vaccinated laboratory workers.

Virucidal efficacy of povidone-iodine-containing disinfectants

The objective of this study was to evaluate virucidal efficacy of the commercially available povidone-iodine formulations Betaisodona solution and Betaseptic Mundipharma (Mundipharma). The quantitative suspension test for virucidal testing of biocides according to the German guideline was used as method. The use of Betaisodona solution resulted in virucidal efficacy, corresponding to >or=10(4)-fold reduction in viral titre, against vaccinia virus, bovine viral diarrhoea virus and polyomavirus SV40 within 0.5 min and adenovirus type 5 within 3-5 min without and with organic load. For inactivation of the most resistant poliovirus type 1, a time interval of >or=60 min was needed. By contrast, Betaseptic Mundipharma inactivated significantly all model viruses for virucidal testing including poliovirus type 1 within 5 min independently from the addition of proteins. Betaisodona solution shows a good efficacy against enveloped model viruses as well as against some nonenveloped human viruses, e.g. adenovirus and polyomavirus. Betaseptic Mundipharma has an excellent virucidal efficacy including the inactivation of the most resistent poliovirus type 1. The findings of this study make Betaseptic Mundipharma suitable for virucidal disinfection of the skin within short time intervals.

Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization

The virucidal activity of 2 hand rubs proposed by the World Health Organization was studied in a quantitative suspension test for chemical disinfectants and antiseptics in human medicine (EN 14476). These formulations are recommended if no hand rubs with declared microbiological activity are available in health care settings. Formulation I, based on ethanol, inactivated bovine viral diarrhea virus (BVDV), hepatitis C virus (HCV), adenovirus, and murine norovirus as a surrogate for human norovirus. Formulation II, based on isopropyl alcohol, was active only against adenovirus and enveloped viruses, such as BVDV and HCV. Both formulations failed to inactivate poliovirus by 4 log(10) steps within 300 seconds.

In vitro cultivation and cryopreservation of duck embryonic hepatocytes

Hepatitis B-virucidal testing of biocides in quantitative suspension tests using duck hepatitis B virus (DHBV) requires primary duck embryonic hepatocytes for viral propagation. To improve the test system and availability of these cells, commercial culture plates with different growth surfaces were tested for cell cultivation and different approaches for cryopreservation of hepatocyte suspension were examined. After 12 days of culture, the largest amounts of hepatocytes were grown in CellBIND and TTP plates and CellBIND surface showed the lowest tendency of monolayer detachment nearly comparable with collagen 1-coated CELLCOAT plates. For cryopreservation of hepatocyte suspension, the use of growth medium supplemented with fetal calf serum (FCS) and dimethyl sulfoxide (ME 2SO), FCS supplemented with ME 2SO or cryosafe-1 as cryoprotective agents provided the highest rates of surviving cells after thawing. The freezing–thawing process did not significantly reduce the susceptibility of hepatocytes to infection with DHBV. In conclusion, plates without collagen 1 such as CellBIND are recommended for cultivation of primary duck embryonic hepatocytes in infectivity experiments of DHBV for virucidal testing of biocides. The use of cryopreserved hepatocytes is possible when freshly isolated cells from the liver of duck embryos are not available.

Effect of lactoperoxidase on the antimicrobial effectiveness of the thiocyanate hydrogen peroxide combination in a quantitative suspension test.

BackgroundThe positive antimicrobial effects of increasing concentrations of thiocyanate (SCN-) and H2O2 on the human peroxidase defence system are well known. However, little is known about the quantitative efficacy of the human peroxidase thiocyanate H2O2 system regarding Streptococcus mutans and sanguinis, as well as Candida albicans. The aim of this study was to evaluate the effect of the enzyme lactoperoxidase on the bactericidal and fungicidal effectiveness of a thiocyanate-H2O2 combination above the physiological saliva level. To evaluate the optimal effectiveness curve, the exposure times were restricted to 1, 3, 5, and 15 min.ResultsThe bactericidal and fungicidal effects of lactoperoxidase on Streptococcus mutans and sanguinis and Candida albicans were evaluated by using two test mixtures of a 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide solution, one without and one with lactoperoxidase. Following the quantitative suspension tests (EN 1040 and EN 1275), the growth of surviving bacteria and fungi in a nutrient broth was measured. The reduction factor in the suspension test without lactoperoxidase enzyme was < 1 for all three tested organisms. Thus, the mixtures of 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide had no in vitro antimicrobial effect on Streptococcus mutans and sanguinis or Candida albicans. However, the suspension test with lactoperoxidase showed a high bactericidal and fungicidal effectiveness in vitro.ConclusionThe tested thiocyanate and H2O2 mixtures showed no relevant antimicrobial effect. However, by adding lactoperoxidase enzyme, the mixtures became not only an effective bactericidal (Streptococcus mutans and sanguinis) but also a fungicidal (Candida albicans) agent.

Development of bactericidal and virucidal testing standards for aquaculture disinfectants

There is a need for standardised comparative data on the efficacy of aquaculture disinfectants to guide their use by farmers and health professionals, as well as Competent Authorities for authorisation or listing purposes. Towards this aim, two already available CEN (Comité Européen de Normalisation) quantitative suspension test standards for the evaluation of bactericidal and virucidal activity of disinfectants and antiseptics for use in the veterinary field were modified by using agents and testing conditions representative of aquaculture conditions. For evaluating bactericidal activity, BS EN1656:2000 was modified to test disinfectant activity against Aeromonas salmonicida subsp. salmonicida (ATCC 14174), Yersinia ruckeri (ATCC 29473), Carnobacterium piscicola (ATCC 35586) and Lactococcus garvieae (NCIMB 702927) for a contact time of 30 min and temperature of 4 °C. Interfering substance was used as described for dirty conditions in the standard (10 g l − 1 yeast extract plus 10 g l − 1 bovine serum albumin solution). The substituted organisms could be stored and readily resuscitated. They were not adversely affected by the assay conditions (except exposure to the disinfectant) and could be readily prepared as suspensions. Four example products, coded A–D, were tested using both methods (including a peroxygen system, a product containing a mixture of hydrogen peroxide and peracetic acid, an acidic iodophore and a Chloramine T containing product). The neutralisation-dilution method, preferred in the standard, was appropriate for all products. Although there was greater test-to-test variation at the higher end of effective dilutions, repeat testing gave reliable results for four products as bactericides. L. garvieae and C. piscicola were the most resistant bacterial pathogens tested, requiring concentrations between 0.1–0.5% of the four products tested to achieve 5 log 10 inactivation. For evaluating virucidal activity, BS EN 14675:2006 was modified to use infectious pancreatic necrosis virus (IPNV), Spjarup serotype, using the same products with testing conditions as for BS EN1656:2000. The acidic iodophore-containing product (C) gave rise to cell cytotoxicity problems with the recommended neutralisation dilution method for titration of remaining viral activity. The other three products were reliably tested for virucidal activity using the modified assay. A further assay modification for virus mixed with Product C involved membrane dialysis for 48 h following test exposure and neutralisation by dilution. This reduced cell cytotoxicity enough to allow titration of the remaining viable IPNV.

Instrument processing with lauryl dimethyl benzyl ammonium bromide: A challenge for patient safety

The quaternary ammonium compound (QUAT) lauryl dimethyl benzyl ammonium bromide (LDBAB) is commercialized in the Caribbean, Central, and South America under label claims to sterilize medical and dental instruments in 30 minutes or less. But QUAT formulations are classified as low-level disinfectants with no sporicidal activity or tuberculocidal efficacy. This study evaluated the presumptive sporicidal and tuberculocidal activities of 2 LDBAB formulations with label claims of high-level disinfectant action used widely in the Americas. In this study, sporicidal activity was evaluated against a challenge inoculum of 10(6)Bacillus atrophaeus spores. Tuberculocidal activity was evaluated against Mycobacterium tuberculosis H37Rv using the quantitative suspension test described in European Standard EN14348:2005. Tested at the recommended product concentrations indicated on their respective labels, both LDBAB solutions failed to demonstrate sporicidal activity and tuberculocidal efficacy. These findings underscore the need for public health authorities, as well as medical and dental professionals, to correctly identify LDBAB as a low-level disinfectant and avoid its use in instrument processing, a practice that may endanger lives.

Substitution of Vaccinia Virus Elstree by Modified Vaccinia Virus Ankara to test the Virucidal Efficacy of Chemical Disinfectants

After the eradication of variola in 1980, the smallpox vaccination was considered to be no longer required and was subsequently abandoned mainly because of possible adverse effects of vaccinia virus especially in first-time vaccinees. Despite a growing number of humans without immunity against vaccinia virus, vaccinia virus Lister Elstree (VACV) is still prescribed for testing virucidal efficacy of chemical disinfectants in the guidelines of the German Veterinary Medical Society [Deutsche Veterinärmedizinische Gesellschaft (DVG)], the German Association for the Control of Virus Diseases [Deutsche Vereinigung zur Bekämpfung der Viruskrankheiten (DVV)] and the Robert Koch Institute (RKI). To evaluate a possible substitution of VACV, with the attenuated modified vaccinia virus Ankara (MVA) the virucidal efficacy of four different DVG-listed commercially available chemical disinfectants representing different groups of chemicals was tested against these two viruses. Quantitative suspension tests and qualitative carrier tests with poplar wood and gauze were performed. Distinction of VACV and MVA was confirmed by cytopathogenic effects, such as differences in plaque morphology. No significant difference in disinfection efficacy between VACV and MVA was observed for any of the disinfectants tested. Implying that vaccinia virus poses a risk after inadvertent inoculation, our results show that MVA, which does not replicate in humans, should replace VACV in the chemical disinfectant testing guidelines.

Application of a Quantitative Carrier Test to Evaluate Microbicides against Mycobacteria

Microbicides for reprocessing heat-sensitive medical devices, such as flexible endoscopes, must be mycobactericidal to reduce the risk of nosocomial infections. Suspension test methods currently used for efficacy evaluation lack the stringency required for assessing inactivation of mycobacteria on surfaces. The quantitative carrier test method reported here is based on mycobacteria-contaminated reference carrier disks of brushed stainless steel. Each disk was contaminated with 10 microL of a suspension of Mycobacterium terrae containing a soil load. Each disk with a dried inoculum was placed in a glass or Teflon vial, and then overlaid with 50 microL of the test formulation or 50 microL saline for the control carriers. Five test and 3 control disks were used in each run. At the end of the contact time, each vial received 9.95 mL neutralizer solution with 0.1% Tween-80 to stop the reaction and perform the initial microbicide dilution. The inoculum was eluted by mixing on a Vortex mixer for 60 s, and the eluates and saline used to subsequently wash the vials and the funnels were membrane-filtered. Filters were placed on plates of Middlebrook 7H11 agar and incubated at 37 degrees C for at least 30 days before colonies were counted and log10 reductions were calculated in colony-forming units. Tests with a range of commercially available products, having claims against mycobacteria, or believed to be broad-spectrum microbicides, showed that the method gave reproducible results. Products used included oxidizing agents (sodium hypochlorite and an iodophore), a phenolic, a quaternary ammonium compound, and ortho-phthalaldehyde. This method represents a much more realistic evaluation than the currently used quantitative suspension test method for the evaluation of mycobactericidal formulations for registration and, when performed at different product concentrations, allows an assessment of any safety margin or risks in using the test formulation in the field.

Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses.

BackgroundA procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses.MethodsWe have studied the activities of three commonly-used alcohol-based hand rubs (hand rub A, based on 45% propan-2-ol, 30% propan-1-ol and 0.2% mecetronium etilsulfate; hand rub B, based on 80% ethanol; hand rub C, based on 95% ethanol) against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test.ResultsAll three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by ≥ 4 log10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load.ConclusionCommonly used alcohol-based hand rubs with a total alcohol concentration ≥ 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test.

In vitro Antibacterial Activity of Lucilia sericata Maggot Secretions

Maggots of the green blowfly, Lucilia sericata, are used as an alternative to surgical intervention and long-term antiseptic therapy for the treatment of chronic wounds. The secretions of maggots are known to have antibacterial properties. To quantify the bactericidal effect of secretions from larvae of L. sericata, an in vitro test model based on the modified European quantitative suspension test (EN 1040) was developed, in which a co-culture of maggots and bacteria (Micrococcus luteus,Escherichia coli, methicillin-sensitive Staphylo-coccus aureus) in tryptic soy broth was tested. The numbers of bacterial colonies with and without maggot exposure were compared after 24, 48 and 72 h of exposure. The mean log₁₀ reduction factor (RF) for bacterial elimination per maggot was >4 at all examined times for all tested bacteria. Thus, maggot secretion fulfilled the required definitions of an antiseptic. In addition, the maggots’ ability to ingest bacteria was also evaluated. Maggots contained viable bacteria after 48 h of contact with the respective organisms. These maggots also continued excreting bacteria. Therefore, maggots should be disposed of after use as they must be regarded as medical waste.

Broad-spectrum microbicidal activity, toxicologic assessment, and materials compatibility of a new generation of accelerated hydrogen peroxide-based environmental surface disinfectant

BackgroundConcerns on human and environmental safety and label claims of many microbicides point to the need for safer, faster acting, and broad-spectrum substitutes. ACCEL TB, a 0.5% accelerated H2O2 (AHP)-based disinfectant described here, is a potential candidate.MethodsACCEL TB was tested for its broad-spectrum microbicidal activity, safety and materials compatibility using internationally accepted protocols. Activity against bacteria (Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and Salmonella choleraesuis) was tested with the AOAC use-dilution method and the first tier of a quantitative carrier test (QCT-1). Mycobactericidal activity was tested against Mycobacterium bovis and Mycobacterium terrae using a quantitative suspension test (QST) and QCT-1, respectively. Fungicidal activity (Trichophyton mentagrophytes) was determined with the AOAC test and QCT-1. Activity against several enveloped and nonenveloped viruses was evaluated using the American Society for Testing and Materials (ASTM) method No. E-1053. Sanitizing action was tested against 7 types of vegetative bacteria with method No. DIS/TSS-10. All microbicidal tests contained an added soil load; in all AOAC tests, it was 5% fetal bovine serum, and, in QCT-1, a mixture of 3 types of proteins in phosphate buffer was used instead. The methods to test for acute oral, dermal, inhalation toxicities, and dermal and eye irritation as well as skin sensitization complied with the requirements of the Organization for Economic Cooperation and Development and the US Environmental Protection Agency (OPPTS 870). Standard methods were also used to test compatibility with metals and plastics.ResultsAt 20°C, the full-strength product was bactericidal and virucidal in 1 minute and mycobactericidal and fungicidal in 5 minutes. It was nonirritating to skin and eyes. The acute oral LD50 (lethal dose 50%) was >5000 mg/kg. It was compatible with 12 types of plastic and 3 out of 4 metals.ConclusionThe tested formulation showed a high safety and materials compatibility profile in addition to being a fast acting, intermediate-level disinfectant.