Determining the Efficacy of Chemicals for the Inactivation of Liquid Waste Containing Gram-Positive Bacteria of Risk Group 2

Abstract

Background: Inactivation of infectious liquid waste can be performed by different means, including autoclaving or chemical inactivation. Autoclaving is most widely used, but cannot always be implemented, so that chemical inactivation is a possible alternative. However, its efficacy has to be proven by in-house validation. Here, we provide an easy-to-implement validation process adapted from existing standards.

Methods: The bactericidal activity of two commercially available disinfectants, containing glucoprotamine or peracetic acid, was tested on two gram-positive bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae (SP). Quantitative suspension tests were adapted from the European standard EN 13727. Membrane filtration was used to remove any trace of the active antiseptic. The stability of working solutions and the effect of different organic loads were also assessed. Further, the aquatic toxicity of glucoprotamine was tested on the viability and behavior of zebrafish larvae.

Results: Peracetic acid was highly efficient against both strains, even at a working concentration of 0.25% for 1 minute S. pneumoniae was also highly sensitive to glucoprotamine, whereas MRSA required either higher concentrations (1%) or longer exposure times (30 min) at lower concentrations. Further, the lethal dosis that kills 50% of the embryos (LD₅₀) for glucoprotamine was 3-3.5 mg/L using the zebrafish Embryo Acute Toxicity Test.

Conclusion: We could confirm the very high efficacy of glucoprotamine- and peracetic-based disinfectants to inactivate liquid waste containing gram-positive bacteria. Also considering aquatic toxicity, this methodology could help laboratories to establish validated procedures for chemical inactivation of infectious liquid wastes relevant to their institution, balancing efficacy, and environmental issues.

Keywords: bacteria; glucoprotamine; inactivation; liquid waste; peracetic acid; validation.

Figure 1.

Graphical representation of the quantitative suspension test procedure for the determination of the bactericidal concentration. Created with BioRender.com. CSBA, Columbia sheep blood agar.

Figure 2.

(A) Filtration control (B) Validation of the disinfectant by the membrane filtration method. N_V: cfu/mL in the validation solution determined by plating on CSBA plates, N_C: cfu/mL of bacteria determined from the membrane after filtering off the disinfectant. The means and SDs of three duplicate independent experiments are shown. Paired t test with significant difference set a p = 0.05 was performed. ns, not statistically significant; SD, standard deviation.

Figure 3.

Efficacy of glucoprotamine in relation to (A) concentration, (B) exposure time, (C) presence of organic load, and (D) stability of the diluted solution after 28 days. For (B–D), only strain MRSA was tested. The means and SDs of three duplicate independent experiments are shown. Dotted lines mark the targeted 5 log level germ reduction (R) required to consider a disinfectant as efficient. For (C) an ordinary one-way ANOVA with assumed Gaussian distribution and Tukey's post-test with significant difference set as p = 0.05 was performed. MRSA, methicillin-resistant Staphylococcus aureus.

Figure 4.

Efficacy of peracetic acid in relation to (A) concentration, (B) influence of organic load, and (C) stability of diluted solution after 1, 3, and 5 days. The means and SDs of three duplicate independent experiments are shown. Dotted lines mark the targeted 5 log level germ reduction (R) required to consider a disinfectant as efficient. For (B) ordinary one-way ANOVA with assumed Gaussian distribution and Tukey's post-test with significant difference set as p = 0.05 was performed. PAA, peracetic acid.

Figure 5.

(A) LC₅₀ of glucoprotamine determined by the acute fish toxicity test. LC₅₀ was found to be between 3 and 3.5 mg/L. For concentrations higher than 5 mg/L, 100% of mortality occurs within the first 24 hours. Further, concentrations lower than 2 mg/L resulted only in sporadic mortality. (B) An alternate light-dark condition assay was used to determine the zebrafish behavior. (C) Zebrafish larvae treated with 3.5 mg/L glucoprotamine showed reduced BLA compared with controls (p = 0.0007). In contrast, larvae treated with 2 and 0.1 mg/L showed increased (BLA) (p < 0.05). *p < 0.05; **p < 0.01; ***p < 0.001. BLA, basal locomotor activity; LC₅₀, 50% lethal concentration.