Validation of biofilm cleaning using a mixture of cleaning agents and microbial communities

Medical YongValidation of biofilm cleaning using a mixture of cleaning agents and microbial communities

Validation of biofilm cleaning using a mixture of cleaning agents and microbial communities：In the medical field, the problem of infections caused by biofilms is becoming increasingly severe, and mixed microbial biofilms have become a difficult point for hospital infection control due to their complex structure and drug resistance. Research has shown that mixed microbial biofilm can increase bacterial resistance by over 1000 times, leading to an 8-fold increase in the risk of sterilization failure. According to monitoring data from the National Health Commission in 2023, 23% of surgical instrument related infections are directly related to biofilm residue, with mixed microbial biofilm accounting for as much as 62%. Therefore, scientifically and accurately verifying the cleaning effect of the mixed microbial community biofilm of medical cleaning agents has become a key link in ensuring medical safety.

At present, the international standard system for the validation of mixed microbial biofilm cleaning is gradually being improved.

ISO 18471:2023 "Evaluation of the removal effect of mixed bacterial biofilm on medical equipment sterilization" establishes a dedicated testing system, requiring that the removal rate of mixed bacterial biofilm on surgical instruments should reach 99.99%, and the log value of luminal instruments should be reduced by ≥ 5.0; GB/T 38502-2020 "Evaluation Method for Antimicrobial Properties of Medical Equipment Biofilms" in China further stipulates that standard biofilms should be prepared using 316L stainless steel carriers (surface roughness Ra 0.8-1.6 μ m), and the initial bacterial count should be controlled at 5-7 log CFU/cm ². The YY/T 0734.4-2025 "Medical Cleaning Agents Part 4: Determination of Mixed Biofilm Clearance" implemented in 2025 innovatively introduces the combined technology of "Fluorescence In Situ Hybridization (FISH) strain identification+selective culture counting", significantly improving the specificity and accuracy of mixed microbial community detection.

The core of the validation of mixed microbial biofilm cleaning lies in constructing a standard biofilm model that is close to clinical practice. Referring to the ratio of clinically isolated strains, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Candida albicans (ATCC 10231) were mixed and inoculated in a ratio of 3:3:2:2. The specific preparation process is as follows: etch the 316L stainless steel carrier (10mm × 10mm) with a 5% nitric acid solution for 30 minutes, followed by ultrasonic cleaning and sterilization; Prepare a mixed bacterial suspension (concentration 1 × 10 ⁸ CFU/mL) using TSB medium (containing 10% fetal bovine serum), immerse the carrier in the bacterial solution, let it stand at 37 ℃ for 24 hours, and then switch to dynamic flow addition culture (flow rate 0.5mL/min) for 72 hours to form a mature biofilm (thickness 20-30 μ m, viable cell count 6-7 log CFU/cm ²).

The detection method adopts a three-level verification system of "qualitative quantitative identification".

Firstly, rapid screening was performed using ATP bioluminescence method. The Hygiena SystemSURE Plus sampling rod was used to wipe back and forth in a 10cm ² area on the carrier surface, with a detection limit of up to 1 × 10 ³ CFU/cm ². The results were expressed in relative light units (RLU), and a threshold set to ≤ 250 RLU was considered preliminary qualified. For ATP positive samples, selective culture counting was performed: the carrier was eluted with ultrasound (power 300W, frequency 40kHz, time 20 minutes), and then inoculated with TSA medium (aerobic culture at 37 ° C for 48 hours, counting the total number of bacteria) and SDA medium (culture at 28 ° C for 72 hours, counting the number of fungi), respectively. At the same time, FISH technology was used to identify residual strains - targeting Staphylococcus aureus (probe sequence 5 '- GCT TTC GTC CCT TGC GCT T-3'), Escherichia coli (5 '- GGT TTA CCA AGT TTC CGT TCG-3'), Pseudomonas aeruginosa (5 '- ACT TGC CGT TCC TTC GGC T-3'), and Candida albicans (5 '- TCC TCC). Design a specific fluorescent probe using AAT CCG TTA CAG-3' and observe the distribution of bacterial strains under a laser confocal microscope (excitation wavelength 488nm/561nm, Z-axis step of 0.5 μ m).

The configuration of key detection instruments must meet the requirements of multidimensional analysis.

The core equipment includes: Olympus FV3000 laser confocal microscope (equipped with Airyscan ultra-high resolution mode), 3M Clean Trace ATP detector (detection range 0-9999 RLU), and Thermo Fisher Varioskan LUX multifunctional enzyme-linked immunosorbent assay (used for XTT metabolic activity determination). Auxiliary equipment includes: Branson CPX3800 ultrasonic cleaner (temperature control ± 1 ℃), ESCO Class II biosafety cabinet, Mettler SevenCompact pH meter. Special attention should be paid to fixing the sample with 4% paraformaldehyde before FISH detection, controlling the hybridization temperature at 46 ℃± 0.5 ℃, and strictly controlling the probe concentration (50ng/μ L) and hybridization time (16h).

The result judgment implements a "graded qualification system": Class I (implants) require a total clearance rate of mixed microbial communities of ≥ 99.99% (log reduction value ≥ 4.0), and the clearance rate of each bacterial species is ≥ 99.9%; Class II (endoscopy) total clearance rate ≥ 99.9% (log reduction value ≥ 3.0), fungal clearance rate ≥ 99%; A total clearance rate of ≥ 99% for Class III (conventional devices) is considered qualified. The 2024 capability verification results of the China National Institute for Food and Drug Control showed that only 7 out of 18 laboratories were able to accurately complete the identification of mixed microbial communities, with the main source of error being the cross interference between fungi and bacteria (Candida mycelium easily wraps around bacteria to form a "protective shell"). It is recommended to add 0.1% chitinase pretreatment during testing, which can increase the dispersion efficiency of fungal biofilm by 40%.

In practical applications, simulating clinical contamination conditions is key to ensuring the effectiveness of detection. Research has shown that adding 10% human serum protein can increase the resistance of mixed biofilms by 50%. It is recommended to prepare the contaminated solution formula in a ratio of "80% mixed bacterial suspension+20% simulated body fluid (containing 3% albumin and 0.5% mucin)". For luminal instruments, a "flow-through biofilm reactor" (inner diameter 2mm, length 30cm) should be used, with a peristaltic pump controlling the flow rate of 1mL/min to form a gradient biofilm inside the lumen (inlet section thickness 35 μ m, outlet section 15 μ m), which is closer to the actual clinical contamination state.

The validation of mixed microbial biofilm cleaning has becomeMedical YongThe 'gold standard' for evaluating the performance of cleaning agents.

By integrating multidimensional techniques such as ATP rapid screening, selective culture counting, and FISH strain identification, combined with the ISO/GB dual standard system, the cleaning agent's removal efficiency on complex biofilms can be comprehensively evaluated. Medical institutions should establish a "biofilm risk classification" system and implement 100% biofilm testing for high-risk devices such as implants; Cleaning agent production enterprises need to optimize the enzyme formulation scheme. The latest research shows that the triple formula of protease, glycosidase, and chitinase can increase the biofilm clearance rate of mixed bacterial communities by 2.3 log values. With the full implementation of GB 32630-2025 "General Requirements for Medical Cleaning Agents", the removal efficiency of mixed microbial biofilm will become a key indicator for market access, promoting the industry's transformation and upgrading from "broad-spectrum cleaning" to "precise membrane removal".