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Detection of Low Bacterial Contamination from Neoprene® Gloves with ICR Swabs

Contact Plates and Swabs Methods to Detect Microbial Contamination

Contact plates and swabs are commonly employed methods for surface monitoring in aseptic filling lines for pharmaceuticals. Both methods are recommended by the current European and US GMP guidance.

The published recovery rates for several swabs and contact plates vary widely due to differences in chosen methods, surfaces, and test microorganisms. This variability is also reported by the USP chapter <1116>. Nevertheless, the methods should be able to detect low levels of microorganisms, especially in grade A cleanrooms or isolators. The manufacturing of drugs in isolators is an obvious trend in the pharmaceutical industry and therefore, microorganisms must be detected from surfaces commonly used in isolators, such as isolator gloves, e.g. Neoprene® gloves.

This article shows the suitability of ICR swabs (Ref. No. 146529) and lockable TSA w. LTHThio contact - ICR+ (Ref. No. 146783) contact plates, to detect low numbers of different bacterial test strains from Neoprene® gloves. The ICR swab is designed to test the presence or absence of microorganisms on dry and hard-to-access surfaces, whereas ICR contact plates are suitable for the enumeration of microorganisms on flat, dry surfaces in cleanrooms and isolators. As a control, an "Agar Overlay" method was chosen to determine the number of surviving microorganisms.

Experimental Conditions for Bacterial Test Strains detection

The Neoprene® glove pieces (5 cm x 5 cm) were cleaned and autoclaved. The surfaces were inoculated with 10 x 10 µL of two different dilutions of 4 microbial suspensions to obtain a homogeneous dispersion on the highly hydrophobic surface. Table 1 lists the chosen microorganisms and the total number of CFU used to inoculate the test surfaces. A higher inoculum level is used for gram-negative bacteria since they are expected to be more sensitive to the drying procedure of the inoculated surface material.

The number of CFU used to inoculate the test surface was controlled using the spread plate method on 90 mm TSA plates.

Table 1.Selected bacterial test stains and CFU inoculated by applying 100 µl of suspension.

Each surface was dried for 60 min under a laminar flow hood without an air stream. The microorganisms were simultaneously detected from the dry surface using 3 different methods and each test was performed in triplicate. The procedures are given below and also shown in Figure 1.

  • Agar Overlay: This method was chosen as a control method. As it was assumed that a high number of microorganisms are killed by the drying process on the surface, “Agar Overlay” method was used to determine the surviving microorganisms on the surface. The molten TSA Agar was poured onto the inoculated and dried surface. All plates were incubated for up to 7 days at 30-35 °C. The colonies were counted every day.
  • Contact plate method: The dried surface was sampled using a TSA w. LTHThio contact - ICR+ contact plate, which was pressed on the test surface for 10 seconds with a standardized pressure of 500 g. All plates were incubated for up to 7 days at 30-35 °C. The colonies were counted every day.
  • Swab method: The dried surface was carefully sampled with the premoistened swab tip of the ICR swab in longitudinal and cross directions. The broth medium from the reservoir was squeezed onto the swab tip afterward, and the swabs were incubated at 30-35 °C for up to 7 days to consider a prolonged lag phase due to desiccation. The tubes were inspected for turbidity every day.

Figure 1.Test procedure for microbial detection Neoprene® glove pieces by "Agar Overlay" method, TSA w. LTHThio contact- ICR+ plates and ICR swabs

Incubation for up to 7 days at 30-35 °C and daily evaluation (CFU on overlay and ICR contact plates, turbidity of Swab media)

1 = e.g. S. aureus (high inoculum) after 48 h incubation


Results and Discussion

 

1) Recovery rates of bacterial test strains: "Agar Overlay" method vs. ICR contact plates

The survival rate of microorganisms after dehydration on the Neoprene® surface was determined by the "Agar Overlay" method.

The detected CFU per method and test strain are shown in Figure 2 and Table 2 shows the approximate recovery rates by percent of inoculum. The recovery rates (Table 2) were calculated for each dilution. The amount listed illustrates the spectrum of both dilutions compared to the inoculum.

The recovery of survived microorganisms was dependent on the type of microorganism. The "Agar Overlay" method recovered fewer CFU than the ICR contact plate in most cases, but the results were still comparable.

Nevertheless, it can clearly be seen that spores of B. subtilis are most resistant to dehydration, as the recovery was high for ICR contact plates with 70% to 85%, followed by S. aureus with 25% to 30%. The gram-negative bacteria E. coli and P. aeruginosa were much more sensitive to dehydration of the test surface and resulted in recovery rates <10% using the ICR contact plate.

All positive results were achieved after an incubation time of ≤ 3 days. The prolonged incubation of up to 7 days did not improve the detection.

recovery-of-microorganisms

Figure 2.Survival of bacterial test strains on Neoprene® after a drying period of 60 minutes in CFU (average of 3 samples each)

Table 2.Recovery rates of test bacterial strains on the Neoprene® surface using the "Agar Overlay" and ICR contact plate methods

2) Detection rates: ICR swabs vs. ICR contact plates from Neoprene® Surfaces

Based on the first part of this study the "Agar Overlay" doesn't seem to be a suitable method of choice to determine the number of viable microorganisms on the surface following dehydration of the microbial suspensions. The CFU numbers derived using the contact plates were higher in most cases, and hence it was selected to determine the suitability of ICR swabs in detecting low numbers of microorganisms on Neoprene® surfaces.

To check if the ICR swab can detect low numbers of microorganisms on Neoprene® surfaces, the overall rate of positive ICR samples was compared to the ICR contact plate. As indicated in Figure 3, all bacteria were detected at low levels from the surface using either TSA+LTHThio cont. – ICR+ contact plates or ICR swabs. The total number of positive and negative samples (22 and 4, respectively) were identical for both ICR swabs and ICR contact plates.

In situations where the TSA w. LTHThio cont.-ICR+ contact plate detected ≥ 2 CFU in all 3 samples for the specified microbial suspension, the ICR swab was also positive (see Table 3).

When at least 1 out of the 3 contact plate samples either show no growth or ≤ 1 CFU single negative, the ICR swab samples showed a positive turbidity result, as in case of P. aeruginosa. In addition, an average of 1 CFU for all contact plates, such as for the detection of E. coli results in 3 positive samples for the ICR swab.

positive-samples-of-contact-plates

Figure 3.Detection rates in positive samples by ICR contact plates and ICR swabs from Neoprene® surfaces with the average CFU detected from the sample surface

Table 3.Recovery of ICR contact plates in CFU from Neoprene® surfaces compared to inoculated CFU and positive sample results of ICR swabs

Suitability of ICR Swabs and contact plate to detect microorganisms from Neoprene® surface

This study demonstrated that ICR swabs and ICR contact plates are both suitable methods for the detection of low numbers of gram-positive and gram-negative microorganisms from Neoprene® gloves and showed comparable growth results. The study also revealed that the overlay method is not the most appropriate method to detect microorganisms on the Neoprene® surfaces. This could be due to the high hydrophobic structure of the Neoprene® surface that prevents the homogeneous spreading of the microorganisms on the surface.

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References

1.
2004. Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice. [Internet]. FDA Guidance for Industry (2004). Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/sterile-drug-products-produced-aseptic-processing-current-good-manufacturing-practice
2.
<1116> Microbiological Control and Monitoring of Aseptic Processing Environments. [Internet]. United States Pharmacopoeia 40 NF 35: Available from: https://www.dcvmn.org/IMG/pdf/usp_1116_em_for_aseptic_processing_3_.pdf
3.
EU GMP Guide (2008): Volume 4 EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use. [Internet]. Annex 1 Manufacture of Sterile Medicinal Products.
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