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Evaluation of Swab in Maintaining Survival Efficiency according to the CLSI M40-A2 Standard
Biomed Sci Letters 2023;29:249-255
Published online December 31, 2023;  https://doi.org/10.15616/BSL.2023.29.4.249
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Hyeokjin Kwon1,4,*, Myeongguk Jeong1,4,*, Yeeun Kim1,4,*, Yunhee Chang2,**, Myeonggi Jeong3,*** and Go-Eun Choi1,4,†,**

1Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea
2Department of Biomedical Laboratory Science, Kyungnam College of Information & Technology, Busan 51767, Korea
3Korea Standard Co., Ltd, Naju 58209, Korea
4Next-Generation Industrial Field-Based Specialist Program for Molecular Diagnostics, Brain Busan 21 Plus Project, Graduate School, Catholic University of Pusan, Busan 46252, Korea
Correspondence to: Go-Eun Choi. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea.
Tel: +82-51-510-0563, Fax: +82-51-510-0568, e-mail: gechoi@cup.ac.kr
*Graduate student, **Professor, ***Researcher.
Received November 9, 2023; Accepted December 1, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Transporting clinical samples for microbiological testing requires a proper transport medium that guarantees the survival of microorganisms. Therefore, the aim of the study was to determine the ability of Amies Transport Medium (ATM) to maintain the viability of microorganisms in clinical specimens and its suitability as a transport medium for microbiological testing. This study evaluated the performance of swab provided by KS Co., Ltd. for three groups of bacteria comprising aerobic and facultative anaerobic bacteria, anaerobic bacteria, and fastidious bacteria, according to the Clinical and Laboratory Standard Institute (CLSI) 8.11.2. The ATM stability test was conducted by dividing the medium into two groups based on the product expiration date of use. All tested media, A and B (the date of manufacture and expiration date are different) showed ≥5 CFU, and there was no significant difference in the result values of Category A and Category B with different serial numbers for each test. The results of this experiment when cross-checked with the guidelines suggest that ATM is a suitable transport medium for microbiological testing, as it maintains the viability of microorganisms and is suitable for overgrowth trials. In addition, compared to the number of CFUs at the origin, the number of CFUs did not increase by more than 1 log after storage. These results have important implications for the development of transport media that can guarantee the survival of microorganisms in clinical specimens.
Keywords : Transport medium, CLSI, Viability, Overgrowth, CFU, Bacteria
INTRODUCTION

Although various devices are available to transport specimens, based on the type of specimen and culture environment of the bacteria being transported, a common method of transport involves a plastic tube containing a bacterial medium and a sterile disposable swab.

Microorganism recovery from clinical specimens requires proper specimen collection and transportation (Tyrrell et al., 2016). Maintaining the viability and relative proportions of all microorganisms in the clinical specimen is essential during transportation (Wayne, 2010). A swab collection and transport device are frequently used in hospitals and other healthcare settings to obtain clinical specimens. It is crucial to collect, transport, and inoculate samples for microbiological testing (Drake et al., 2005; Gizzie and Adukwu, 2016).

If testing cannot be performed promptly after collection, the sample should be stored in a suitable environment or moved to a location where microbiological testing is possible. However, it may take several to 24 h from sample collection to the microbial laboratory; if the transportation is delayed, microbial isolation may fail depending on the state of the preserved sample.

The Amies Transport Medium (ATM, KS Co., Ltd. Naju, Korea) is an in vitro diagnostic medical device used for maintaining and preserving the viability of bacteria while transporting samples collected from patients to the laboratory for bacterial testing. Therefore, in this study, we aim to use ATM to evaluate the viability of aerobic, facultative anaerobic, anaerobic, and fastidious bacteria according to the Clinical and Laboratory Standard Institute (CLSI), M40-A2 guidelines (Perry, 1997; Farhat et al., 2001; Hindiyeh et al., 2001; Van Horn et al., 2008; Namekar et al., 2013). In addition, we conducted a stability test for ATM to determine whether the bacterial culture remains active until the expiration date of the transport media. There are two methods of bacterial inoculation. First, the initial inoculation method is to collect the bacteria by the Medschenker's nylon flocked swab and inoculate it immediately (5 min) at 20~25꼦. Another method is inoculation after storage, in which the bacteria are smeared on the MedSchenker's Flocked swab, stored for 24 or 48 h at 4~8꼦, and then inoculated into the medium (Wayne, 2010).

MATERIAL AND METHOD

The test was performed using a single colony after culturing the standard strain on a media plate for 24~48 h using Medschenker's nylon flocked swabs. Absorbance was measured at a wavelength of 600 nm according to the guidelines of a 0.5 McFarland turbidity standard in the range 0.08~1 (approximately cell density 1.5 횞 108 CFUs/mL). Qualitative criteria are as follows: the inoculum concentration was prepared according to the guidelines of the CLSI 8.12.1., and three bacterial groups (aerobic bacteria, anaerobic bacteria, and picky bacteria) were evaluated. There are two methods of bacterial inoculation. First, the initial inoculation method is to collect the bacteria by the Medschenker's nylon flocked swab and inoculate it immediately (5 min) at 20~25꼦. Another method is inoculation after storage, in which the bacteria are smeared on MedSchenker's Flocked swab, stored for 24 or 48 hr at 48꼦, and then inoculated into the medium. The experiment was re-peated thrice in the same manner. For the ATM stability tests until the expiration date, we used different serial numbers for each test (Table 1).

To confirm the qualitative criteria, the detailed number of tests is shown in the table below

Serial number (Lot No.) Features Organism Dilution ratio Bacterial inoculation Number of quantity

Category A

Solid type with charcoal (KLA21G1)

Liquid type (KLB21G2)

Category B

Solid type with charcoal (KLA22D1)

Liquid type (KLB22D2)

Category A

Manufacture date

2021. 07. 01.

Category B

Manufacture date

2022. 04. 01.

P. aeruginosa

S. pyogenes

S. pneumoniae

H. influenzae

B. fragilis

P. anaerobius

F. nucleatum

C. acnes

P. melaninogenica

N. gonorrhoeae

103

Initial inoculation immediately: 3

Inoculation after storage: 3

270
104

Initial inoculation immediately: 3

inoculation after storage: 3

105

Initial inoculation immediately: 3

Inoculation after storage: 3

P. aeruginosa

S. pyogenes

S. pneumoniae

H. influenzae

B. fragilis

P. anaerobius

F. nucleatum

C. acnes

P. melaninogenica

N. gonorrhoeae

103

Initial inoculation immediately: 3

Inoculation after storage: 3

270
104

Initial inoculation immediately: 3

Inoculation after storage: 3

105

Initial inoculation immediately: 3

Inoculation after storage: 3



Cell line

This study was conducted in the Biosafety Level 2 laboratory in the Department of Clinical Pathology, Catholic University of Pusan. Test strains are the standard strains presented in the (CLSI) guidelines. This study was conducted by purchasing standard strains from Korean Collection for Type Cultures (KCTC, Daejeon, Korea) and American Type Culture Collection (ATCC, Virginia, USA) (Table 2).

The standard strains used in the experiment

Organism Strain Inoculum (CFU) Medium Incubation temperature (꼦) Incubation atmosphere
Aerobic, facultative anaerobic bacteria
Pseudomonas aeruginosa KCTC 22074 103 ~ 105 TSA + 5% sheep blood 35짹2 Aerobic
Streptococcus pyogenes ATCC 19615 103 ~ 105 TSA + 5% sheep blood 35짹2 5% CO2
Streptococcus pneumoniae ATCC 6305 103 ~ 105 TSA + 5% sheep blood 35짹2 5% CO2
Haemophilus influenzae ATCC 10211 103 ~ 105 Chocolate agar 35짹2 5% CO2
Anaerobic bacteria
Bacteroides fragilis KCTC 5013 103 ~ 105 BHI agar 35짹2 Anaerobic
Peptostreptococcus anaerobius ATCC 27337 103 ~ 105 BHI agar 35짹2 Anaerobic
Fusobacterium nucleatum KCTC 2640 103 ~ 105 BHI agar 35짹2 Anaerobic
Cutibacterium acnes ATCC 6919 103 ~ 105 BHI agar 35짹2 Anaerobic
Prevotella melaninogenica KCTC 5457 103 ~ 105 BHI agar 35짹2 Anaerobic
Facultative anaerobic bacteria
Neisseria gonorrhoeae ATCC 43069 103 ~ 105 Chocolate agar 35짹2 5% CO2


Study isolates

According to the CLSI 8.11.2., three groups of bacteria (aerobic and facultative anaerobic bacteria, anaerobic bacteria, and fastidious bacteria) were evaluated. This collection of three groups of bacteria test isolates consisted of four aerobic and facultative anaerobic bacteria reference isolates (Pseudomonas aeruginosa KCTC 22074, Streptococcus pyogenes ATCC 19615, Streptococcus pneumoniae ATCC 6305, Haemophilus influenzae ATCC 10211), five anaerobic bacteria reference isolates (Bacteroides fragilis KCTC 5013, Peptostreptococcus anaerobius ATCC 27337, Fusobacterium nucleatum KCTC 2640, Cutibacterium acnes ATCC 6919, Prevotella melaninogenica KCTC 5457) and one fastidious bacteria reference isolates (Neisseria gonorrhoeae ATCC 43069) that are difficult to culture were used.

CLSI M40-A2 roll-plate method

The experiment was conducted using the roll-plate method. Microorganisms grown for 24~48 hr were diluted with 0.85% physiological saline (pH 6.8~7.2) to approximately

1.5 횞 108 CFU (equivalent to 0.5 McFarland turbidity stand-ard) inoculation solution concentration. The inoculated solution is subjected to 1:10 serial dilution four times and adjusted such that the final concentration falls within 1.5 횞 107~1.5 횞 104 CFUs/mL. For each microbial group to be evaluated, 100 關L is added to 96-well for each diluent of 1.5 횞 104, 1.5 횞 105, and 1.5 횞 106 and inoculated using a cotton swab. The final inoculations absorbed by the swab were 1.5 횞 103, 1.5 횞 104, and 1.5 횞 105 CFUs/mL. Two types of bacterial swabs were used to inoculate into different types of ATM of A (liquid type) and B (solid type with charcoal) with varying dates of expiration. First, the initial inoculation method is to collect the bacteria by the Medschenker's nylon flocked swab and inoculate it immediately (5 min) at 20~25꼦. Another method is inoculation after storage, in which the bacteria are smeared on MedSchenker's Flocked swab, stored for 24 or 48 hr at 4~8꼦, and then inoculated into the medium. The experiment was repeated thrice in the same manner.

Viability test and check for overgrowth

After incubation, a viability test was performed by counting the CFUs and calculating the average. Each medium was evaluated according to the inoculation time and dilution factor. The final CFUs were calculated as the average of the CFUs of the three culture plates. The plate of origin medium closest to 250 CFUs was used to qualify for compliance in a viability test. The viability test was performed with an appropriate dilution factor and storage time. If the result is 돟 5 CFUs, it indicates that it meets the criteria as suitable. The check for overgrowth is the average number of colonies in the origin plate that should be 5~50 CFUs to effectively perform the overgrowth experiment. For the results of an overgrowth test to be considered acceptable, the number of CFUs after storage should not increase by more than 1 log compared to the number of CFUs measured in the plate of origin. For example, if the plate of origin had 15 CFUs, it is considered acceptable if the overgrowth plate test has less than 150 CFUs after storage.

RESULTS

In this study, the CLSI M40-A2 roll plate method was used; two types of ATM were used for transport systems, viz., liquid type and solid type with charcoal. Bacteria used in the study were three groups of bacteria (aerobic and facultative anaerobic bacteria, anaerobic bacteria, and fastidious bacteria). A total of 10 bacteria were cultured in two media types with different manufacture dates. This process was repeated thrice (Table 2).

Recovery of Liquid type transport system

Of the two Amies Transport Medium (ATM) systems, the liquid-type transport system showed acceptable recovery from all ten isolates according to CLSI standards. Recoveries of the aerobic and facultative anaerobic bacteria after storage at room temperature and refrigeration are summarized in Table 3. The final CFUs were calculated as the average of the CFUs of the three plates. If the result is 돟 5 CFUs, it indicates that it meets the criteria as suitable in a viability test. To consider it acceptable in the overgrowth test, the number of CFUs after storage should not increase by more than 1 log compared to the number of CFUs at the origin. As a result, 돟 5 CFUs was found in all tested media, and accordingly all ten isolates showed acceptable recovery, and overgrowth did not increase by more than 1 log.

Viability and overgrowth tests after storage at refrigerated and room temperature (The Liquid type)

Organism Type Recovery at concentration of a
105 CFU/mL at (h) 104 CFU/mL at (h) 103 CFU/mL at (h)
5 min 24 hr, 48 hr 5 min 24 hr, 48 hr 5 min 24 hr, 48 hr
RT 4꼦 RT 4꼦 RT 4꼦
Aerobic, facultative anaerobic bacteria
P. aeruginosa A 725 655 142 120 12 13
B 815 648 155 135 15 14
S. pyogenes A 302 316 35 32 4 3
B 319 267 42 30 4 3
S. pneumoniae A 608 758 74 90 9 9
B 749 971 90 107 10 11
H. influenzae A 1389 1311 213 174 20 19
B 1230 1281 183 171 17 17
Anaerobic bacteria
B. fragilis A 1429 1412 313 313 29 28
B 1442 1358 321 314 30 27
P. anaerobius A 1513 1453 226 216 24 23
B 1421 1435 212 216 24 23
F. nucleatum A 1212 1264 244 244 21 20
B 1181 1103 241 205 20 18
C. acnes A 1258 1170 288 253 30 28
B 1241 1162 257 222 25 21
P. melaninogenica A 1111 1419 180 204 20 22
B 1375 1391 213 221 24 24
Fastidious bacteria
N. gonorrhoeae A 1227 956 251 228 29 27
B 1060 869 216 211 27 29

aAverage of triplicate tests in CFU/100 關L. RT, room (ambient) temperature

A and B the date of manufacture and expiration date are different



Recovery of Solid type with charcoal transport system

Of the two ATM systems, the solid type with the charcoal transport system showed acceptable recovery from all ten isolates according to CLSI standards. Recoveries of the anaerobic bacteria after storage at room temperature and refrigeration are summarized in Table 4. The final CFUs were calculated as the average of the CFUs of the three plates. If the result is 돟 5 CFUs, it indicates that it meets the criteria as suitable in a viability test. To consider it acceptable in the overgrowth test, the number of CFUs after storage should not increase by more than 1 log compared to the number of CFUs at the origin. As a result, 돟 5 CFUs was found in all tested media, and accordingly all five isolates showed acceptable recovery, and overgrowth did not increase by more than 1 log.

Viability and overgrowth tests after storage at refrigerated and room temperature (The solid type with charcoal)

Organism Type Recovery at concentration of a
105 CFU/mL at (h) 104 CFU/mL at (h) 103 CFU/mL at (h)
5 min 24 hr, 48 hr 5 min 24 hr, 48 hr 5 min 24 hr, 48 hr
RT 4꼦 RT 4꼦 RT 4꼦
Aerobic, facultative anaerobic bacteria
P. aeruginosa A 907 819 160 157 14 13
B 820 764 152 150 13 12
S. pyogenes A 327 252 30 24 3 3
B 350 273 44 26 4 2
S. pneumoniae A 809 837 104 112 9 9
B 937 854 122 115 10 9
H. influenzae A 1525 1400 221 184 18 18
B 1413 1418 180 196 20 19
Anaerobic bacteria
B. fragilis A 1545 1450 329 294 32 29
B 1522 1360 322 296 31 28
P. anaerobius A 1530 1521 247 240 26 24
B 1490 1571 243 254 26 26
F. nucleatum A 1282 1135 251 211 23 21
B 1347 1251 286 234 25 22
C. acnes A 1554 1447 327 309 31 31
B 1508 1461 301 317 35 34
P. melaninogenica A 1557 1677 253 252 23 22
B 1353 1607 228 248 22 22
Fastidious bacteria
N. gonorrhoeae A 1062 974 218 187 27 29
B 993 931 208 177 29 25

aAverage of triplicate tests in CFU/100 關L. RT, room (ambient) temperature

A and B the date of manufacture and expiration date are different


DISCUSSION

It is essential to collect, transport, and inoculate samples for microbiological testing. The CLSI M40-A2 provides standardized methods for both roll plate method and elution, to aid manufacturers and laboratories in determining the performance characteristics of swab transport devices. If testing cannot be performed promptly after collection, the sample should be stored in a suitable environment or moved to a location where microbiological testing is possible. However, it may take several to 24 h from sample collection to the microbial laboratory; if the transportation is delayed, microbial isolation may fail depending on the state of the preserved sample. Therefore, the transport medium must ensure the viability of microorganisms in clinical specimens, and the performance of the transport medium is very important for the accurate diagnosis of clinical trials.

The criteria set by the CLSI M40-A2 standard for the roll plate method is as follows: all samples stored at 4꼦 and room temperature for viability compliance must yield 돟 5 CFUs after the specified storage period. The collected samples were evaluated for the ability to maintain the viability of bacteria in the two types (liquid type and solid type with charcoal) of transport medium, which is the ATM provided by KS (Avolio and Camporese, 2015). Three groups of bacteria (aerobic and obligate anaerobic bacteria, anaerobic bacteria, and fastidious bacteria) were evaluated according to CLSI 8.11.2 (Rishmawi et al., 2007). For ATM stability tests until expiration, we used Category A and B with different serial numbers for each test.

As a result, 돟 5 CFU was found in the tested media A and B (the date of manufacture and expiration date are different). According to the guidelines, the results of this experiment were proved to be suitable for the viability test. In addition, compared to the number of CFUs at the origin, the number of CFUs did not increase by more than 1 log after storage. It has also proven to be suitable in overgrowth trials.

In conclusion, the ATM system is an acceptable swab transport system for aerobic, anaerobic, and picky bacteria. This system met CLSI acceptance criteria for all aerobic and anaerobic isolates when tested under refrigerated storage conditions. Therefore, it is considered that this result is suitable as a transport medium for transport to a place where microbiological testing is possible.

ACKNOWLEDGEMENT

This research was supported by the BB21plus funded by Busan Metropolitan City and Busan Techno Park.

Compliance with ethical standards

This study was conducted by purchasing standard strains from KCTC (Daejeon, Korea) and ATCC (Virginia, USA). As such, no consent was required from study subjects.

CONFLICT OF INTEREST

The authors declare that they have no commercial or financial relationships that could be perceived as a potential conflict of interest in conducting this research.

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