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Comparative Evaluation of Biomed InTray® Colorex MRSA with BD ESwab Collection Kit/ BBL™ CHROMagar® MRSA II
ISSN: 2161-0703

Journal of Medical Microbiology & Diagnosis
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Comparative Evaluation of Biomed InTray® Colorex MRSA with BD ESwab Collection Kit/ BBL™ CHROMagar® MRSA II

Kiran H Bijlani, Marcela Gomez, Rowena Matias, Ana Najafi, Ron Najafi and Sridhar Arumugam*
Emery Pharma Cell Biology and Microbiology, CA, USA
*Corresponding Author: Sridhar Arumugam, Director of Cell and Microbiology, Emery Pharma Cell Biology and Microbiology, 1000 Altantic Avenue Suite 110 Alameda, CA 94501, USA, Tel: 0015108998828, Email: [email protected]

Received Date: Feb 08, 2018 / Accepted Date: Feb 15, 2018 / Published Date: Feb 19, 2018

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most dangerous antibiotic resistant pathogens and a common cause of most health-care acquired infections. MRSA causes skin, wound and bloodstream infections that can cause sepsis and ultimately lead to death. CDC and WHO have listed MRSA as a serious threat infection and included in The National Action Plan for Combating Antibiotic-resistant Bacteria. Early, reliable, and accurate diagnosis of MRSA in a clinical setting is critical for the treatment and control of infection in hospitals and the community. We comparatively evaluated the efficacy of two commercial diagnostic systems, Biomed InTray® Colorex and BDTM ESwab Regular Collection Kit/ BBL™ CHROMagar® (ESwab + CHROMagar®) to recover 51 MRSA clinical isolates. The percentage recovery of MRSA clinical isolates in InTray® and in ESwab + CHROMagar® was 99% and 75%, respectively. Our findings suggest that InTray® was more efficient than ESwab + CHROMagar® in recovering MRSA clinical isolates.

Keywords: Methicillin-resistant S. aureus (MRSA); Diagnostic test; InTray®; Eswab+CHROMagar®

Introduction

Antimicrobial resistance has developed over time, usually through evolution of the bacteria, however it has been further accelerated because of misuse and overuse of antibiotics. As a result, the antimicrobial drug becomes ineffective in killing the bacteria, resulting in persistent infections, prolonged illness and ultimately increasing the cost of health care across the globe. Such bacteria are also referred to as “Superbugs” because infections caused by them are extremely difficult to treat [1]. Many strains of S. aureus have developed resistance to methicillin, oxacillin and nearly all the beta-lactam antibiotics by producing an alternative penicillin-binding protein known as PBP2a that are collectively referred to as “Methicillin-resistant Staphylococcus aureus” or “MRSA” [2]. PBP2a is encoded by the mecA gene and has a low affinity to many beta-lactam antibiotics [3].

MRSA is one of the most dangerous antibiotic-resistant pathogens and one of the leading causes of hospital acquired infections (HAIs), in addition, MRSA is rapidly spreading within the community. The typical sites of colonization of the bacteria are the nostrils and mucous membranes of the upper respiratory tract. It can cause lethal infections such as infective endocarditis (IE), skin and soft tissue infection (SSTI), and hospital-acquired/ventilator acquired pneumonia (HAP) [4,5]. The Center for Disease Control (CDC) and the World Health Organization (WHO) have listed MRSA as a “serious threat” and it is on high priority list on health challenges faced globally by practitioners. In the United States, the National Health Care Safety Network estimates that hospitalized patients acquire 2 million HAIs per year, out of which a large percentage are related to MRSA infections [6]. The costs are estimated in the billions of dollars, making the economic burden of disease very high. According to the CDC, MRSA leads to 10,000 deaths in the U.S. per year [6].

To provide effective and targeted treatment for MRSA to the patient, the first step is identifying the infection as MRSA from the multitude of infectious bacterium. Screening usually involves swabbing of the nasal passages in a conventional selective media (Amies media) and then transporting to the clinical laboratory for bacterial culture. Molecular diagnostic methods such as PCR [7], which may provide rapid detection, have been cited with reports of false positive results where patients have been diagnosed as MRSA positive [8]. Other concerns regarding this method include the associated costs of trained technicians, specialized machinery, and consumables [9]. The ease at which conventional diagnosis methods are performed in laboratories with limited resources continues to underscore the importance of nonmolecular diagnostic testing as reliable and cost-efficient. Thus, we comparatively evaluated two commercially available non-molecular diagnostics systems, BioMed InTray® Colorex (InTray®) and the conventional BDTM ESwab Collection Kit/ BBL™ CHROMagar® (ESwab + CHROMagar®).

Materials and Methods

Test articles

InTray® Colorex MRSA (InTray®) Lot # 6VA139X; Expiration: 11-12- 2016) was provided by Biomed Diagnostics, Inc. Conventional products, BDTM ESwab collection kit (ESwab) [Lot # 1MMJ45; Expiration: 05-31-2017) and BBL™ CHROMagar® MRSA II (CHROMagar) (Lot # 6244574; Expiration: 11-18-2016) were purchased from Becton, Dickinson and Company.

Bacterial strains

This study was carried out to compare the recovery of MRSA clinical isolates on the InTray® and ESwab + CHROMagar® diagnostic systems. All the clinical isolates acquired from Eurofin Medinet, Inc. (Herndon, VA) were Oxacillin resistant and 100% susceptible to Vancomycin, Daptomycin, and Linezolid. ATCC 29213 which is a Wild type QC susceptible strain of S. aureus was used as a negative control. ATCC 29213 was obtained from the American Type Culture Collection (Manassas, VA). The strains were maintained as frozen glycerol stocks at -80°C. Working stocks were prepared by thawing a glycerol stock, streaking onto a Tryptic Soy Agar plate and incubated at 37°C for 24 hours.

Methods

Antimicrobial susceptibility testing was performed on 51 clinical isolates as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Overnight cultures for each of the isolates were used to dilute stock solutions of microorganism’s equivalent to a 0.5 McFarland standard. These were prepared in cation-adjusted Mueller-Hinton broth (CAMHB) to achieve an inoculum of 1.5 × 108 CFU/mL [10]. This inoculum was diluted further in CAMHB to achieve 3 × 103 CFU/mL for each strain and 50 μL of this inoculum containing 150 CFU was plated on the InTray® and the ESwab + CHROMagar®. The test was performed in triplicates. To determine the bacterial recovery in the InTray™, 50 μL of inoculum containing 150 CFU was plated, incubated at room temperature for 48 hours and then incubated at 37°C for additional 24 hours. This 48 hour incubation at room temperature was done to simulate shipping conditions while samples are in transit before they reach the clinical lab testing facilities. To test the ESwab + CHROMagar®, 1 mL of Amies media was inoculated with 50 μL of inoculum containing 150 CFU. The inoculum was incubated at room temperature for 48 hours, then plated on CHROMagar® plates, and incubated at 37°C for additional 24 hours. After the incubation, the recovery efficacy was determined by enumerating the number of colonies in the InTray® and CHROMagar® plates. Growth of pinkpigmented colonies was considered as positive (indicating MRSA) and no growth of colonies, or with other colors, was considered as negative.

Results

The 51 clinical isolates were categorized based on the susceptibility or resistance to the following 5 antibiotics: Ciprofloxacin, Clindamycin, Erythromycin, Imipenem and Sulfamethoxazole/Trimethoprim. The antibiogram shown in Table 1 confirms that MRSA isolates are multidrug resistant. In addition, clinically important strains ATCC 33591 and ATCC BAA- 1717 USA 300 were included to further validate the study.

Serial # Organism Isolate # Antibiotic Resistance Ciprofloxacin Clindamycin Erythromycin Imipenem Trimethoprim/ Sulfamethoxazole
No. of Antibiotics that are Ineffective MIC (μg/mL) CLSI interpretation MIC (μg/mL) CLSI interpretation MIC (μg/mL) CLSI interpretation MIC (μg/mL) CLSI interpretation MIC (μg/mL) CLSI interpretation
1 Staphylococcus aureus 1974126 4 >16 R >4 R >8 R >32 R ≤ 0.25 S
2 Staphylococcus aureus 1974131 4 >16 R >4 R >8 R 16 R ≤ 0.25 S
3 Staphylococcus aureus 1974129 3 16 R >4 R >8 R 0.5 S ≤ 0.25 S
4 Staphylococcus aureus 1974133 3 16 R >4 R >8 R 2 S ≤ 0.25 S
5 Staphylococcus aureus 1974136 3 >16 R >4 R >8 R 0.5 S ≤ 0.25 S
6 Staphylococcus aureus 1974140 3 >16 R >4 R >8 R 0.5 S ≤ 0.25 S
7 Staphylococcus aureus 1974143 2 16 R 0.12 S >8 R 0.25 S ≤ 0.25 S
8 Staphylococcus aureus 1974119 2 >16 R 0.12 S >8 R 0.25 S ≤ 0.25 S
9 Staphylococcus aureus 1974120 2 >16 R 0.12 S >8 R 0.25 S ≤ 0.25 S
10 Staphylococcus aureus 1974121 4 >16 R >4 R >8 R 32 R ≤ 0.25 S
11 Staphylococcus aureus 1974135 4 >16 R >4 R >8 R 32 R ≤ 0.25 S
12 Staphylococcus aureus 1974148 3 >16 R 0.25 S >8 R 16 R ≤ 0.25 S
13 Staphylococcus aureus 1974155 3 16 R >4 R >8 R 2 S ≤ 0.25 S
14 Staphylococcus aureus 1974161 3 >16 R >4 R >8 R 0.25 S ≤ 0.25 S
15 Staphylococcus aureus 1974171 3 >16 R >4 R >8 R 0.125 S ≤ 0.25 S
16 Staphylococcus aureus 1974175 2 16 R 0.12 S >8 R 1 S ≤ 0.25 S
17 Staphylococcus aureus 1974122 2 >16 R 0.12 S >8 R 0.12 S ≤ 0.25 S
18 Staphylococcus aureus 1974123 2 0.5 S >4 R >8 R 2 S ≤ 0.25 S
19 Staphylococcus aureus 1974125 2 16 R 0.12 S >8 R 0.25 S ≤ 0.25 S
20 Staphylococcus aureus 1974127 2 >16 R 0.12 S >8 R 0.5 S ≤ 0.25 S
21 Staphylococcus aureus 1974132 2 >16 R 0.12 S >8 R 0.06 S ≤ 0.25 S
22 Staphylococcus aureus 1974137 2 4 R 0.12 S >8 R 0.06 S ≤ 0.25 S
23 Staphylococcus aureus 1974138 2 16 R 0.12 S >8 R 0.12 S ≤ 0.25 S
24 Staphylococcus aureus 1974139 2 >16 R 0.12 S >8 R 0.5 S ≤ 0.25 S
25 Staphylococcus aureus 1974144 2 16 R 0.12 S >8 R 0.12 S ≤ 0.25 S
26 Staphylococcus aureus 1974145 2 >16 R 0.25 S >8 R 0.25 S ≤ 0.25 S
27 Staphylococcus aureus 1974152 2 16 R 0.12 S >8 R 0.5 S ≤ 0.25 S
28 Staphylococcus aureus 1974153 2 8 R 0.06 S >8 R 8 I ≤ 0.25 S
29 Staphylococcus aureus 1974156 1 0.5 S 0.12 S >8 R 0.5 S ≤ 0.25 S
30 Staphylococcus aureus 1974128 1 0.5 S 0.12 S >8 R 0.5 S ≤ 0.25 S
31 Staphylococcus aureus 1974141 1 0.5 S 0.12 S >8 R 0.12 S ≤ 0.25 S
32 Staphylococcus aureus 1974142 1 0.5 S 0.12 S >8 R 0.12 S ≤ 0.25 S
33 Staphylococcus aureus 1974146 1 0.5 S 0.12 S >8 R 0.12 S ≤ 0.25 S
34 Staphylococcus aureus 1974147 1 0.5 S 0.12 S >8 R 0.5 S ≤ 0.25 S
35 Staphylococcus aureus 1974150 1 16 R 0.12 S 0.25 S 0.06 S ≤ 0.25 S
36 Staphylococcus aureus 1974162 1 >16 R 0.12 S 0.5 S ≤ 0.06 S ≤ 0.25 S
37 Staphylococcus aureus 1974169 1 >16 R 0.12 S 0.5 S ≤ 0.06 S ≤ 0.25 S
38 Staphylococcus aureus 1974170 1 >16 R 0.12 S 0.5 S ≤ 0.06 S ≤ 0.25 S
39 Staphylococcus aureus 1974176 1 >16 R 0.12 S 0.5 S 0.5 S ≤ 0.25 S
40 Staphylococcus aureus 1974177 1 >64 R 0.12 S >4 R ≤ 0.06 S ≤ 0.25 S
41 Staphylococcus aureus 1974184 1 0.125 S 0.12 S >4 R ≤ 0.06 S ≤ 0.25 S
42 Staphylococcus aureus 1974199 1 >64 R 0.12 S >4 R ≤ 0.06 S ≤ 0.25 S
43 Staphylococcus aureus 1974201 1 4 R 0.12 S >4 R ≤ 0.06 S ≤ 0.25 S
44 Staphylococcus aureus 1974202 1 0.5 S 0.12 S 0.5 S 1 S ≤ 0.25 S
45 Staphylococcus aureus 1974130 0 0.25 S 0.12 S 0.25 S 0.12 S ≤ 0.25 S
46 Staphylococcus aureus 1974134 0 0.12 S 0.12 S 0.5 S 0.25 S ≤ 0.25 S
47 Staphylococcus aureus 1974149 0 0.12 S 0.12 S 0.5 S 0.25 S ≤ 0.25 S
48 Staphylococcus aureus ATCC 33591 1 0.125 S >64 R >64 R 1 S 1 S
49 Staphylococcus aureus USA300 1 0.125 S 0.125 S 32 R ≤ 0.06 S 0.25 S
50 Staphylococcus aureus 1974151 0 0.5 S 0.12 S 0.5 S 0.12 S ≤ 0.25 S
51 Staphylococcus aureus 1974158 0 0.5 S 0.12 S 0.5 S 0.5 S ≤ 0.25 S

Clinical Isolates obtained from Eurofins Medinet, Inc. Herndon, VA except USA 300 and ATCC 33591 were obtained from ATCC.NOTE: All isolates listed were oxacillin resistant and 100% susceptible to vancomycin, daptomycin, and linezolid.

Table 1: Antibiogram of methicillin resistant S. aureus (MRSA) clinical isolates used in the current study. This confirms that MRSA isolates used in the study are indeed multi-drug resistant.

We evaluated the recovery of 51 MRSA strains in InTray® and ESwab + CHROMagar® diagnostic systems. Out of the 51 MRSA clinical isolates tested, 50 MRSA isolates were recovered in InTray® whereas only 38 strains were recovered in ESwab + CHROMagar® (Table 2). Figure 1 is a representative picture showing recovery of MRSA strain (Eurofin isolate # 1974134) in InTray® but not in ESwab + CHROMagar®. Recovery of MRSA strains were consistent within the triplicates in InTray®. In contrast, there was variation in recovery within the triplicates in ESwab + CHROMagar®. Figure 2 is a representative result where the MRSA strain (Eurofin isolate # 1974135) was recovered within all the InTray® triplicates whereas one of the triplicates in ESwab + CHROMagar® did not recover the bacteria. One MRSA strain (Eurofin isolate # 1974140) was not recovered in InTray® or in ESwab + CHROMagar®. ATCC 29213 which is a Wild type QC susceptible strain of S. aureus was used as a negative control and it did not grow on both the InTray® and CHROMagar®, confirming that both the systems are selective to MRSA isolates.

- In Tray CHROMagar
Serial # Isolate # # of Antibiotic
Resistant
1 2 3 Average 1 2 3 Average
1 1974126 4 51 TNTC TNTC TNTC TNTC 250 89 TNTC
2 1974131 4 TNTC TNTC TNTC TNTC TNTC TNTC TNTC TNTC
3 1974129 3 57 42 51 50 0 0 0 0
4 1974133 3 43 50 54 49 44 62 TNTC TNTC
5 1974136 3 49 54 50 51 1 0 0 1
6 1974140 3 0 0 0 0 0 0 0 0
7 1974143 3 TNTC TNTC TNTC TNTC 0 0 0 0
8 1974119 2 TNTC 37 56 TNTC 0 0 0 0
9 1974120 2 26 33 31 30 0 0 0 0
10 1974121 2 46 18 41 35 TNTC 10 TNTC TNTC
11 1974135 4 42 36 23 34 13 TNTC 0 TNTC
12 1974148 4 42 57 40 46 TNTC TNTC TNTC TNTC
13 1974155 3 76 61 48 662 TNTC TNTC TNTC TNTC
14 1974161 3 12 17 27 19 TNTC TNTC TNTC TNTC
15 1974171 3 47 46 50 48 TNTC TNTC TNTC TNTC
16 1974175 3 43 46 50 46 TNTC TNTC TNTC TNTC
17 1974122 2 38 40 54 44 0 0 0 0
18 1974123 2 56 46 55 52 0 0 0 0
19 1974125 2 39 40 47 42 0 0 0 0
20 1974127 2 55 55 127 79 0 0 0 0
21 1974132 2 30 26 60 39 0 48 29 26
22 1974137 2 34 45 58 46 1 0 1 1
23 1974138 2 35 41 25 34 TNTC TNTC TNTC TNTC
24 1974139 2 57 53 67 59 0 0 0 0
25 1974144 2 46 56 47 50 TNTC TNTC TNTC TNTC
26 1974145 2 46 35 60 47 0 0 0 0
27 1974152 2 34 33 36 34 TNTC TNTC TNTC TNTC
28 1974153 2 28 51 57 45 0 0 0 0
29 1974156 2 8 1 2 4 TNTC TNTC TNTC TNTC
30 1974128 1 27 31 28 29   1 0 0
31 1974141 1 51 36 60 49 0 11 0 4
32 1974142 1 55 74 73 67 2 1 0 1
33 1974146 1 68 78 40 62 0 0 0 1
34 1974147 1 55 57 39 50 2 2 2 2
35 1974150 1 85 75 98 86 3 TNTC TNTC TNTC
36 1974162 1 34 53 42 43 TNTC 2 1 1
37 1974169 1 61 38 56 52 0 TNTC TNTC TNTC
38 1974170 1 43 56 43 47 TNTC TNTC TNTC TNTC
39 1974176 1 26 34 32 31 TNTC TNTC TNTC TNTC
40 1974177 1 40 46 56 47 TNTC TNTC TNTC TNTC
41 1974184 1 43 38 34 40 TNTC TNTC TNTC TNTC
42 1974199 1 34 45 38 39 TNTC TNTC TNTC TNTC
43 1974201 1 34 36 51 40 TNTC TNTC TNTC TNTC
44 1974202 1 47 42 41 43 TNTC TNTC TNTC TNTC
45 1974130 0 48 39 44 44 1 1 1 1
46 1974134 0 28 40 61 43 0 0 0 0
47 1974149 0 67 43 64 58 0 6 11 6
48 ATCC33591 1 30 35 50 38 TNTC TNTC TNTC TNTC
49 USA 300 1 37 26 29 31 0 1 74 25
50 1974151 0 83 59 56 66 5 6 5 5
51 1974158 0 64 62 49 58 TNTC TNTC TNTC TNTC
Total Number of Strains Recovered In Tray   CHROMagar
50/51 38/51
Percentage of Recovery 99% 75%
  Positive for bacterial growth   Negative for bacterial growth
TNTC Too Numerous to count >300 colonies

Table 2: Recovery data for 51 MRSA clinical isolates in InTray® and ESwab + CHROMagar®.

medical-microbiology-diagnosis-good-recovery

Figure 1: Representative result showing good recovery of MRSA strain (Eurofin isolate # 1974134) in InTray® but not in ESwab + CHROMagar®. The study was carried out in triplicates. Pink colonies represent positive for MRSA growth.

medical-microbiology-diagnosis-MRSA-strain

Figure 2: Representative result where the MRSA strain (Eurofin isolate # 1974135) was recovered within all the triplicates in InTray®, whereas one of the triplicates in ESwab + CHROMagar® did not recover the bacteria. The study was carried out in triplicates. Pink colonies represent positive for MRSA growth.

Discussion

The impact of MRSA on hospitals and the community remains a burden in both developed and developing countries. Recent data reported to the National Healthcare Safety Network (NHSN) on MRSA bloodstream infections in the United States, indicates that by the end of 2015 there was little change in the average facility Standardized Infection Ratio (0.988), compared to a 2010-2011 baseline and is significantly increased compared to the previous year statistics [6,11]. Hence, an effective, easy to use and simple diagnostic system is much needed.

The current study was carried out to compare and evaluate the recovery efficacy of Biomed InTray® Colorex MRSA with a conventional product, BDTM ESwab collection kit and BBL™ CHROMagar® MRSA II. The current study evaluated the limit of detection in both the diagnostic systems by using a low inoculum size and determine if these diagnostic systems were effective in recovering the MRSA isolates.

The conventional method for MRSA diagnosis usually involves swabbing the patient sample using ESwab, which is then incubated with Amies media and then transported to a clinical laboratory, where the patient sample is then streaked for isolation on a selective media such as CHROMagar® [6,12,13]. In developing countries, the clinical laboratory may not be accessible and hence the transit time may take 1-2 days before the patient’s sample can be cultured and streaked to a selective agar for diagnosis. This may lead to significant loss of viability of the bacteria present in the patient sample. There have been few studies that have measured bacterial cell viability in Amies media, e.g. Robinson et al. demonstrated that 10 MRSA isolates used in their study could survive in Amies media at room temperature up to 14 days [14]. However, our results demonstrate that not all MRSA clinical isolates used in this study could be recovered on ESwab + CHROMagar® suggesting potential loss of viability of low bacterial load in transportation media. The percentage recovery of MRSA isolates in ESwab + CHROMagar® was only 75%. In contrast, InTray® demonstrated a higher percentage recovery of 99%.

Additionally, performing the testing with InTray® was easier and faster than ESwab + CHROMagar® which makes the InTray® system practically more “user friendly” with less room for error and contamination. The InTray® system has significant benefits over the ESwab + CHROMagar®. The InTray® system consists of a re-closable outer seal containing an optically clear, anti-fog window, which creates an airtight 2” diameter chamber providing a large enough area to streak for isolation. It is convenient to use as it combines collection, culture, and observation into one device. Minimal laboratory procedures and equipment are needed, and the device is easier to store. Because it is fully enclosed, the InTray® system prevents contamination and it also easy to see presence or absence of growth. There have been reports that newer molecular diagnostics such as rapid PCR testing have some issues with false-positive results, in which a high proportion (12.9%) of the patients were wrongly determined to be MRSA positive patients [8]. Hence, there is a need for a reliable diagnostic tool, and our data suggest that the InTray® system offers a robust, non-molecular solution for screening MRSA.

Conclusion

This study demonstrates that, in comparison with ESwab + CHROMagar®, the InTray® diagnostic system efficiently recovered more MRSA clinical isolates. The percentage of MRSA strains recovered in InTray® was 99% and in ESwab + CHROMagar® was 75%, suggesting InTray® may provide improved efficiency, however, additional studies are needed to confirm these findings in a clinical setting.

Acknowledgements

This study was funded by Biomed Diagnostics, Inc. (White City, OR).

References

Citation: Bijlani KH, Gomez M, Matias R, Najafi A, Najafi R, et al. (2018) Comparative Evaluation of Biomed InTray® Colorex MRSA with BD ESwab Collection Kit/ BBL™ CHROMagar® MRSA II. J Med Microb Diagn 7: 273. DOI: 10.4172/2161-0703.1000273

Copyright: © 2018 Bijlani KH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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