Oligotrophic Media Compared with a Tryptic Soy Agar or Broth for the Recovery of Burkholderia cepacia Complex from Different Storage Temperatures and Culture Conditions원문보기
Ahn, Youngbeom
(Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration)
,
Lee, Un Jung
(Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration)
,
Lee, Yong-Jin
(Department of Biological Sciences, Albany State University)
,
LiPuma, John J.
(Department of Pediatrics, University of Michigan)
,
Hussong, David
(Eagle Analytical Services)
,
Marasa, Bernard
(Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, U.S. Food and Drug Administration)
,
Cerniglia, Carl E.
(Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration)
The Burkholderia cepacia complex (BCC) is capable of remaining viable in low-nutrient environments and harsh conditions, posing a contamination risk in non-sterile pharmaceutical products as well as a challenge for detection. To develop optimal recovery methods to detect BCC, three oligotrophic medi...
The Burkholderia cepacia complex (BCC) is capable of remaining viable in low-nutrient environments and harsh conditions, posing a contamination risk in non-sterile pharmaceutical products as well as a challenge for detection. To develop optimal recovery methods to detect BCC, three oligotrophic media were evaluated and compared with nutrient media for the recovery of BCC from autoclaved distilled water or antiseptic solutions. Serial dilutions ($10^{-1}$ to $10^{-12}CFU/ml$) of 20 BCC strains were inoculated into autoclaved distilled water and stored at $6^{\circ}C$, $23^{\circ}C$ and $42^{\circ}C$ for 42 days. Six suspensions of Burkholderia cenocepacia were used to inoculate aqueous solutions containing $5{\mu}g/ml$ and $50{\mu}g/ml$ chlorhexidine gluconate (CHX) and $10{\mu}g/ml$ benzalkonium chloride (BZK), and stored at $23^{\circ}C$ for a further 199 days. Nutrient media such as Tryptic Soy Agar (TSA) or Tryptic Soy Broth (TSB), oligotrophic media (1/10 strength TSA or TSB, Reasoner's $2^{nd}$ Agar [R2A] or Reasoner's $2^{nd}$ Broth [R2AB], and 1/3 strength R2A or R2AB) were compared by inoculating these media with BCC from autoclaved distilled water and from antiseptic samples. The recovery of BCC in water or antiseptics was higher in culture broth than on solid media. Oligotrophic medium showed a higher recovery efficiency than TSA or TSB for the detection of 20 BCC samples. Results from multiple comparisons allowed us to directly identify significant differences between TSA or TSB and oligotrophic media. An oligotrophic medium pre-enrichment resuscitation step is offered for the United States Pharmacopeia (USP) proposed compendial test method for BCC detection.
The Burkholderia cepacia complex (BCC) is capable of remaining viable in low-nutrient environments and harsh conditions, posing a contamination risk in non-sterile pharmaceutical products as well as a challenge for detection. To develop optimal recovery methods to detect BCC, three oligotrophic media were evaluated and compared with nutrient media for the recovery of BCC from autoclaved distilled water or antiseptic solutions. Serial dilutions ($10^{-1}$ to $10^{-12}CFU/ml$) of 20 BCC strains were inoculated into autoclaved distilled water and stored at $6^{\circ}C$, $23^{\circ}C$ and $42^{\circ}C$ for 42 days. Six suspensions of Burkholderia cenocepacia were used to inoculate aqueous solutions containing $5{\mu}g/ml$ and $50{\mu}g/ml$ chlorhexidine gluconate (CHX) and $10{\mu}g/ml$ benzalkonium chloride (BZK), and stored at $23^{\circ}C$ for a further 199 days. Nutrient media such as Tryptic Soy Agar (TSA) or Tryptic Soy Broth (TSB), oligotrophic media (1/10 strength TSA or TSB, Reasoner's $2^{nd}$ Agar [R2A] or Reasoner's $2^{nd}$ Broth [R2AB], and 1/3 strength R2A or R2AB) were compared by inoculating these media with BCC from autoclaved distilled water and from antiseptic samples. The recovery of BCC in water or antiseptics was higher in culture broth than on solid media. Oligotrophic medium showed a higher recovery efficiency than TSA or TSB for the detection of 20 BCC samples. Results from multiple comparisons allowed us to directly identify significant differences between TSA or TSB and oligotrophic media. An oligotrophic medium pre-enrichment resuscitation step is offered for the United States Pharmacopeia (USP) proposed compendial test method for BCC detection.
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문제 정의
Results from this study clearly demonstrate the utility of using diluted media and enriched cultures with a pre-enrichment step as a superior strategy in the compendial test method USP [60] for BCC recovery and detection [45]. Furthermore, this study provides a comprehensive data set that supports the USP inclusion of lower nutrient, resuscitative media as suggested previously [18, 20].
In addition, the recovery of microorganisms from distilled water samples and antiseptics increased by using a pre-enrichment medium. Results in this study provide confirmatory data for USP in support of its proposed compendial chapter for BCC detection. Results from this study clearly demonstrate the utility of using diluted media and enriched cultures with a pre-enrichment step as a superior strategy in the compendial test method USP [60] for BCC recovery and detection [45].
In this study, the 1/10 × TSA/B, 1/3 × R2A/B, and R2A/B is referred to as oligotrophic medium. The aim of this study was to assess the recovery of BCC using oligotrophic media as compared to the TSA or TSB at different storage temperatures in autoclaved distilled water samples as well as at different antiseptic concentrations.
The manuscript is dedicated to the late Mr. Walter Bond for his seminal research contributions on the recovery of Burkholderia cepacia from nebulizer fluids that stimulated the research presented in this paper as well as to the development of the USP chapter on this subject. The views presented in this article do not necessarily reflect those of the Food and Drug Administration.
제안 방법
BCC survival was tested at 6°C (initial serial dilution 10-2, 10-3, 10-4, 10-5, and 10-6), 23°C (initial serial dilution 10-8, 10-9, and 10-10), and 42°C (initial serial dilution 1, 10-1, 10-2, and 10-3) for 42 days.
Number of positive wells (number of wells set up) of B. cepacia complex at 10-2-10-12 dilutions in nonselective broth media at 6°C, 23°C, and 42°C for 42 days.
Results in this study provide confirmatory data for USP in support of its proposed compendial chapter for BCC detection. Results from this study clearly demonstrate the utility of using diluted media and enriched cultures with a pre-enrichment step as a superior strategy in the compendial test method USP [60] for BCC recovery and detection [45]. Furthermore, this study provides a comprehensive data set that supports the USP inclusion of lower nutrient, resuscitative media as suggested previously [18, 20].
The TSB, 1/10 × TSB, 1/3 × R2AB, and R2AB broth media were evaluated for their ability to recover B. cenocepacia in 5 and 50 μg/ml of CHX or 10 μg/ml of BZK.
The lag phase (λ; A, B, and C) and growth rate (μ; D, E, and F) of B. cenocepacia AU19236 inoculated in TSB, 1/10 × TSB, 1/3 × R2AB and R2AB medium stored at 6°C (A and D), 25°C (B and E) and 42°C (C and F) for 42 days using the Baranyi growth model.
To evaluate the effect of nutrient content on growth kinetics, TSB, 1/10 × TSB, R2AB and 1/3 × R2AB broth media were used.
대상 데이터
TSA, R2A, and diluted agar medium (1/10 × TSA and 1/3 × R2A) containing 15 g agar in deionized water were used.
데이터처리
A Tukey’s HSD test on ANOVA was performed to examine pairwise differences of the proportion of the colony counts between different agar media.
A Tukey’s honest significant difference (HSD) test on ANOVA was applied to examine pairwise differences between groups for continuous variables.
A Tukey-type multiple comparison on Pearson’s χ2 -test was performed to evaluate pairwise difference of the recovered BCC between different broth media.
Differences between media groups were analyzed using the one-way analysis of variance (ANOVA) followed by Tukey’s HSD test.
For data analysis from broth media, a macro, CHISQ_MC, was implemented to perform a Tukey-type multiple comparison on Pearson’s χ2-test for categorical variables [34].
Summary of descriptive statistics and results of post hoc tests using Tukey’s HSD test on ANOVA (A) and Tukey-type multiple comparison on Pearson’s χ2 test (B).
The number of colony forming units for BCC was statistically evaluated by the Mann-Whitney Rank Sum Test using SigmaPlot software (GraphPad Software, Inc. USA). The number of positive broth media for BCC was statistically evaluated by the χ2-test using Social Science Statistics web-based software (https:// www.
Univariate analyses were performed using ANOVA for continuous variables and Pearson’s χ2-test for categorical variables to explore significant differences among groups.
이론/모형
Table 1. Formulation of enrichment media used to evaluate the B. cepacia complex culture method.
성능/효과
B. cenocepacia AU19236 had a significantly longer lag phase at 6°C (17.6–27.4 h) than at 23°C and 42°C (10.1–14.2 h).
Evaluation of the four broths inoculated with the lowest colony numbers (10-12) indicated that 1/10 × TSB, 1/3× R2AB, and R2AB supported the most efficient recovery of BCC from 23°C distilled water for 42 days.
In this investigation, the survival of 20 BCC strains in distilled water samples was observed at 6°C and 23°C over a 42-day period, which slightly surpasses the previously examined 40-day period (see Table S1 to S6).
The differences between oligotrophic media and TSB or TSA medium for growth or survival in this study were due to the favorable nutritional composition of oligotrophics, compared to a high nutrition medium. On the basis of our experiments, R2A and dilutions of TSA constituted better enrichment media for the recovery of BCC from distilled water and antiseptics. In addition, the recovery of microorganisms from distilled water samples and antiseptics increased by using a pre-enrichment medium.
The Mann-Whitney Rank Sum Test was performed to compare the difference between TSA and 1/10 × TSA, 1/3 × R2A, and R2A, and a significant difference was not found (p <0.05).
The Mann-Whitney Rank Sum Test was performed to compare the difference between TSA and 1/10 × TSA, and a significant difference was not found (p < 0.05).
The Mann-Whitney Rank Sum Test was performed to compare the difference between TSA and R2A, and a significant difference was found (p < 0.041).
In addition, the number of bacteria recovered with different broth media indicated that oligotrophic broth media provide for the highest recovery of BCC. The differences between oligotrophic media and TSB or TSA medium for growth or survival in this study were due to the favorable nutritional composition of oligotrophics, compared to a high nutrition medium. On the basis of our experiments, R2A and dilutions of TSA constituted better enrichment media for the recovery of BCC from distilled water and antiseptics.
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