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Journal of Clinical Microbiology, January 2008, p. 145-149, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01769-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Influence of Type of Culture Medium on Characterization of Mycobacterium avium subsp. paratuberculosis Subtypes{triangledown}

N. Cernicchiaro, S. J. Wells, H. Janagama, and S. Sreevatsan*

Veterinary Population Medicine Department, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota 55108

Received 5 September 2007/ Returned for modification 9 October 2007/ Accepted 14 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We evaluated the effects of culture and/or enrichment methods on the selection of Mycobacterium avium subsp. paratuberculosis subtypes. M. avium subsp. paratuberculosis isolates from bovine fecal samples processed using a centrifugation protocol were investigated in both liquid (MGIT ParaTb tubes) and solid (Herrold's egg yolk medium) culture media. For this evaluation, M. avium subsp. paratuberculosis subtyping was based on the sequence variation in two of the most discriminatory short sequence repeat loci, i.e., mononucleotide G and trinucleotide GGT, in isolates from liquid and solid cultures. This study identified the existence of one major predominating fingerprint (>13G-5GGT) in bovine fecal samples, regardless of the type of medium used for isolation. Matched-pair analysis of subtypes showed that 69% of samples presented unique subtypes in the two culture media used, while 31% shared the same G-GGT allele. Furthermore, the liquid culture method appeared to select for a more genotypically diverse subtype population than the solid culture method. The variety of subtypes observed between liquid and solid cultures obtained from the same fecal samples suggests that the culture method could provide a "microbiological" bias and lead to a discrepancy in the growth of M. avium subsp. paratuberculosis strains. In conclusion, this study identified that these two types of culture media determined differential growth of M. avium subsp. paratuberculosis strains and that this should be considered in evaluating detection capabilities of diagnostic tests or interpreting data from molecular epidemiological studies performed using conventional solid fecal culture or automated liquid medium culture methods.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Differentiation of bacterial strains is expected to provide important information for molecular epidemiologic analysis and to assist in providing an understanding about the dynamics of transmission of Johne's disease. DNA-based molecular subtyping techniques, such as multiplex PCR for IS900 integration loci (12), restriction fragment length polymorphism analysis (15), amplified fragment length polymorphism analysis (8, 12), randomly amplified polymorphic DNA analysis (16), and pulsed-field gel electrophoresis (19), have been applied to investigate genetic variation in Mycobacterium avium subsp. paratuberculosis. Although these methods have been used extensively, they are generally not capable of resolving M. avium subsp. paratuberculosis isolates into meaningful epidemiologic groups due to the apparently restricted genetic diversity within subspecies and to the fact that these techniques may not be able to contribute to proper selection or discrimination of M. avium subsp. paratuberculosis subtypes (1, 14).

Differentiation and subtyping of bacterial pathogens can be performed by using repetitive elements of bacterial DNA, such as short sequence repeats (SSRs) or variable-number tandem repeats (VNTRs), as markers. The analysis of the whole genome sequence of M. avium subsp. paratuberculosis strain K-10 identified 185 SSRs consisting of three or fewer nucleotides per repeat unit (11). Even though there are numerous SSRs in the M. avium subsp. paratuberculosis genome, strain typing is typically based on two highly polymorphic SSR loci, namely, G and GGT repeats (1), and several M. avium subsp. paratuberculosis studies, representing different host species and geographic areas, have been conducted using these loci (1, 2, 4, 5, 12, 13).

The restricted diversity of M. avium subsp. paratuberculosis subtypes observed in previous studies may have been due to the fact that a very restrictive culture method was used. Our hypothesis is that the microbial cultivation method employed might lead to differential selection of M. avium subsp. paratuberculosis subtypes, leading to a microbiologic bias. The microbial growth and the constraints provided by each method would limit or allow growth of certain subtypes, and sensitive subtyping methods such as those based on SSRs should be able to resolve this bias. Although we and others (17) have observed different rates of M. avium subsp. paratuberculosis recovery between solid and liquid culture methods, investigations about the ability of strains to grow in different media have not been reported to date. Molecular characterization of M. avium subsp. paratuberculosis isolates from diverse culture media may lead to a better understanding of these relationships. Therefore, the aim of this study was to evaluate the effects of liquid and solid culture on the selection of M. avium subsp. paratuberculosis subtypes by using two of the most discriminatory SSR loci, i.e., locus 1 (G residue repeat) and locus 8 (GGT repeats).


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Samples. Fifty-two fecal samples were selected based upon detectable growth in both liquid and solid culture media. These samples were selected from a set of 100 well-characterized individual bovine fecal samples from existing frozen banks (–70°C) at the Minnesota Veterinary Diagnostic Laboratory. Of the 100 known samples, 75 previously culture-positive fecal samples with different levels of bacterial load from infected Minnesota dairy herds were included. These samples were processed using a centrifugation protocol followed by inoculation into Herrold's egg yolk medium slants and MGIT ParaTb liquid medium culture tubes. Figure 1 outlines the workflow of the methods employed in the study.


Figure 1
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  		FIG. 1. Workflow of the methods employed in this study. HEYM, Herrold's egg yolk medium.

 
Centrifugation method. Fecal samples were processed using the Pennsylvania centrifugation procedure described by Janet Payeur et al. in the Manual for the Isolation and Identification of M. avium subsp. paratuberculosis (15a) and modified by Becton Dickinson laboratories (as specified by the manufacturer's insert for BD BACTEC MGIT ParaTB medium [revised package insert draft 1-20-06]).

MGIT 960 system. After being processed, one hundred microliters of inoculum was placed in liquid culture tubes (BBL MGIT tubes; BD Laboratories). All manufacturer instructions were followed, and the instrument was set to a 49-day protocol. The system was checked twice a week during the 7-week protocol. Liquid culture results were expressed in terms of time to diagnosis or time to positive result, in days, according to the instrument signal. Whenever a sample signaled positive on the MGIT system, it was removed from the instrument, vortexed, and reentered. If the reentered tube signaled positive again, it was removed from the system and a sample was obtained to confirm the presence of M. avium subsp. paratuberculosis by performing acid-fast bacillus (AFB) staining and PCR tests. Tubes were not reentered into the system more than once. In cases of first removal, samples were reentered within a 5-hour interval. Tubes that signaled positive at 42 days or longer were not reentered into the system but were vortexed instead, and AFB staining (Ziehl-Neelsen method) and PCR tests were performed after 72 h. After the tubes signaled positive and were removed from the machine, they were placed in a 37°C incubator to continue the remaining incubation time. At the end of the protocol (49 days), the remaining tubes were pulled out of the MGIT system to perform AFB staining and ISMav2 Taqman PCR (21) tests for a final report of all samples.

DNA extraction. DNA material was extracted from liquid culture tubes as well as from colonies present in the corresponding solid culture slants. Broth samples were collected in a microcentrifuge tube containing 1 ml of 0.85% saline by taking 1 ml of the liquid culture medium from the bottom of the tube. The contents of the tubes were vortexed, and tubes were kept at –20°C until processed. Cell pellets from 1 ml of broth samples or bacteria harvested from solid slants were placed in a microcentrifuge tube containing 200 µl of deionized water. Tubes were centrifuged at 13,000 rpm for 10 min, and the pellets were employed for DNA extraction by using a QIAamp DNA mini blood kit (Qiagen Inc., Valencia, CA) following the manufacturer's procedure. Two other methods (QIAamp DNA mini blood kit with zirconium beads and zirconium beads together with proteinase K and subsequent phenol-chloroform extraction) were used for DNA extraction from bacterial cells isolated from a set of broth samples where G repeats could not be amplified (3, 6).

PCR amplification of G and GGT repeats. Amplification of loci carrying the G and GGT repeats was performed by separate PCRs. The 50-µl PCR master mix included 5 µl of 10x buffer, 2 µl of MgCl2, 2.5 µl of dimethyl sulfoxide, 1 µl of each primer at a concentration of 10 µM, 4 µl of deoxynucleoside triphosphates, 0.25 µl of hot Taq polymerase, and 0.4 µl of bovine serum albumin (10 mg/ml), and the remaining volume was completed with distilled water. A volume of 5 µl of genomic DNA was employed in each reaction mix. The thermocycler conditions were set at an initial 95°C incubation step for 15 min, with denaturation at 94°C for 20 seconds, annealing at 55°C for 20 seconds, and extension at 72°C for 20 seconds and with a final extension step at 72°C for 7 min. In some samples where the amplification of G repeats could not be accomplished by using external primers (VNTR-F/R), a nested PCR was employed. The VNTR-F primer amplified the locus and the VNTR-F2 primer bound within the first PCR product, producing a shorter PCR product. Table 1 shows the sequences of the primers employed in the study. Visualization of the amplification products was performed on a UV transilluminator after gel electrophoresis at 150 V for 30 min in 1% agarose prestained with 5% ethidium bromide.


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  		TABLE 1. Primers employed in the study

 
SSR analysis and DNA sequencing. Detectable PCR products for both repeats in 1% agarose gel were purified using a QIAquick PCR purification kit (Qiagen Inc., Valencia, CA) following the manufacturer's recommendations. Purified PCR products were sequenced by using an ABI PRISM Big Dye Terminator, version 3.1, cycle sequencing kit and were analyzed in an automated DNA sequencer (ABI PRISM 3730 xl DNA analyzer) at the Biomedical Genomics Center (University of Minnesota). Sequences were aligned against sequences in the GenBank database and analyzed by using the Editseq and MegAlign programs (DNASTAR, Inc., Madison, WI). The fingerprints were designated by the number of G repeats followed by the number of GGT residue repeats.

Analysis. Subtypes were identified by their numbers of G and GGT residues. When the total number of G repeats was 13 or more, the subtype was identified as >13G because above this number, it is more difficult to differentiate clear G residue repeat peaks in the chromatogram. The frequencies of distribution of M. avium subsp. paratuberculosis subtypes from all liquid and solid culture isolates were estimated, and a cluster analysis including all identified subtypes was conducted by the unweighted-pair group method using average linkages (UPGMA) (18), using MEGA, version 3.0, software (10). Additionally, Simpson's index of diversity, as a simple mathematical measure that characterizes the diversity of subtypes in liquid and solid culture isolates, was estimated using the equation described by Hunter and Gaston (7). Subtyping results for matched pairs of liquid and solid culture isolates were compared by type of culture medium, using contingency tables. The kappa statistic was estimated to determine the level of agreement (sharing the same G-GGT subtype) between solid and liquid isolates obtained by paired testing of samples.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
From the 52 selected samples, we were able to amplify G repeats from 27 broth and 50 solid medium isolates and GGT repeats from 46 broth and 52 solid medium isolates. Overall, 27 broth and 50 solid culture isolates with G-GGT genotype information were included in the analysis. Accordingly, of the 52 initial samples, 26 were available to perform matched comparisons of subtypes between isolates from liquid and solid media.

Ten G-GGT alleles were identified among all isolates. Nine alleles with 7 to >13 G residues and 4 or 5 GGT repeats were identified in 27 broth isolates. The 7G-5GGT and >13G-5GGT alleles were each present in 24% of M. avium subsp. paratuberculosis isolates. Eight alleles with 7 to >13 G residues and 4 or 5 GGT repeats were identified among the 50 solid culture isolates. The >13G-5GGT allele was present in 49% of M. avium subsp. paratuberculosis isolates. A total of 29 subtypes were obtained from the 27 broth isolates, and 2 isolates had evidence of mixed subtypes (11G-5GGT/7G-5GGT and 11G-5GGT/8G-5GGT). Fifty-one subtypes were found in 50 solid culture isolates, with one sample showing mixed subtypes (12G-5GGT/7G-5GGT). Figure 2 shows the frequencies of distribution of G-GGT subtypes identified among all liquid and solid culture isolates.


Figure 2
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  		FIG. 2. Cluster analysis and frequency distribution of Mycobacterium avium subsp. paratuberculosis subtypes from liquid and solid culture isolates. A UPGMA tree was generated based on G-GGT repeats. The frequencies of fingerprints identified among all liquid and solid culture isolates are represented next to each subtype.

 
Cluster analysis using UPGMA created a rooted tree that reflected similarities between subtypes under the assumption of a constant evolutionary rate. This method used a sequential clustering algorithm that identified subtypes in order of similarity, and the dendrogram was constructed in a stepwise manner. Subtypes with the highest similarity were identified and clustered. Subtype profiles were displayed along with their frequencies among liquid and solid culture isolates. 7G-4GGT was identified as the common ancestor genotype, and from it, all the remaining subtypes are presented individually in ascending order, except for a single cluster including genotypes 13G-4GGT and 13G-5GGT, which represent the most recent descendants of the common ancestor (Fig. 2).

Simpson's indices of diversity were 0.83 and 0.71 for broth and solid culture isolates, respectively. Of the 26 matched-pair samples, 18 (69%) produced unique fingerprints in both medium types, whereas 8 (31%) shared the same G-GGT fingerprint. Six of the eight samples that shared the same alleles had the >13G-5GGT fingerprint, while the other two showed subtypes 8G-4GGT and 10G-5GGT (Table 2). The kappa statistic for comparing subtypes by type of culture medium was 0.13.


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  		TABLE 2. Comparison of M. avium subsp. paratuberculosis strains present in liquid and solid culture isolates (n = 26)

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study represents the first evaluation of M. avium subsp. paratuberculosis subtypes by the type of culture medium used to culture bovine fecal samples. Some investigators have suggested that bacterial culture using liquid media has greater analytical sensitivity than that using solid media (20, 22), but the influence of the type of culture medium on differential growth of certain M. avium subsp. paratuberculosis strains has never been addressed. The present study describes the distribution of subtypes present in isolates cultured in two commonly used types of medium. Medium reagents as well as the state of medium in the bacteriologic culture methods employed may have a significant role in differential growth of M. avium subsp. paratuberculosis strains obtained from bacterial culture.

G-GGT subtypes of a set of matched-pair isolates were analyzed and compared, but the inability of amplifying G repeats in several samples considerably reduced the initial sample size of the study. Three different methods of DNA extraction, external and internal primers, and bovine serum albumin (9) were employed to facilitate PCR amplification of some problematic samples. In this study, it was more difficult to amplify G repeats from broth samples than from solid medium samples. The presence of PCR inhibitors, such as egg yolk, and the difficulty of amplifying these mononucleotide repeats may have caused the reduced sensitivity of many PCRs. A further evaluation of the procedures and reagents applied to amplify SSRs from broth culture should be performed.

This study has identified the existence of one major fingerprint (>13G-5GGT) predominating in bovine fecal samples, regardless of the type of medium used. In addition, multiple subtypes were identified at different frequencies among broth and solid culture isolates.

Cluster analysis provided a quick guide to identifying differences among subtypes. The rooted tree displaying all subtypes showed that they were distinct from each other and were logically ordered (in order of lineage). The farther, in terms of increasing numbers of G and GGT residue repeats, the subtypes were from the ancestor (7G-4GGT), the more recent they were evolutionarily. Moreover, broth isolates had more diverse subtypes than did solid culture isolates. Broth isolate subtypes contributed mainly to the richness of the subtype population, given the larger number of identified fingerprints and the mixed subtypes observed in some samples. Even though solid culture isolates showed a lower Simpson's index of diversity than did broth isolates, they contributed more to the evenness or equitability of subtypes due to the larger numbers of isolates showing the same subtypes.

The majority of matched-pair samples showed different subtypes in both culture media, and 31% of samples shared the same G-GGT fingerprint. Actual differences due to sequencing artifacts could be elucidated in a further study by using a third locus to break down the subtypes identified by the first two loci.

Fecal samples may carry more than one subtype of the organism, and depending on the culture method, we might or might not select for recovery of specific subtypes. Disease transmission dynamics, which are dependent on animal movement, animal raising, and some other management factors, may also be involved in the presence of different subtypes in a sample. We were not able to draw conclusions from our findings given the small number of isolates presenting more than one genotype, but if mixed infections are identified more frequently from broth than from solid culture isolates, then liquid culture systems could be employed for further molecular epidemiologic and population genetic analyses, given their higher resolution in identifying a wider spectrum of subtypes. Further investigation is needed to confirm these findings.

The application of SSR analysis allowed differentiation of M. avium subsp. paratuberculosis strains between liquid and solid culture isolates. The different subtypes observed for liquid and solid isolates obtained from the same samples suggest that different media could lead to differential growth of M. avium subsp. paratuberculosis strains, which was corroborated by the different recovery rates found when isolating M. avium subsp. paratuberculosis using Herrold's egg yolk medium slants and a liquid culture system (MGIT).

In conclusion, this study has shown that two types of culture medium determined differential growth of M. avium subsp. paratuberculosis strains. This should be considered in evaluating detection capabilities of diagnostic tests or interpreting data from molecular epidemiological studies performed using conventional solid fecal culture or automated liquid culture methods.


   ACKNOWLEDGMENTS
 
This work was supported by the Minnesota Board of Animal Health and the Johne's Disease Integrated Project, funded by USDA-CSREES NRI.

We thank Xiaochun Zhu and Meetu Seth for their technical support.


   FOOTNOTES
 
* Corresponding author. Mailing address: Veterinary Population Medicine Department, University of Minnesota, 136G Andrew Boss Laboratory—Meat Hygiene, 1354 Eckles Avenue, St. Paul, MN 55108. Phone: (612) 625-3769. Fax: (612) 624-4906. E-mail: sreev001{at}umn.edu Back

{triangledown} Published ahead of print on 24 October 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Journal of Clinical Microbiology, January 2008, p. 145-149, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01769-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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