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Journal of Clinical Microbiology, March 1999, p. 565-569, Vol. 37, No. 3
Departments of Biochemistry and Molecular
Biology1 and
Medicine,2 New York Medical College,
Valhalla, New York 10595
Received 12 August 1998/Returned for modification 13 October
1998/Accepted 17 November 1998
Two hundred seventeen isolates of Borrelia burgdorferi
originally cultured from skin biopsy samples or blood of early Lyme disease patients were genetically characterized by PCR-restriction fragment length polymorphism (RFLP) typing of the 16S-23S ribosomal DNA
intergenic spacer. Three major RFLP types were observed. Of the
cultured isolates, 63 of 217 (29.0%) were type 1, 85 of 217 (39.2%)
were type 2, and 58 of 217 (26.7%) were type 3; mixtures of two RFLP
types were obtained in 6.0% (13 of 217) of the cultures. Comparison of
typing of B. burgdorferi performed directly on 51 patient
skin specimens with typing of cultures originally isolated from the
same tissue revealed that a much larger proportion of direct tissue
samples had mixtures of RFLP types (43.1% by direct typing versus
5.9% by culture [P < 0.001). In addition, identical RFLP types were observed in only 35.5% (11 of 31) of the paired samples. RFLP type 3 organisms were recovered from blood at a significantly lower rate than were either type 1 or type 2 strains. These studies demonstrate that the genetic diversity of B. burgdorferi patient isolates as determined by cultivation differs
from that assessed by PCR performed directly on patient tissue.
Lyme disease, the most prevalent
vector-borne disease in the United States, is caused by infection with
the spirochete Borrelia burgdorferi (4, 5, 26,
30). Inoculation of the spirochete into humans occurs during
feeding of certain Ixodes ticks (4, 26, 27).
Early Lyme disease is manifested by a characteristic skin rash,
erythema migrans, and is frequently accompanied by other
systemic symptoms (e.g., fatigue, arthralgia, myalgia, headache, fever, and stiff neck) (20).
B. burgdorferi was originally characterized as a single
species. However, in recent years it has become clear that the broad grouping of spirochetes referred to as B. burgdorferi sensu
lato is composed of a number of distinct species and genomic groups (3, 13, 14, 18, 31). Of these, only B. burgdorferi sensu stricto, Borrelia garinii,
Borrelia afzelii, and group 25015 organisms have been
isolated from Lyme disease patients (1, 5, 28, 29). Thus,
the pathogenic potential of the other B. burgdorferi sensu
lato species remains uncertain. Furthermore, several studies have
suggested a possible correlation between a specific species and
particular disease manifestations (1, 2, 6, 29, 32).
B. burgdorferi sensu lato is distributed throughout the
northern hemisphere, but in North America virtually all
characterized isolates are B. burgdorferi sensu
stricto (3, 15, 19). Recently, several studies have
demonstrated that there is significant genetic heterogeneity among
North American B. burgdorferi sensu stricto isolates
(8, 15, 19). Many of those studies involved tick-derived
isolates, and virtually all analyses were carried out with cultured
organisms rather than those analyzed directly in clinical specimens. In
an earlier report, we determined the genetic diversity among clinical
isolates of B. burgdorferi by a PCR-restriction fragment
length polymorphism (RFLP) typing method targeted at the 16S-23S
ribosomal DNA (rDNA) spacer region and found predominance of one
specific RFLP type among 93 cultured clinical isolates investigated
(17).
In the present study, this analysis has been extended to a total of 217 cultured clinical isolates. In addition, typing of B. burgdorferi directly in skin biopsy tissue was performed, and for
some of these specimens, paired samples of tissue and culture were analyzed.
Skin biopsy and culture.
Skin biopsy samples (2 mm) were
obtained from the advancing border of primary erythema migrans lesions
from patients enrolled in a prospective study at the Lyme Disease
Diagnostic Center of the Westchester Medical Center, as previously
described (25). Biopsy specimens were placed in a transport
medium for later processing in the laboratory. Tissues were transferred
to 0.5 ml of BSK-II medium lacking rabbit serum and gelatin and ground
in a Spectrum Brand microtissue grinder. A 0.1-ml portion of this
suspension was introduced into 6 ml of complete BSK-II medium
supplemented with 6% rabbit serum and 1.2% gelatin and incubated at
34°C for 2 to 8 weeks. The remainder of the suspension (0.4 ml) was
processed for PCR. Spirochetes present in whole blood, plasma, or serum were cultured essentially as described elsewhere with minor
modifications (21, 33).
DNA isolation.
DNA from tissue biopsy specimens, 0.3 ml of
EDTA-treated whole blood, or 0.2 ml of B. burgdorferi
cultures (either primary or at passage 1 or 2) was prepared with a
commercial nucleic acid extraction kit (IsoQuick; Orca Research,
Bothell, Wash.). Prior to DNA extraction, the macerated skin biopsy
tissue was separated from the BSK-II medium and was solubilized by
suspension in 0.1 ml of lysis buffer (10 mM Tris-HCl [pH 7.4], 0.5%
Nonidet P-40, 0.5% Tween 20, 0.1 mg of proteinase K per ml),
incubation overnight at 55°C, and boiling for 15 min. DNA was
extracted from both the medium and the digested skin sample by means of
the Isoquick extraction kit as described elsewhere (24).
Purified DNA was resuspended into a total volume of 50 µl of water,
and 10 µl was employed for PCR.
PCR amplification.
A 941-bp region of the B. burgdorferi 16S-23S rDNA spacer region was amplified by PCR by a
modification of a previously reported protocol (17). The
most crucial refinement was the use of a nested-PCR procedure which
resulted in an increased yield of product by direct PCR from clinical
material, obviating the necessity of culture. First-round amplification
employed PA (5'-GGTATGTTTAGTGAGGG-3'; positions
1465 to 1481 in the mature 16S rRNA sequence) as the forward primer and
P95 (5'-GGTTAGAGCGCAGGTCTG-3'; positions 941 to
924 of the spacer) as the reverse primer. PCR amplification results in
a 1,014-bp product. Ten microliters of a 1/1,000 dilution of the
first-round PCR product was employed as template in a second PCR with
PB (5'-CGTACTGGAAAGTGCGGCTG-3'; positions 1505 to 1524 in the mature 16S rRNA sequence) as the forward primer and
P97 (5'-GATGTTCAACTCATCCTGGTCCC-3'; positions
908 to 886 of the spacer) as the reverse primer. PCR amplification was
performed in 50 µl containing 100 mM (each) deoxynucleoside
triphosphates, 1.5 U of Taq DNA polymerase (Boehringer
Mannheim), and 30 pmol of each primer in a Perkin-Elmer model 9600 thermocycler. The amplification profile for both first- and
second-round PCR consisted of 35 cycles of denaturation at 94°C for
30 s, annealing at 52°C for 30 s, and extension at 72°C
for 30 s.
RFLP analysis.
Ten-microliter aliquots of the nested-PCR
amplification products were subjected to RFLP analysis by digestion
with 2 U of either HinfI or MseI, and digested
fragments were resolved by agarose gel electrophoresis in
Tris-borate-EDTA buffer as previously described (17).
Statistical analysis.
Statistical analyses were performed
with True Epistat software (version 5.1; Richardson, Tex.). All
analyses were two tailed. Categorical variables were analyzed with the
log-likelihood ratio test. P values less than 0.05 were
considered statistically significant.
Typing of B. burgdorferi isolates obtained by
culture.
In earlier reports, we described molecular typing of 93 clinical isolates cultured from Lyme disease patients by PCR-RFLP analysis of a segment of the 16S-23S rDNA spacer (17). We
have now extended this analysis to an additional 124 clinical isolates (a total of 217). Table 1 contains the
distribution of RFLP types from these B. burgdorferi
cultures obtained from either skin or blood of patients with early Lyme
disease evaluated in Westchester County, N.Y., during the 7-year period
1991 to 1997. The data confirmed the presence of the three major RFLP
types previously described (17). Of 183 skin isolates, 46 (25.1%) were type 1, 70 (38.3%) were type 2, and 55 (30.1%) were
type 3; the remaining 6.6% (12 of 183) were mixed cultures composed of
at least two genotypically distinct isolates. Type 2 isolates were more
frequently cultured from skin biopsy specimens than were either of the
other types (P = 0.07 for comparison of type 1 and type
2; P = 0.013 for comparison of type 2 and type 3). Of
the 34 blood isolates analyzed, 91% were RFLP types 1 and 2. The
number of RFLP type 3 cultures was significantly underrepresented in
this group of specimens (P = 0.0003 for comparison of
type 3 with either type 1 or type 2). A log-likelihood ratio analysis
showed a significant difference in the distribution of RFLP types
between skin and blood (P = 0.024). The number of RFLP
type 3 cultures was significantly lower in blood than in skin
(P = 0.004). In contrast, blood specimens yielded
significantly more RFLP type 1 isolates in culture than did skin
specimens (P = 0.033).
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Genetic Diversity of Borrelia
burgdorferi in Lyme Disease Patients as Determined by Culture
versus Direct PCR with Clinical Specimens
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
TABLE 1.
PCR-RFLP typing of B. burgdorferi clinical
isolates cultured from skin or blood of Lyme disease patients
|
Typing of B. burgdorferi directly in tissue.
In
order to extend this technique for typing of B. burgdorferi
directly in patient tissue, a nested-PCR procedure which was sensitive
enough to facilitate typing of B. burgdorferi without the
requirement for prior culture was developed. Data for 58 specimens (51 skin and 7 blood samples) analyzed in this manner are presented in Table 2. In contrast to the findings
in culture, the predominant RFLP type detected in tissue was type 1 (P = 0.0074 and P < 0.0001 for
comparison with type 2 or type 3, respectively).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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The results of the present study indicate that a genotypically heterogeneous group of B. burgdorferi infects patients in Westchester County, N.Y. In addition, the distribution of each of these genotypes in patient tissue or the cultures derived from them is nonrandom. RFLP type 2 was the predominant isolate in cultures from skin biopsy specimens, whereas RFLP type 1 was most frequently detected by PCR directly in skin tissue. Mixtures of RFLP types were demonstrated significantly more frequently by direct PCR analysis of tissue than by original cultures (43.1 versus 5.9% [P < 0.0001). The observed differences in distribution of RFLP types between tissue and culture samples suggest that a bias is introduced by in vitro propagation. A similar conclusion was reached based on an analysis of B. burgdorferi species present in Ixodes spinipalpis ticks collected in Colorado. The Colorado study demonstrated that the frequency of p66 and ospA alleles determined by PCR and single-strand conformation polymorphism analysis was significantly different between cultured and uncultured spirochetes (22). In our study, it was possible to address culture bias by comparison of culture and direct typing for a subset of 51 skin samples since both tests were applied to the identical specimen (see above). This analysis revealed no significant difference in culture recovery for any RFLP type; study of a larger number of paired assays on the same tissue sample is warranted.
The direct comparison analysis of the 51 skin specimens did establish, however, that mixtures of RFLP types are observed much less frequently in culture than by direct PCR testing. Forty-three percent (22 of 51) of the skin specimens directly analyzed by PCR-RFLP contained mixtures of two different RFLP types, whereas only 5.9% (3 of 51) of the cultures of these same skin specimens contained a mixture of RFLP types (P < 0.0001). The most common finding (45%) was a mixture of RFLP types detected by direct analysis and one of these growing out in culture (Table 3). All cultures were analyzed either as the original inoculate or at passage 1 or 2. This implies that in vitro culture conditions efficiently eliminate the propagation of certain individuals in the mixed specimen and result in outgrowth of a specific subtype. This could be due to differential acquisition or assimilation by certain B. burgdorferi RFLP types of required nutrients in BSK-II medium, thereby altering the growth success of those cells. This may be addressed by analyzing genomic differences or alterations in gene expression among the different RFLP types.
The distribution of RFLP types obtained by direct analysis of patient tissue probably reflects the spirochete population distribution originally deposited in skin by the feeding tick. A number of studies have indicated that Ixodes ticks are infected by multiple B. burgdorferi genotypes (11, 23). This is also true of the local Ixodes scapularis population in Westchester County, N.Y. PCR-RFLP analysis showed that 52% of 27 ticks contained a mixture of RFLP types (16).
For certain specimens (19%), a particular RFLP type was observed only in culture and not by direct analysis of the skin tissue (Table 3). The appearance of RFLP types in culture which are not detected by PCR analysis suggests that the isolate which ultimately grows out in culture was present in tissue at levels below the detection capabilities of the PCR-RFLP typing method. Under the experimental conditions employed in this study, RFLP type isolates representing only 5% of the total spirochetes in a mixture could be readily detected (data not shown). This suggests that even minor components of a spirochete population mixture can become the predominant species after only one or two passages in culture.
The possibility of generating one RFLP type from another is not very likely. The typing scheme employed in our analysis assesses the presence (or absence) of a single HinfI site and two MseI sites (17). Sequencing of the 941-bp spacer for three representative isolates of each RFLP type revealed a maximum of 19 nucleotide differences between members of the same type but a minimum of 36 nucleotide differences between the most closely related isolates of different RFLP types. This confirms that the RFLP analysis is indicative of significant genotypic (i.e., sequence) variation in the rDNA spacer. The simultaneous mutation of three or more nucleotides which would be required to generate one RFLP type from another in the relatively short period of adaptation to culture conditions seems highly implausible.
What might the differences in recovery of certain RFLP types by culture or their distribution in patient specimens reveal regarding their potential for infectivity, invasion, or pathogenesis? A number of studies have suggested that specific B. burgdorferi sensu lato species may be responsible for different manifestations of Lyme disease, B. garinii being more often associated with neuroborreliosis and B. afzelii being more often associated with chronic skin manifestations (1, 6, 29). It is interesting to note the underrepresentation of RFLP type 3 in blood both by culture and by direct analysis in the present study. RFLP type 3 isolates were found in 31.7% of skin biopsy cultures (Table 1) and 29.4% of skin biopsy specimens tested directly by PCR (Table 2). In contrast, only 8.8% of the blood cultures contained this RFLP type (P = 0.004), and it was not detected in any of the blood samples by direct analysis. Thus, genetic heterogeneity may be responsible for the wide degree of variation in symptomatology and severity of Lyme disease encountered in the United States (20).
At present, the properties of the distinct RFLP types which may be responsible for different biological activities are not known. It should be noted that the typing method employed here is based on a noncoding spacer region within the rRNA gene cluster of B. burgdorferi (10) which is unlikely to have any role in invasion or pathogenesis. We have recently characterized a subset of 36 of the isolates analyzed in the current study by whole-genome RFLP and plasmid content (by pulsed-field gel electrophoresis of MluI-digested DNA or undigested total DNA) and found that there is a greater than 90% correspondence between rDNA spacer RFLP and the other two typing methods (12). These results suggest that typing by rDNA spacer RFLP analysis is an accurate reflection of genomic heterogeneity among different B. burgdorferi sensu stricto isolates.
Recent publication of the B. burgdorferi genome sequence yielded little information with respect to possible factors which may be responsible for virulence and/or pathogenicity (9). With current technology, investigations of possible B. burgdorferi virulence determinants must be carried out with cultured organisms. The present study indicates that the diversity of genotypes infecting human tissue is underestimated by culture and, to a lesser extent, by direct PCR analysis. This implies that isolates may be selected for by cultivation conditions rather than by their pathogenic potential. This possibility should be considered in any attempt to correlate properties of cultured B. burgdorferi isolates with their observed effects in patients, ticks, and wildlife reservoirs.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grants AR41508 and AR41511 from the National Institutes of Health and Cooperative Agreements U50/CCU210280 and U50/CCU210286 from the Centers for Disease Control and Prevention.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595. Phone: (914) 594-4658. Fax: (914) 594-4058. E-mail: schwartz{at}nymc.edu.
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References
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Journal of Clinical Microbiology, March 1999, p. 565-569, Vol. 37, No. 3
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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78: 806-810
[Abstract] [Full Text] -
Ogden, N. H., Lindsay, L. R., Hanincova, K., Barker, I. K., Bigras-Poulin, M., Charron, D. F., Heagy, A., Francis, C. M., O'Callaghan, C. J., Schwartz, I., Thompson, R. A.
(2008). Role of Migratory Birds in Introduction and Range Expansion of Ixodes scapularis Ticks and of Borrelia burgdorferi and Anaplasma phagocytophilum in Canada. Appl. Environ. Microbiol.
74: 1780-1790
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Hanincova, K., Ogden, N. H., Diuk-Wasser, M., Pappas, C. J., Iyer, R., Fish, D., Schwartz, I., Kurtenbach, K.
(2008). Fitness Variation of Borrelia burgdorferi Sensu Stricto Strains in Mice. Appl. Environ. Microbiol.
74: 153-157
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Embers, M. E., Wormser, G. P., Schwartz, I., Martin, D. S., Philipp, M. T.
(2007). Borrelia burgdorferi Spirochetes That Harbor Only a Portion of the lp28-1 Plasmid Elicit Antibody Responses Detectable with the C6 Test for Lyme Disease. CVI
14: 90-93
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Jones, K. L., Glickstein, L. J., Damle, N., Sikand, V. K., McHugh, G., Steere, A. C.
(2006). Borrelia burgdorferi Genetic Markers and Disseminated Disease in Patients with Early Lyme Disease. J. Clin. Microbiol.
44: 4407-4413
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Bockenstedt, L. K., Liu, N., Schwartz, I., Fish, D.
(2006). MyD88 Deficiency Enhances Acquisition and Transmission of Borrelia burgdorferi by Ixodes scapularis Ticks. Infect. Immun.
74: 2154-2160
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Ojaimi, C., Mulay, V., Liveris, D., Iyer, R., Schwartz, I.
(2005). Comparative Transcriptional Profiling of Borrelia burgdorferi Clinical Isolates Differing in Capacities for Hematogenous Dissemination. Infect. Immun.
73: 6791-6802
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Aguero-Rosenfeld, M. E., Wang, G., Schwartz, I., Wormser, G. P.
(2005). Diagnosis of Lyme Borreliosis. Clin. Microbiol. Rev.
18: 484-509
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Wormser, G. P., McKenna, D., Carlin, J., Nadelman, R. B., Cavaliere, L. F., Holmgren, D., Byrne, D. W., Nowakowski, J.
(2005). Brief Communication: Hematogenous Dissemination in Early Lyme Disease. ANN INTERN MED
142: 751-755
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Clark, K., Hendricks, A., Burge, D.
(2005). Molecular Identification and Analysis of Borrelia burgdorferi Sensu Lato in Lizards in the Southeastern United States. Appl. Environ. Microbiol.
71: 2616-2625
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Derdakova, M., Dudioak, V., Brei, B., Brownstein, J. S., Schwartz, I., Fish, D.
(2004). Interaction and Transmission of Two Borrelia burgdorferi Sensu Stricto Strains in a Tick-Rodent Maintenance System. Appl. Environ. Microbiol.
70: 6783-6788
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Wang, G., Iyer, R., Bittker, S., Cooper, D., Small, J., Wormser, G. P., Schwartz, I.
(2004). Variations in Barbour-Stoenner-Kelly Culture Medium Modulate Infectivity and Pathogenicity of Borrelia burgdorferi Clinical Isolates. Infect. Immun.
72: 6702-6706
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Dolan, M. C., Piesman, J., Schneider, B. S., Schriefer, M., Brandt, K., Zeidner, N. S.
(2004). Comparison of Disseminated and Nondisseminated Strains of Borrelia burgdorferi Sensu Stricto in Mice Naturally Infected by Tick Bite. Infect. Immun.
72: 5262-5266
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Courtney, J. W., Kostelnik, L. M., Zeidner, N. S., Massung, R. F.
(2004). Multiplex Real-Time PCR for Detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J. Clin. Microbiol.
42: 3164-3168
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Bunikis, J., Garpmo, U., Tsao, J., Berglund, J., Fish, D., Barbour, A. G.
(2004). Sequence typing reveals extensive strain diversity of the Lyme borreliosis agents Borrelia burgdorferi in North America and Borrelia afzelii in Europe. Microbiology
150: 1741-1755
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Ranka, R., Bormane, A., Salmina, K., Baumanis, V.
(2004). Identification of Three Clinically Relevant Borrelia burgdorferi Sensu Lato Genospecies by PCR-Restriction Fragment Length Polymorphism Analysis of 16S-23S Ribosomal DNA Spacer Amplicons. J. Clin. Microbiol.
42: 1444-1449
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Wang, G., Liveris, D., Brei, B., Wu, H., Falco, R. C., Fish, D., Schwartz, I.
(2003). Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States. Appl. Environ. Microbiol.
69: 4561-4565
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Iyer, R., Kalu, O., Purser, J., Norris, S., Stevenson, B., Schwartz, I.
(2003). Linear and Circular Plasmid Content in Borrelia burgdorferi Clinical Isolates. Infect. Immun.
71: 3699-3706
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Terekhova, D., Sartakova, M. L., Wormser, G. P., Schwartz, I., Cabello, F. C.
(2002). Erythromycin Resistance in Borrelia burgdorferi. Antimicrob. Agents Chemother.
46: 3637-3640
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Liveris, D., Wang, G., Girao, G., Byrne, D. W., Nowakowski, J., McKenna, D., Nadelman, R., Wormser, G. P., Schwartz, I.
(2002). Quantitative Detection of Borrelia burgdorferi in 2-Millimeter Skin Samples of Erythema Migrans Lesions: Correlation of Results with Clinical and Laboratory Findings. J. Clin. Microbiol.
40: 1249-1253
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Iyer, R., Liveris, D., Adams, A., Nowakowski, J., McKenna, D., Bittker, S., Cooper, D., Wormser, G. P., Schwartz, I.
(2001). Characterization of Borrelia burgdorferi Isolated from Erythema Migrans Lesions: Interrelationship of Three Molecular Typing Methods. J. Clin. Microbiol.
39: 2954-2957
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Wang, G., Ojaimi, C., Iyer, R., Saksenberg, V., McClain, S. A., Wormser, G. P., Schwartz, I.
(2001). Impact of Genotypic Variation of Borrelia burgdorferi Sensu Stricto on Kinetics of Dissemination and Severity of Disease in C3H/HeJ Mice. Infect. Immun.
69: 4303-4312
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Ornstein, K., Berglund, J., Nilsson, I., Norrby, R., Bergström, S.
(2001). Characterization of Lyme Borreliosis Isolates from Patients with Erythema Migrans and Neuroborreliosis in Southern Sweden. J. Clin. Microbiol.
39: 1294-1298
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De Michelis, S., Sewell, H.-S., Collares-Pereira, M., Santos-Reis, M., Schouls, L. M., Benes, V., Holmes, E. C., Kurtenbach, K.
(2000). Genetic Diversity of Borrelia burgdorferi Sensu Lato in Ticks from Mainland Portugal. J. Clin. Microbiol.
38: 2128-2133
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Gorbacheva, V. Y., Godfrey, H. P., Cabello, F. C.
(2000). Analysis of the bmp Gene Family in Borrelia burgdorferi Sensu Lato. J. Bacteriol.
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Ling, C L, Joss, A W L, Davidson, M M, Ho-Yen, D O
(2000). Identification of different Borrelia burgdorferi genomic groups from Scottish ticks. Mol. Pathol.
53: 94-98
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Caimano, M. J., Yang, X., Popova, T. G., Clawson, M. L., Akins, D. R., Norgard, M. V., Radolf, J. D.
(2000). Molecular and Evolutionary Characterization of the cp32/18 Family of Supercoiled Plasmids in Borrelia burgdorferi 297. Infect. Immun.
68: 1574-1586
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Iyer, R., Hardham, J. M., Wormser, G. P., Schwartz, I., Norris, S. J.
(2000). Conservation and Heterogeneity of vlsE among Human and Tick Isolates of Borrelia burgdorferi. Infect. Immun.
68: 1714-1718
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Wang, G., van Dam, A. P., Schwartz, I., Dankert, J.
(1999). Molecular Typing of Borrelia burgdorferi Sensu Lato: Taxonomic, Epidemiological, and Clinical Implications. Clin. Microbiol. Rev.
12: 633-653
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