Emerg Infect DisEmerging Infect. DisEIDEmerging Infectious Diseases1080-60401080-6059Centers for Disease Control and Prevention16102309332051204-135410.3201/eid1108.041354ResearchResearchCoxiella burnetii GenotypingCoxiella burnetii genotypingGlazunovaOlga*1RouxVéronique*1FreylikmanOlga*†SekeyovaZuzana*‡FournousGhislain*TyczkaJudith§TokarevichNikolai†KovacovaElena‡MarrieThomas J.¶RaoultDidier*Unité des Rickettsies, Marseille, France; Pasteur Institute of Epidemiology and Microbiology, Saint Petersburg, Russia;Institute of Virology SAS, Bratislava, Slovak Republic;University of Giessen, Giessen, Germany;University of Alberta Department of Medicine, Edmonton, Alberta, CanadaAddress for correspondence: Didier Raoult, Unité des Rickettsies, Faculté de Médecine, CNRS UMR 6020, IFR48, 27 Boulevard Jean Moulin, 13385 Marseille, Cedex 05, France; fax: 33-491-32-03 90. email: didier.raoult@medecine.univ-mrs.fr8200511812111217
Multispacer sequence typing is the first reliable method for typing Coxiella burnetii isolates.
Coxiella burnetii is a strict intracellular bacterium with potential as a bioterrorism agent. To characterize different isolates of C. burnetii at the molecular level, we performed multispacer sequence typing (MST). MST is based on intergenic region sequencing. These regions are potentially variable since they are subject to lower selection pressure than the adjacent genes. We screened 68 spacers in 14 isolates and selected the 10 that exhibited the most variation. These spacers were then tested in 159 additional isolates obtained from different geographic areas or different hosts or were implicated in different manifestations of human disease caused by C. burnetii. The sequence analysis yielded 30 different allelic combinations. Phylogenic analysis showed 3 major clusters. MST allows easy comparison and exchange of results obtained in different laboratories and could be a useful tool for identifying bacterial strains.
Coxiella burnetii is a strict intracellular microorganism, included in the γ subdivision of the Proteobacteria phylum (1). It is found in close association with arthropod and vertebrate hosts, and it causes Q fever in humans and animals. Cattle, goats, and sheep are the primary reservoirs of human infection. In humans, the disease may appear in 2 forms, acute and chronic (2). Acute Q fever may be asymptomatic or appear as atypical pneumonia, granulomatous hepatitis, or self-limited febrile illness. In some persons, the immune system is unable to control the infection and chronic Q fever occurs. The manifestations of chronic Q fever are endocarditis, hepatitis, osteomyelitis, or infected aortic aneurysms. C. burnetii is highly infectious by the aerosol route and can survive for long periods in the environment.
Previous studies have shown that C. burnetii isolates differed respect to their plasmid type (QpH1, QpRS, QpDG, and QpDV) (3–6), lipopolysaccharide profiles (7), and analysis of endonuclease-digested DNA separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8) or pulsed-field gel electrophoresis (PFGE) (9–11). Differentiation was also obtained by sequence determination of the isocitrate dehydrogenase gene (12), com1 gene, and mucZ gene, which was renamed djlA when the whole genome of C. burnetii was sequenced (13,14).
Several other methods have been used to type different isolates of the same species, in particular, multilocus enzyme electrophoresis (15) and multilocus sequence typing (MLST) (16). Many bacterial species have been studied by using these approaches (17–19).
Recently, the whole genome of the C. burnetii Nine Mile strain was sequenced (14). We decided to investigate parts of the genome located between 2 open reading frames (ORFs) because they are considered potentially variable since they are subject to lower selection pressure than the adjacent genes. The 16S/23S ribosomal spacer region has been widely used to genotype bacteria (20–23). We investigated the utility of multispacer sequence typing (MST) with 173 C. burnetii isolates. After screening, we selected 10 variable spacers and showed that the combination of the different sequences allowed us to characterize 30 different genotypes. Phylogenetic analysis inferred from compiled sequences characterized 3 monophyletic groups, which could be subdivided into different clusters.
MethodsBacterial Strains
The C. burnetii strains included in this study are listed in Table A1. All the strains were propagated on Vero cell monolayers (ATCC CRL 1587). Minimal essential medium (MEM) (Invitrogen, Cergy-Pontoise, France) supplemented with 4% fetal bovine serum (Invitrogen) and 1% L-glutamine (Invitrogen) was used for cultivation. Infected cells were maintained in a 5% CO2 atmosphere at 35°C. C. burnetii cells were harvested, pelleted, resuspended in 200 μL MEM, and mixed with 500 μL Chelex 100 20% (Bio-Rad, Ivry sur Seine, France). The preparation was boiled for 30 min, centrifuged at 10,000 × g for 30 min (24), and the supernatant containing DNA was transferred to a clean Eppendorf tube and stored at 4°C or –20°C.
Multispacer Sequence Typing
The whole genome of C. burnetii was accessible in the NCBI server (GenBank NC 002971). We kept spacers that were 300–700 bp in length. Primers were chosen in neighboring genes to allow polymerase chain reaction (PCR) amplification at 57°C and are listed in Table 1. Each PCR was carried out in a T3 Thermocycler Biometra (Biolabo, Archamps, France). Two microliters of the DNA preparation was amplified in a 50-μL reaction mixture containing 200 μmol/L of each primer, 200 μmol/L (each) dATP, dCTP, dGTP, and dTTP (Invitrogen), 1.5 U Taq DNA polymerase (Roche, Meylan, France) in 1× Taq buffer. Amplifications were carried under the following conditions: initial denaturation of 10 min at 95°C, followed by 37 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 57°C, and extension for 1 min at 72°C. PCR products were purified and sequenced as previously described (25).
Primers used for PCR amplification and sequencing of Coxiella burnetii gene spacers
Spacer name
ORF
Nucleotide sequence (5´–3´)*
Amplified fragment length (bp)
Cox2
Hypothetical protein
Cox20766 CAACCCTGAATACCCAAGGA
397
Hypothetical protein
Cox21004 GAAGCTTCTGATAGGCGGGA
Cox5
Sulfatase domain protein
Cox77554 CAGGAGCAAGCTTGAATGCG
395
Entericidin, putative
Cox77808 TGGTATGACAACCCGTCATG
Cox18
Ribonuclease H
Cox283060 CGCAGACGAATTAGCCAATC
557
DNA polymerase III, epsilon subunit
Cox283490 TTCGATGATCCGATGGCCTT
Cox20
Hypothetical protein
Cox365301 GATATTTATCAGCGTCAAAGCAA
631
Hypothetical protein
Cox365803 TCTATTATTGCAATGCAAGTGG
Cox22
Hypothetical protein
Cox378718 GGGAATAAGAGAGTTAGCTCA
383
Amino acid permease family protein
Cox378965 CGCAAATTTCGGCACAGACC
Cox37
Hypothetical protein
Cox657471 GGCTTGTCTGGTGTAACTGT
463
Hypothetical protein
Cox657794 ATTCCGGGACCTTCGTTAAC
Cox51
Replicative DNA helicase, intein-containing
Cox824598 TAACGCCCGAGAGCTCAGAA
674
Conserved hypothetical protein – Uridine kinase
Cox825124 GCGAGAACCGAATTGCTATC
Cox56
OmpA-like transmembrane domain protein
Cox886418 CCAAGCTCTCTGTGCCCAAT
479
Conserved hypothetical protein
Cox886784 ATGCGCCAGAAACGCATAGG
Cox57
Rhodanese-like domain protein
Cox892828 TGGAAATGGAAGGCGGATTC
617
Hypothetical protein
Cox893316 GGTTGGAAGGCGTAAGCCTTT
Cox 61
Dioxygenase, putative
Cox956825 GAAGATAGAGCGGCAAGGAT
611
Hypothetical protein
Cox957249 GGGATTTCAACTTCCGATAGA
*The numbers are beginning or end locations of the genes where the primers were chosen.
PCR products were cloned in PGEM-T Easy Vector (Promega, Charbonnières, France) according to the manufacturer's instructions. Ten clones were cultivated in LB medium (USB, Cleveland, OH, USA) overnight, and PCR and sequencing were performed as described previously.
Plasmid Sequence Type, <italic>com1</italic> Type, and <italic>djlA</italic> Type Determination
PCR for QpH1 and QpRS sequence plasmids were performed with the primers previously described QpH11/12 and QpRS01/02 (5). PCR was carried out as described for MST, except that annealing temperature was 55°C and cycle number was 35. PCR primers for QpDV and QpRS sequence plasmid amplification were chosen after comparison of the entire sequence of the 2 plasmids. The primers were QpDV1f and QpDV1r. PCR amplification was carried out at 63°C for 30 cycles. PCR was performed as previously described for com1 and djlA (13) (Table A2).
Data Analysis
Statistical analyses were performed by using the chi-square test in the program EpiInfo 6 (26). The spacer sequences were compiled and aligned by using the multisequence alignment program ClustalX (1.8). The phylogenetic relationships between the C. burnetii isolates were determined by using Mega version 2.0 (27). A matrix of pairwise differences in allele profiles was constructed, and the similarities between the allelic profiles of the isolates were assessed by cluster analysis using the unweighted pair-group method with arithmetic mean (UPGMA). Another analysis of the results was performed by using the BURST algorithm (http://www.mlst.net), which defines clonal complexes in which every isolate shares at least 5 identical alleles with at least 1 other isolate (Cox2, Cox5, Cox18, Cox20, Cox37, Cox56, and Cox57 were kept for the analysis) and characterizes ancestral genotypes. C. burnetii MST database was entered at the following website: http://ifr48.timone.univ-mrs.fr, and ST determination by sequence comparison is possible at this site.
ResultsChoice of Spacers for Typing and Analysis by MST
Initially 14 isolates were chosen to test the genetic diversity of the spacers: Nine Mile, Priscilla, Q212, Heizberg, Brasov, Dog ut Ad, CB15, CB20, CB26, CB28, CB33, CB35, CB114, and CB115. We chose 68 spacers, but we retained only 51 spacers for which PCR amplification was obtained for all the isolates. We kept 10 spacers (Cox2, Cox5, Cox18, Cox20, Cox22, Cox37, Cox51, Cox56, Cox57, and Cox61) (Table 1) because they were representative of the results found when we analyzed the entire test set of 51 spacers. For each spacer, the number of variable sites in the sequences was determined, and the percentage of variability was calculated. They were, respectively, 1.1, 1.4, 1.9, 0.7, 2.3, 1.2, 1.4, 2.5, 1.7, and 2.1. We kept Cox18, Cox22, Cox51, Cox56, Cox57, and Cox61 because the percentage of variability in these spacers was high compared with the other spacers. We kept Cox2, Cox5, Cox20, and Cox37 because they allowed the characterization of CB35, CB15, CB26 and CB28, and Nine Mile respectively. To test the reliability of the spacers we kept, chi-square value was determined by using the value of 1% as the threshold value. The Fisher value was found to be statistically significant (9 × 10–4). We then added 159 other isolates. Sequences were obtained for all the isolates with spacers Cox2, Cox18, Cox20, Cox22, Cox37, Cox51, and Cox57. Mixed sequences were obtained with the isolate Poker Cat with spacers Cox5, Cox56, and Cox61. We cloned the PCR products and showed that several sequences were present after PCR amplification, including insertions or deletions. Allele distribution of the different gene spacers are described in Table 2. Each of the different sequences in a locus defined a distinct genotype, even if it differed from the others by only a single nucleotide. Thirty different sequence types (STs) were identified by using MST.
Alleles of 10 spacers which allow the definition of the different Coxiella burnetii sequence types
COX
2
5
18
20
22
37
51
56
57
61
ST
1
5
6
3
4
6
5
8
1
5
6
2
5
6
3
5
6
5
8
1
5
6
3
5
6
3
4
6
7
8
1
5
6
4
5
6
3
2
6
5
8
1
5
6
5
4
6
3
5
6
2
8
2
5
6
6
4
3
3
5
6
5
8
2
5
6
7
4
6
3
5
6
5
8
2
5
6
8
5
4
2
5
1
5
3
3
4
4
9
1
4
2
5
1
5
2
3
4
6
10
5
4
2
5
1
5
2
3
2
6
11
6
5
1
6
5
4
5
4
3
2
12
3
5
1
6
5
4
5
4
3
2
13
3
5
1
6
5
4
5
5
3
2
14
7
5
1
6
5
6
9
4
3
2
15
7
5
1
6
5
6
9
6
3
2
16
3
7
5
3
4
1
6
7
6
5
17
3
7
5
7
4
1
10
8
6
7
18
3
7
1
6
3
4
7
9
6
3
19
3
2
7
8
5
4
11
9
6
5
20
3
2
6
1
5
4
4
10
6
5
21
2
1
4
6
2
3
1
11
1
1
22
3
7
1
6
3
8
7
9
6
8
23
3
7
1
6
3
8
7
9
6
3
24
3
5
1
6
5
4
5
12
3
9
25
3
7
1
6
3
4
7
9
7
3
26
9
4
8
5
8
5
2
3
4
6
27
3
5
1
6
5
4
5
12
3
2
28
8
4
8
5
7
5
2
3
4
6
29
3
7
1
9
3
4
7
9
6
3
30
5
6
9
5
6
5
8
13
8
6
The nucleotide sequence accession numbers are noted in Table A3. Accession numbers for Poker Cat isolate clones are, respectively, AY619726, AY619728, and AY619729, and AY619721 for Cox5, Cox56, and Cox61.
Computer Analysis of MST Data
The dendrogram in the Figure was constructed from a matrix of pairwise allelic differences between the compiled sequences of the 30 STs. We identified 3 monophyletic groups within the tree. The first group, representing 13 different STs, included isolates from France, Spain, Russia, Kyrgyzstan, Namibia, Kazakhstan, Ukraine, Uzbekistan, and the United States. It was divided in 2 subgroups. The first one included 36 isolates representing 8 different STs (ST1 to ST7 and ST30). Nineteen were represented by ST1. The second subgroup included 39 isolates which represented 5 different STs (ST8, ST9, ST10, ST26, and ST28). Twenty-eight were represented by ST8.
Dendrogram of the genetic relatedness among the 30 different sequence types defined by multispacer sequence type (MST) analysis. The dendrogram was constructed by unweighted pair-group method with arithmetic mean. Plasmid sequence type, com1 group, and djlA group corresponding to each ST are indicated on the right of the figure. The 3 monophyletic groups defined by MST analysis are indicated on the left.
The second group included isolates from Europe (France, Germany, Switzerland, Romania, Italy, Greece, Austria, Slovakia), the United States, Russia, Africa (Central Africa and Senegal), and Asia (Kazakhstan, Uzbekistan, Mongolia, and Japan). It was divided into 4 subgroups. The first one included 26 isolates, which represented 7 different STs (ST11, ST12, ST13, ST14, ST15, ST24, and ST27). The second subgroup included 34 isolates that were included in ST18, ST22, ST23, ST25, and ST29 groups. The third subgroup included 18 isolates (ST16 and ST17), and the fourth subgroup included 10 isolates (ST19 and ST20).
The third group consisted of only 1 ST, ST21, and included the 7 Canadian isolates, 2 isolates from France (CB4 and CB7), and 1 isolate from the United States (Scurry). The clusters determined by the BURST algorithm were consistent with those determined by the phylogenetic analysis. Five groups were defined. The first one included ST1 to ST7; the putative ancestral genotype in this group was ST1. ST8 (putative ancestral genotype), ST9, ST10, ST26, and ST28 were included in the second group; ST11, ST12 (putative ancestral genotype), ST13, ST14, ST15, and ST24 in the third group, ST16 and ST17 in the fourth group; and ST18 (putative ancestral genotype), ST22, ST23, ST25, and ST29 in the fifth group. ST19, ST20, ST21, and ST30 were considered as singletons.
Sequence Type Determination and Correlation with Pathology
In the monophyletic group 1, the sequence of plasmid QpRS was found for isolates included in ST4, ST5, ST6, ST7, ST8, ST9, ST10, ST26, ST28, and ST30. The QpDV plasmid sequence was amplified for isolates included in ST1, ST2, ST3, and ST4. In the monophyletic group 2, the QpH1 plasmid sequence was found in all the isolates. In the monophyletic group 3, the QpH1 plasmid sequence or none of the searched plasmid sequences was detected. Sequence comparison of djlA generated 4 different groups. Group I included all STs included in the monophyletic group 2 defined by MST analysis. Group II included ST1, ST2, ST3, and ST4. Group III included ST5, ST6, ST7, ST30, ST26, ST28, ST8, ST9, and ST10. Group IV corresponded to ST21. Com1 sequence comparison generated 6 different groups. Group I included all the STs included in the monophyletic group 2 defined by MST analysis except ST14 (group V) and ST20 (groupVI). Group II included ST1, ST2, ST3, and ST4. Group III included ST5, ST6, ST7, ST30, ST26, ST28, ST8, ST9, and ST10. Group IV corresponded to ST21. When com1 typing was used, only 1 strain was not in accordance with MST typing results. This strain, CB95, was included in ST8 but exhibited a group II com1 sequence.
QpDV plasmid presence in human isolates was correlated with the acute form of the disease (p = 2 × 10–7), and QpRS plasmid presence was correlated with the chronic form of the disease (p = 2 × 10–4). The acute form of the disease was correlated with ST1 (p = 10–3), ST4 (p = 7 × 10–4) ST16 (p = 3 × 10–3), ST18 (p = 10–2), and the chronic form of the disease was correlated with ST8 (p = 2 × 10–3).
Modifications in ORFs Surrounding Studied Spacers
As primers were chosen in ORFs surrounding the studied spacers, mutations, deletions, or insertions were noted in the protein sequences. Mutations were noted in the hypothetical protein (gi29653385) for ST11; in the hypothetical protein (gi29653385) for ST9 and ST26; in entericin (gi29653446) for ST20, in ribonuclease H (gi29653667) in ST1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 21, 26, 28, and 30; in amino acid permease family protein (gi29653908) in ST28; in hypothetical protein (gi29654047) in ST1, 2, 4, 5, 6, 7, 8, 9, 10, 26, 28, and 30. In CB118 (ST3), a stop codon appeared which shortened the length of the ORF. Mutations were noted in uridine kinase (gi29654198) in ST18, ST22, ST23, ST25, and ST29; in ompA-like transmembrane domain protein (gi29654257), in ST20; in rhodanese-like domain protein (gi29654263) in ST20 (the protein was longer by 2 amino acids); in dioxygenase (gi29654325) in ST21 and ST22; in hypothetical protein (gi29732244), in ST17.
Insertions or deletions were noted in hypothetical protein (gi29653386) in ST5, 6, and 7; in hypothetical protein (gi29653755) in ST1 and ST3 (insertion of a base G in the DNA sequence made the protein sequence longer of 22 amino acids); in the amino acid permease family protein (gi29653772) in ST8, 9, and 10 (deletion of a base A in the DNA sequence made the protein sequence longer of 24 amino acids); in ompA-like transmembrane domain protein (gi29654257) in ST11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 27, and 29.
Discussion
Q fever in humans and animals, caused by C. burnetii, is found worldwide. In humans, it causes a variety of diseases such as acute flulike illness, pneumonia, hepatitis, and chronic endocarditis. In animals, C. burnetii is found in the reproductive system, both uterus and mammary glands and may cause abortion or infertility.
Molecular methods are now almost universally used to characterize strains and to determine the relatedness between isolates causing diseases in different contexts. The most discriminative approach used for C. burnetii isolates until this study was PFGE. Twenty different restriction patterns were distinguished after NotI restriction of total C. burnetii DNA and PFGE (11). Comparison of PFGE profiles is sometimes difficult because good separation of the different fragments is required. For example, the isolate Heizberg was classified in group 1 by Thiele et al. (10) and in group 2 by Jäger et al. (11). This fact highlights the difficulty of comparing results obtained by this technique. Moreover, in some species, rapid genomic rearrangements occur because of repeats or insertion sequences, so even if isolates descended from a common ancestor that arose several decades ago, they may not readily be seen to be minor variants of the same clone. In these cases, PFGE does not contribute to tracing of isolates. The great advantage of MST over PFGE as a typing method is the lack of ambiguity and the portability of sequence data, which allow results from different laboratories to be compared without exchanging strains. This work is the first to include so many isolates in a rigorous examination of molecular epidemiology. The study of this bank of sequences will contribute to understanding the propagation mode of the bacteria as variations accumulate relatively slowly, thus making it an ideal tool for global epidemiology. For example, in ST16 we characterized isolates that were obtained from 1935 (Nine Mile) to 1991 (CB25).
Most of the French isolates were included in monophyletic group 1. Nineteen were included in ST1, and 24 were included in ST8. Thus, an isolate has a geographic distribution even if genetic modifications appear (insertions, deletions or mutations) over time, giving rise to a new ST that is related to the ancestor isolate. This fact was highlighted when the analysis of the STs was performed by using the BURST algorithm. ST1 and ST8 were described as the ancestral genotypes and for example, ST9 and ST10 corresponded to SLVs of ST8 (isolates that differ at only 1 of the 7 loci) and ST26 and ST28 corresponded to DLVs of ST8 (double locus variants). But some types were not delineated on the basis of geographic origin because they were isolated from different parts of the world. This distribution in distant countries is likely related to movements of infected patients, animals, or ticks. This is particularly true for ST16 isolates that were encountered on 4 different continents, America, Europe, Asia, and Africa. The homology of the Canadian isolates from Nova Scotia should be noted. Q fever is just as endemic in Nova Scotia as in France. This may indicate rapid and recent spreading of a single strain. The association between ST21 and Canada is significant as tested with the chi-square test with a Fisher value <10–8. Notably, patient CB115, who had Q fever endocarditis, was living in Edmonton, Alberta (≈3,000 miles from Nova Scotia) when this illness was diagnosed. He grew up in Nova Scotia, and the molecular epidemiologic findings show that he acquired his disease there. Q fever is uncommon in Alberta. Most of the STs are found in Europe. A sample bias could exist as most of the isolates tested were from this continent, but the results obtained may also indicate that C. burnetii originated from the Old World and spread later in the New World, excluding New Zealand.
Concordant results were found when MST was compared with com1 and djlA sequences comparison (Figure) However MST was more discriminant. Plasmid profile investigation of C. burnetii detected 4 different plasmids QpH1, QpRS, QpDV, and QpDG and 1 group of plasmidless isolates. QpH1 was first found in the Nine Mile tick isolate (28). QpRS was first found in the goat isolate Priscilla (29). QpDG was described from isolates obtained from feral rodents near Dugway, Utah (8). QpDV was found in French and Russian isolates (5,6). Another not-well-characterized plasmid type was described in China (30). The existence of a plasmidless C. burnetii isolate, Scurry Q217 was described (31), but a chromosomally integrated plasmid-homologous DNA fragment was found in this isolate by hybridization (32,33). Plasmid type sequence detection was also correlated with MST. Group 2 included isolates that PCR amplification found to be positive with primers specific for QpH1. Group 3 included 3 isolates, 2 from France (CB4 and CB7) and 1 from Nova Scotia (Poker Cat), in which plasmid sequence type of QpH1 was detected. No such sequence was detected in the other isolates of Nova Scotia origin included in group 3. Group 1 included isolates that were positive by PCR amplification with primers specific for QpRS (47/77). QpDV plasmid was described in isolates from France, Spain, Ukraine, and Kyrgyzstan. In fact, regions shared by QpH1, QpRS, and QpDV were termed "core plasmid sequences" and encompassed 25 kb. QpH1, QpRS, and QpDV are, respectively, 37 kb, 39 kb, and 33 kb in size. Integrated sequences in American isolate represent 18 kb. Differences in plasmid size and sequence can be explained by notable sequence rearrangements, such as deletions, insertions, or duplications, because several repeat sequences have been identified through which such rearrangements might have occurred. For CB13, we were able to characterize sequences for plasmids QpH1 and QpDV, which can be caused by several situations: this isolate may have 1) 2 different plasmids, 2) a QpH1 plasmid and sequences of QpDV integrated in the chromosome, or 3) a new plasmid that arose from combination of QpH1 and QpDV. All these hypotheses are in agreement with the presence of QpH1 plasmid in the ancestor of C. burnetii isolates. This plasmid was lost by some of them (monophyletic group 3) but genetic information of crucial importance for the organism was integrated in the chromosome. For other isolates, QpH1 plasmid evolved to QpRS plasmid, in some isolates QpRS plasmid evolved to QpDV plasmid.
This study showed a correlation between QpDV and acute infections, between QpRS and chronic infections, and an association between some genotypes and disease type. A bias in sampling exists since acute disease is 20 times more frequent than chronic disease, but in this study, most of the human isolates were from chronic disease patients, and the isolates from acute infections were mainly obtained from France. These facts reflect the difficulty in isolating the bacteria. A genomic typing method such as MST could be applied directly to samples to obtain a more precise idea of how C. burnetii is spreading in the environment and the pathogenetic implications in acute and chronic forms of Q fever.
Comparison of DNA sequences is the best approach to investigate bacterial evolution. MLST in association with BURST analysis has been used to type isolates of many species. But this method is useful only if housekeeping gene diversity exists in the studied species. For example, in the species Yersinia pestis no diversity was found in the housekeeping genes studied (34). With the MST approach, differentiation of the 3 biovars Antiqua, Medievalis, and Orientalis was possible (25), which shows that the discriminatory power of MST is higher than that of MLST and is comparable to that of tandem repeats analysis (35). Low variability was found in C. burnetii housekeeping genes such as 16S rRNA (36) and rpoB (37). MST is the first method that allows a rapid and reliable typing of C. burnetii isolates during investigations of outbreaks by sequencing the PCR product obtained from the 10 spacers described. We did not test isolates from Australia and only 8 from the United States. Two isolates from Africa (Namibia and CB119) were considered as singletons in the BURST analysis denoting lack of closely related isolates. In the future, isolates that were not available in our laboratory during this study must be tested so the missing links in our phylogenetic analysis can be determined. The constitution of a database in a website will allow isolates from all the countries in the world to be compared and increase understanding of the propagation of the isolates of C. burnetii.
Suggested citation for this article: Glazunova O, Roux V, Freylikman O, Sekeyova Z, Fournous G, Tyczka J, et al. Coxiella burnetii genotyping. Emerg Infect Dis [serial on the Internet]. 2005 Aug [date cited]. http://dx.doi.org/10.3201/eid1108.041354
Dr. Glazunova and Dr. Roux contributed equally to this work.
Acknowledgments
We are grateful to Marie-Laure Birg and Jean-Yves Patrice for their technical assistance in culture of C. burnetii isolates.
This work was supported by a grant from the French Ministry of Research (ACI Microbiologie 2003) and a grant of the Pasteur Institute (2003-11).
Isolates of Coxiella burnetii studied
Isolate
Origin
Disease, symptoms, clinical status
Geographicsource
ST (sequence type)
Plasmid sequence type
CB108
Human blood
Acute
Marseille, France, 2001
1
QpDV
CB1
Human heart valve
Chronic
Istres, France, 1989
1
QpDV
CB3
Human heart valve
Chronic
Marseille, France
1
QpDV
CB36
Human placenta
Abortion
Martigues, France, 1992
1
QpDV
CB38
Human blood
Acute
Marseille, France, 1992
1
QpDV
CB41
Human heart valve
Chronic
Marseille, France, 1993
1
QpDV
CB60
Human blood
Acute
Marseille, France, 1996
1
QpDV
CB63
Human heart valve
Chronic
Marseille, France, 1994
1
QpDV
CB75
Human blood
Chronic
Marseille, France, 1998
1
QpDV
CB82
Human blood
Acute
Marseille, France, 1999
1
QpDV
CB86
Human blood
Chronic
Marseille, France, 1999
1
QpDV
CB87
Human placenta
Abortion
Martigues, France, 1999
1
QpDV
CB89
Human placenta
Abortion
Martigues, France, 2000
1
QpDV
CB94
Human blood
Acute
Aix en Provence, France, 2000
1
QpDV
CB97
Human blood
Acute
Marseille, France, 2000
1
QpDV
CB110
Human blood
Chronic
Marseille, France, 2002
1
QpDV
CB28
Human blood
Acute
Salon de Provence, France, 1992
1
QpDV
CB26
Human blood
Acute
Marseille, France, 1992
1
QpDV
CB64
Human blood
Acute
Martigues, France, 1996
1
QpDV
CB39
Human blood
Acute
Marseille, France, 1992
2
QpDV
RT-1140
Human blood
Pneumonia
Krimea, Ukrain 1954
2
QpDV
RT-Schperling
Human blood
Fever
Kyrgyzstan, 1955
2
QpDV
CB118
Human heart valve
Chronic
Marseille, France, 2004
3
QpDV
CB62
Human blood
Acute
Martigues, France, 1996
4
QpDV
CB20
Human blood
Acute
Salon de Provence, France, 1991
4
QpRS
CB51
Human placenta
Abortion
Madrid, Spain, 1996
4
QpDV
CB12
Human blood
Acute
Aix en Provence, France
4
QpDV
CB57
Human blood
Acute
Martigues, France, 1996
4
QpDV
CB54
Human blood
Acute
Aix en Provence, France, 1996
4
QpDV
CB111
Human heart valve
Chronic
Marseille, France, 2003
5
QpRS
CB35
Human heart valve
Chronic
Paris, France, 1992
5
QpRS
CB45
Human heart valve
Chronic
Paris, France, 1993
6
QpRS
CB43
Human heart valve
Chronic
Paris, France, 1993
7
QpRS
Leningrad-2
Human blood
-
Leningrad, Russia, 1955
7
QpRS
Leningrad-4
Human blood
-
Leningrad, Russia, 1957
7
QpRS
CB10
Human heart valve
Chronic aneurysm
Grenoble, France
8
QpRS
CB9
Human blood
Chronic
Lyon, France
8
QpRS
CB31
Human heart valve
Chronic
Marseille, France, 1992
8
QpRS
CB34
Human blood
Chronic
Marseille, France, 1992
8
QpRS
CB44
Human heart valve
Chronic
Créteil, France, 1993
8
QpRS
CB53
Aneurysm
Chronic
Marseille, France, 1995
8
QpRS
CB61
Valvular prosthesis
Chronic
Marseille, France, 1996
8
QpRS
CB70
Human heart valve
Chronic
Grenoble, France, 1997
8
QpRS
CB71
Valvular prosthesis
Chronic
Saint-Laurent du Var, France, 1997
8
QpRS
CB73
Valvular prosthesis
Chronic
Marseille, France, 1998
8
QpRS
CB81
Human heart valve
Chronic
Madrid, Spain, 1999
8
QpRS
CB91
Valvular prosthesis
Chronic
Marseille, France, 2000
8
QpRS
CB93
Human placenta
Abortion
Dreux, France, 2000
8
QpRS
CB95
Human blood
Chronic
Marseille, France, 2000
8
QpRS
CB116
Aneurysm
Chronic
Marseille, France, 2003
8
QpRS
CB107
Human heart valve
Chronic
Tours, France, 2001
8
QpRS
CB96
Human heart valve
Chronic
Marseille, France, 2000
8
QpRS
CB114
Human heart valve
Chronic
Marseille, France, 2003
8
QpRS
CB15
Human heart valve
Chronic
Lyon, France, 1991
8
QpRS
CB47
Human heart valve
Chronic
Barcelone, Spain, 1994
8
QpRS
CB99
Human heart valve
Chronic
Marseille, France, 2000
8
QpRS
CB106
Human heart valve
Chronic
Toulouse, France, 2001
8
QpRS
CB79
Human heart valve
Chronic
Paris, France, 1999
8
QpRS
CB98
Human heart valve
Chronic
Marseille, France, 2000
8
QpRS
CB83
Goat placenta
Abortion
Newfoundland, USA, 1999
8
QpRS
CB8
Human heart valve
Chronic
Marseille, France, 1990
8
QpRS
Priscilla
Aborted goat
Abortion
Montana, USA, 1980
8
QpRS
CB117
Human heart valve
Chronic
Marseille, France, 2004
8
QpRS
CB32
Human heart valve
Chronic
Lyon, France, 1992
9
QpRS
CB92
Human heart valve
Chronic
Marseille, France, 2000
9
QpRS
CB68
Pigeon excrement
–
Marseille, France, 1996
9
QpRS
CB49
Human heart valve
Chronic
Marseille, France, 1994
9
QpRS
CB65
Human heart valve
Chronic
Marseille, France, 1996
10
QpRS
CB103
Ewe placenta
Abortion
Marseille, France, 2001
10
QpRS
CB13
Human blood
Chronic
Paris, France
11
QpH1/QpDV
CB40
Human heart valve
Chronic
Paris, France, 1993
11
QpH1
CB46
Valvular prosthesis
Chronic
Paris, France, 1993
11
QpH1
CB5
Human blood
Chronic
Paris, France, 1990
12
QpH1
CB6
Human blood
Chronic
Paris, France
12
QpH1
CB42
Valvular prosthesis
Chronic
Toulouse, France, 1993
12
QpH1
CB52
Valvular prosthesis
Chronic
Paris, France, 1995
12
QpH1
CB56
Human heart valve
Chronic
Paris, France, 1996
12
QpH1
CB58
Spleen abscess
–
Lyon, France, 1996
12
QpH1
CB76
Human heart valve
Chronic
Paris, France, 1998
12
QpH1
CB105
Human heart valve
Chronic
Montpellier, France, 2001
12
QpH1
CB112
Human heart valve
Chronic
Zurich, Switzerland, 2003
12
QpH1
CB109
Human heart valve
Chronic
Berlin, Germany, 2002
12
QpH1
CB113
Goat placenta
Abortion
Albi, France, 2003
12
QpH1
CB33
Human heart valve
Chronic
Clermont-Ferrand, France, 1992
12
QpH1
CB55
Vegetation
Chronic
Paris, France, 1996
12
QpH1
CB2
Human blood
Immunodepression
Toulouse, France,
13
QpH1
CB69
Vegetation
Chronic
Toulouse, France, 1996
13
QpH1
CB85
Human blood
Chronic
Tours, France, 1999
14
QpH1
CB74
Valvular prosthesis
Chronic
Toulouse, France, 1998
14
QpH1
CB59
Aneurysm
Chronic aneurysm
Saint-Etienne, France, 1996
14
QpH1
CB80
Node
Chronic
Niort, France, 1999
14
QpH1
Z3055
Ewe placenta
Abortion
Germany
14
QpH1
CB102
Valvular prosthesis
Chronic
Poitiers, France, 2001
15
QpH1
CB11
Human blood
Acute
Marseille, France
16
QpH1
CB23
Human blood
Chronic
Clermont-Ferrand, France, 1988
16
QpH1
Brasov
Human
Acute
Romania
16
QpH1
Bangui
Human blood
Acute
Central Africa
16
QpH1
California
Cow milk
Persistent
California, USA, 1947
16
QpH1
CB25
Human blood
Acute
Paris, France, 1991
16
QpH1
Dyer
Human blood
Acute
USA, 1938
16
QpH1
Ohio
Cow milk
Persistent
Ohio, USA, 1956
16
QpH1
Nine Mile
Tick
–
Montana, USA, 1935
16
QpH1
CS-KL 9
Ixodes ricinus
–
Slovakia, 1989
16
QpH1
Z-2775/90
Cow placenta
Abortion
Germany, 1990
16
QpH1
J-1
Cow milk
–
Japan
16
QpH1
J-3
Cow milk
–
Japan
16
QpH1
J-27
Cow milk
–
Japan
16
QpH1
J-60
Cow milk
–
Japan
16
QpH1
J-82
Cow milk
–
Japan
16
QpH1
Hardthof
Cow milk
–
Germany, 1990
16
QpH1
CB77
Human heart valve
Chronic
Paris, France, 1998
17
QpH1
CB100
Human blood
Chronic
Strasbourg, France
18
QpH1
Henzerling
Human blood
Acute
Italy/Slovakia, 1945
18
QpH1
Balaceanu
Human
Acute
Romania
18
QpH1
Geier
Human
Acute
Romania
18
QpH1
Heizberg
Human
Acute
Greece
18
QpH1
Cs-Florian
Human blood
–
Slovakia, 1956
18
QpH1
Z-3464/92
Goat placenta
Abortion
Germany, 1992
18
QpH1
Z-4488/93
Ewe placenta
Abortion
Germany, 1993
18
QpH1
Z-349-36/94
Ewe placenta
–
Germany, 1994
18
QpH1
München
Sheep
–
München, Germany, 1969
18
QpH1
CB119
Human heart valve
Chronic
Senegal, 2004
19
QpH1
CB48
Human placenta
Abortion
Grenoble, France, 1994
20
QpH1
CB50
Valvular prosthesis
Chronic
Paris, France, 1994
20
QpH1
CB66
Aneurysm
Chronic aneurysm
Marseille, France, 1996
20
QpH1
CB72
Valvular prosthesis
Chronic
Paris, France, 1996
20
QpH1
CB78
Valvular prosthesis
Chronic
Marseille, France, 1998
20
QpH1
CB88
Human heart valve
Chronic
Lyon, France, 1999
20
QpH1
CB90
Human heart valve
Chronic
Lyon, France,2000
20
QpH1
Z-3567/92
Cow placenta
Abortion
Germany, 1992
20
QpH1
Dugway 5J108-111
Rodent
–
Utah, USA, 1958
20
QpH1
CB4
Human blood
Chronic
Montpellier, France,1988
21
QpH1
CB7
Human heart valve
Chronic Aneurysm
Marseille, France
21
QpH1
Q229
Human heart valve
Chronic
Nova Scotia, Canada, 1982
21
QpH1
Dog CB
Dog uterus
–
Nova Scotia, Canada, 1995
21
Poker Cat
Cat
–
Nova Scotia, Canada, 1986
21
CBNSC1
Cat
–
Nova Scotia, Canada, 1986
21
QpH1
Dog ut Ad
Dog uterus
–
Nova Scotia, Canada, 1989
21
CB115
Human heart valve
Chronic
Nova Scotia, Canada, 2003
21
Q212
Human heart valve
Chronic
Nova Scotia, Canada, 1981
21
Scurry Q217
Human liver biopsy
Hepatitis
Rocky Mountain, USA
21
48
Haemaphysalis punctata
–
Slovakia, 1970
22
QpH1
Irkutsk
Tick
–
Irkutsk, Russia1969
23
QpH1
Uzbekistan
Cow placenta
–
Uzbekistan, 1971
23
QpH1
1894
Liver and spleen of wild bird
–
Czechoslovakia, 1954
23
QpH1
Kazakhstan-6
Dermacentor marginatus
–
Kazakhstan, 1969
23
QpH1
K-261-Louga
Cow milk
–
Leningrad, Russia, 1959
23
QpH1
Kazakhstan -5
Dermacentor hirundinis
–
Kazakhstan, 1969
23
QpH1
Russet mouse
Russet mouse viscera
–
Pskov, Russia
23
QpH1
II/IA
Dermacentor marginatus
–
Slovakia, 1972
23
QpH1
IXO
Ixodes ricinus
–
Czech Republic, 1957
23
QpH1
Mongolia-2
Dermacentor nuttalli
–
Mongolia, 1985
23
QpH1
CS-27
Dermacentor marginatus
–
Slovakia, 1972
23
QpH1
Oufa-1
Human blood
–
Oufa, Russia
23
QpH1
Oufa-2
Ewe placenta
Abortion
Oufa, Russia
23
QpH1
Louga-3
Ixodes ricinus
–
Leningrad, Russia, 1962
23
QpH1
Louga-2
Ixodes ricinus
–
Leningrag, Russia, 1959
23
QpH1
Louga-1
Cimex lecturalius
–
Leningrad, Russia, 1959
23
QpH1
Louga rodent
Apodemus flavicollis viscera
–
Leningrad, Russia, 1958
23
QpH1
Mongolia-1
Dermacentor silvarum
–
Mongolia, 1984
23
QpH1
Vologda-2
Human blood
–
Vologda, Russia, 1987
23
QpH1
8931 F10
Cow placenta
Abortion
Germany
24
QpH1
Grita
-
–
Germany, 1940-1945
25
QpH1
M-44
Vaccine
–
Russia
25
QpH1
Kazakhstan-4
Fly
–
Kazakhstan, 1965
25
QpH1
Termez
Human blood
–
Uzbekistan, 1952
26
QpRS
Z2534
Goat placenta
Abortion
Austria
27
QpH1
Kazakhstan-1
Ewe placenta
Abortion
Kazakhstan
28
QpRS
Kazakhstan-2
Cow milk
–
Kazakhstan, 1962
28
QpRS
Kazakhstan-3
Hyalomma tick
–
Kazakhstan, 1962
28
QpRS
Tcheredov
Human blood
–
Kazakhstan, 1965
28
QpRS
Henzerling-r *
Human blood
–
Italy, 1945 / Slovakia,
29
QpH1
Namibia
Goat
–
Namibia, 1991
30
QpRS
*Strain resistant to chlortetracycline obtained by passages in embryonated hen's eggs in presence of increasing doses of chlortetracycline.
Primer used in djlA, com1 and QpH1 and QpRS plasmid targeted sequences for PCR amplification and sequencing
Primer
Nucleotide sequence 5´ → 3´
Position in the gene
djlA-1*†
CGGTGATGAACTGGATTGG
–5 – 15
djlA-2†
ATTGACCTGACGCGCTTGACG
266 –247
djlA-3†
GGCAACGCAAGACCCCCGTG
579 – 598
djlA-4*†
AACCATGCTTCGCACCTTAC
810 –791
com-1*†
CGTGAAGAACCGTTTGACTG
3 – 22
com-2†
TGAGGATTGCCTGCCACTGG
284 – 265
com-3†
GCGCTGCTCAGTGTCGACGG
490 – 509
com-4*†
CTTTTCTACCCGGTCGATTTC
759 – 739
Primer
Nucleotide sequence 5´ → 3´
Position in the plasmid
ORF
QpH11
TGACAAATAGAATTTCTTCATTTTGATG
QpH1 (gb:AE016829) 15332 –15359
Spacer between two hypothetical proteins
QpH12
GCTTATTTTCTTCCTCGAATCTATGAAT
QpH1 (gb:AE016829) 168348 – 16375
Spacer between two hypothetical proteins
QpRSO1
CTCGTACCCAAAGACTATGAATATATCC
QpRS gb:Y15898) 14761 –14734
Hypothetical protein
QpRS02
CACATTGGGTATCGTACTGTCCCT
QpRS gb:Y15898) 14398 – 14321
Hypothetical protein
QpDV1f
ATGAGAGAAGAGCAGCCGCT
QpRS (gb:Y15898) 9889 – 9908
Hypothetical protein
QpDV1r
TCAATGATCCGATGTGCGTTT
QpH1 (gb:Y15898) 10634 –10614
Hypothetical protein
*Oligonucleotide primer used for PCR amplification. †Oligonucleotide primer used for sequencing.
Accession numbers of nucleotide sequences deposited in GenBank
Cox 2
Cox 5
Cox 18
Cox 20
Cox 22
Cox 37
Cox 51
Cox 56
Cox 57
Cox 61
ST1 (CB26)
AY492067
AY495357
AY502819
AY502857
AY502899
AY502623
AY502735
AY502777
AY502674
AY512785
ST2 (CB39)
AY574327
AY574328
AY574329
AY574330
AY574331
AY574332
AY574333
AY574334
AY574335
AY574336
ST3 (CB118)
AY574337
AY574338
AY574339
AY574340
AY574341
AY574342
AY574343
AY574344
AY574345
AY574346
ST4 (CB20)
AY492065
AY495355
AY502817
AY502855
AY502897
AY502621
AY502733
AY502775
AY502673
AY512784
ST5 (CB35)
AY494735
AY495360
AY502822
AY502860
AY502902
AY502626
AY502738
AY502780
AY502677
AY512788
ST6 (CB45)
AY494734
AY502720
AY502841
AY502881
AY502921
AY502645
AY502761
AY502801
AY502709
AY512819
ST7 (CB43)
AY574307
AY574308
AY574309
AY574310
AY574311
AY574312
AY574313
AY574314
AY574315
AY574316
ST8 (Priscilla)
AY596174
AY502715
AY502837
AY502875
AY502883
AY502642
AY502752
AY502797
AY502681
AY596175
ST9 (CB68)
AY494733
AY502721
AY502842
AY502882
AY502922
AY502646
AY502762
AY502802
AY502710
AY512820
ST10 (CB103)
AY492058
AY495348
AY502810
AY502850
AY502890
AY502613
AY502728
AY502770
AY502688
AY512805
ST11 (CB13)
AY575668
AY575669
AY575670
AY575671
AY575672
AY575673
AY575674
AY575675
AY575676
AY575677
ST12 (CB33)
AY492069
AY495359
AY502821
AY502859
AY502901
AY502625
AY502737
AY502779
AY502676
AY512787
ST13 (CB2)
AY575653
AY575654
AY575655
AY575656
AY575657
AY575658
AY575659
AY575660
AY575661
AY575662
ST14 (CB85)
AY575643
AY575644
AY575645
AY575646
AY575647
AY575648
AY575649
AY575650
AY575651
AY575652
ST15 (CB102)
AY492057
AY495347
AY502809
AY502849
AY502889
AY502612
AY502755
AY502769
AY502687
AY512794
ST16 (Ohio)
AY494725
AY502712
AY502834
AY502872
AY502916
AY502640
AY502748
AY502794
AY502707
AY512816
ST17 (CB77)
AY574325
AY574326
AY574317
AY574318
AY574319
AY574320
AY574321
AY574322
AY574323
AY574324
ST18 (Henzerling)
AY494723
AY495369
AY502832
AY502870
AY502914
AY502638
AY502746
AY502792
AY502701
AY512814
ST19 (CB119)
AY575633
AY575634
AY575635
AY575636
AY575637
AY575638
AY575639
AY575640
AY575641
AY575642
ST20 (CB88)
AY492072
AY502719
AY502825
AY502863
AY502905
AY502629
AY502759
AY502783
AY502696
AY512798
ST21 (Q212)
AY494729
AY502716
AY502838
AY502876
AY502918
AY502643
AY502753
AY502798
AY502682
AY512793
ST22 (48)
AY864229
AY864230
AY864231
AY864232
AY864233
AY864234
AY864235
AY864236
AY864237
AY864238
ST23 (Irkutsk)
AY864239
AY864240
AY864241
AY864242
AY864243
AY864244
AY864245
AY864246
AY864247
AY864248
ST24 (8931 F10)
AY864199
AY864200
AY864201
AY864202
AY864203
AY864204
AY864205
AY864206
AY864207
AY864208
ST25 (Grita)
AY864209
AY864210
AY864211
AY864212
AY864213
AY864214
AY864215
AY864216
AY864217
AY864218
ST26 (Termez)
AY864219
AY864220
AY864221
AY864222
AY864223
AY864224
AY864225
AY864226
AY864227
AY864228
ST27 (Z2534)
AY864249
AY864250
AY864251
AY864252
AY864253
AY864254
AY864255
AY864256
AY864257
AY864258
ST28 (Kazakhstan-2)
AY864259
AY864260
AY864261
AY864262
AY864263
AY864264
AY864265
AY864266
AY864267
AY864268
ST29 (Henzerling-r)
AY864269
AY864270
AY864271
AY864272
AY864273
AY864274
AY864275
AY864276
AY864277
AY864278
ST30 (Namibia)
AY864279
AY864280
AY864281
AY864282
AY864283
AY864284
AY864285
AY864286
AY864287
AY864288
Dr. Glazunova obtained her doctorate in life science at the Russian Medical State University (Moscow) and Irkutsk State University. She is currently a postdoctoral researcher at the Medical Faculty of Marseille. Her specialty is the molecular identification of bacteria.
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