Next Article in Journal
Hepatitis C Virus Down-Regulates the Expression of Ribonucleotide Reductases to Promote Its Replication
Previous Article in Journal
Whole Genome Sequencing and Comparative Genomic Analysis of Chlamydia gallinacea Field Strains Isolated from Poultry in Poland
Previous Article in Special Issue
Orientia tsutsugamushi Infection in Wild Small Mammals in Western Yunnan Province, China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 12
Issue 7
10.3390/pathogens12070893
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

A Novel Marine Mammal Coxiella burnetii—Genome Sequencing Identifies a New Genotype with Potential Virulence

by
Brett R. Gardner
1,*,
Nathan L. Bachmann
2,
Adam Polkinghorne
2,
Jasmin Hufschmid
1,
Mythili Tadepalli
3,
Marc Marenda
1,
Stephen Graves
3,
John P. Y. Arnould
4 and
John Stenos
3
1
Melbourne Veterinary School, The University of Melbourne, Werribee, VIC 3030, Australia
2
Major Mitchell Consulting, Buderim, QLD 4556, Australia
3
Australian Rickettsial Reference Laboratory, University Hospital Geelong, Geelong, VIC 3220, Australia
4
School of Life and Environmental Sciences, Deakin University, Burwood, VIC 3125, Australia
*
Author to whom correspondence should be addressed.
Pathogens 2023, 12(7), 893; https://doi.org/10.3390/pathogens12070893
Submission received: 10 May 2023 / Revised: 23 June 2023 / Accepted: 26 June 2023 / Published: 29 June 2023
(This article belongs to the Collection Updates on Rickettsia and Coxiella)
Browse Figures
Versions Notes

Abstract

:
The obligate intracellular bacterial pathogen Coxiella burnetii has been identified in a few species of marine mammals, some of which are showing population declines. It has been hypothesized that C. burnetii in marine mammals is a distinct genotype that varies significantly from the typical terrestrial genotypes. It appears to lack an IS1111. Isolates originating from Australian marine animals have a distinctly non-Australian profile of multiple-locus variable-number tandem-repeat analysis (MLVA). Extracted Coxiella DNA of Australian fur seal placental origin was sequenced using the Novaseq platform. Illumina 150 bp paired-end reads were filtered and trimmed with Trimgalore. The microbial community present in the sequenced genome was evaluated with Kraken and Bracken software using the NCBI database. A phylogenetic analysis was performed using 1131 core genes. Core genes were identified using Panaroo and inputted into Iqtree to determine the maximum-likelihood tree. A second phylogenetic tree was created using Rickettsiella grylii and using seven housekeeping genes. Results were compared with the C. burnetii Nine Mile RSA439 virulent genome. This new Australian marine mammal isolate of Coxiella (PG457) appears to be a novel genotype that lacks IS1111 and has a distinct MLVA signature (ms26, ms27, ms28, ms30, and ms31). The presence of genes for multiple virulence factors appears to give this genotype sufficient pathogenicity for it to be considered a possible causative agent of abortion in Australian fur seals as well as a potential zoonotic risk.

1. Introduction

Coxiella burnetii is the intracellular, Gram-negative, bacterial causative agent of Q fever. It has adapted exceptionally well to an obligate intracellular existence within the normally inhospitable host-protective phagolysosome [1]. The lack of genetic diversity across all known C. burnetii genotypes is considered evidence of this pathogen only recently emerging from one of the typical Coxiella-like endosymbionts of ticks [2]. Q fever is known to cause disease in both humans and other mammals [3]. Ruminants, especially goats and cattle, are considered some of the most important reservoirs of this zoonotic pathogen, especially in Australia [4,5]. Historically considered predominantly a disease of workers in the livestock industry, especially those working in abattoirs, it has recently become a disease concern in wildlife rehabilitators [5,6]. C. burnetii is also considered an emerging disease in wildlife, where it has been shown to cause reproductive failure in multiple species [7]. There is a considerable paucity of data in wildlife globally and within Australia [5,7]. Concerns over the impact of C. burnetii in wildlife have recently extended to marine mammals with the detection of this pathogen in several species of pinnipeds, some of which are undergoing population declines [8,9]. Beyond the conservation impacts, the presence of this pathogen in wildlife is also of interest given the potential for wildlife to act as reservoirs of C. burnetii infection in humans and domesticated animals [10].
Most genotypes that have been isolated from wildlife appear to cluster into genomic groups I–III, based predominantly on studies from Europe [10]. Despite C. burnetii being detected in more than 109 wild mammal species, there is still a significant lack of sequencing data to investigate the importance of this pathogen in wildlife and the zoonotic spillover risk [7]. It has been hypothesized that wildlife may harbor genetic variations that could allow for the emergence of further pathogenic variations of the organism [10]. Within an Australian context, the macropods appear to include some of the best-known potential reservoir species [11,12]. Most studies in Australian wildlife have focused on serology and molecular prevalence. There do not appear to be any sequencing data available for C. burnetii in terrestrial Australian wild mammals, and to the best of the authors’ knowledge, no genotyping or sequencing data are available for C. burnetii from any marine mammals.
It has nevertheless been hypothesized that C. burnetii in marine mammals is a unique genotype with key features such as the absence of IS1111 (insertion sequence 1111) [13,14]. A recent isolate of C. burnetii from Australian fur seals (Arctocephalus pusillus doriferus) was analyzed using multiple-locus variable-number tandem-repeat analysis (MLVA) for three loci critical to the Australian genotypes (ms24, ms28, and ms33) [15], but only ms28 was amplified [14]. At present, it is unknown whether the C. burnetii identified in northern fur seals (Callorhinus ursinus), Steller sea lions (Eumetopias jubatus), Pacific harbor seals (Phoca vitulina richardsi), harbor porpoises (Phocoena phocoena), and Australian fur seals is the same organism between all the marine mammals [8,9,16]. Nor is it currently known whether it is a definitive cause of abortion and reproductive loss or what exactly the manifestations are of infection in pinnipeds [13,14].
Certain virulence factors are considered essential for the pathogenesis of C. burnetii, as this intracellular pathogen requires both seamless macrophage invasion and the ability to survive in the defensive host phagolysosome [17]. Two of the most important systems genetically encoded for the pathogenesis of C. burnetii are the lipopolysaccharide (LPS) and the genes affecting the type-4 secretory system (T4SS) [18]. The presence or absence of the genes encoding the O-antigen is used to distinguish between virulent and avirulent strains [18]. There is a considerable data deficiency with regard to the presence and absence of virulence factors in C. burnetii in terrestrial wildlife, and no published data for marine mammals. When considering the difficulty of studying this organism’s disease ecology and pathobiology using conventional microbiology and molecular methods, it becomes apparent that genome sequencing is essential to understanding these processes [1].
The main aims of this study were to use whole-genome sequencing to confirm the previous identification of C. burnetii in Australian fur seals and to gain insight into the phylogenetic relationship of this isolate to other pathogenic C. burnetii strains detected in terrestrial environments. The presence of other well-characterized markers of Coxiella diagnosis and genetic diversity were also examined alongside a preliminary investigation into the pathogenic potential of this genotype, as indicated by the presence of previously characterized C. burnetii virulence factors.

2. Materials and Methods

2.1. Whole-Genome Sequencing and Assembly

Genomic DNA was extracted from an Australian fur seal placental swab sample collected from Kanowna Island (39°15′ S, 146°30′ E) in 2021. The placenta was produced from a presumed full-term birth at the peak of the normal pupping season for the species. No clinical data are available for the dam or the pup. This specimen had previously tested positive for C. burnetii DNA and undergone molecular typing as previously described [14].
Following library construction, genome sequencing was performed using the Novaseq platform (Illumina Australia and New Zealand, Melbourne, Australia) by the Australian Genome Research Facility (https://www.agrf.org.au/, accessed on 18 July 2022). Raw sequencing data were uploaded to the Short Read Archive (PRJNA962036).
The Illumina 150 bp paired-end reads were filtered and trimmed with Trimgalore (Version 0.4.5, https://github.com/FelixKrueger/TrimGalore, accessed on 8 November 2022) to identify and remove sequencing adapters and low-quality stretches of bases. Kraken (Version 2.0.8) [19] and Bracken [20] were utilized for assessment of the composition of the microbial communities present in the placental DNA extract using the NCBI genome and nucleotide databases (downloaded 15 April 2021).
Quality-filtered Coxiella reads identified by Kraken were subsequently assembled using SPAdes v 3.15.0 [21]. Assembled contigs were functionally annotated using Prokka3 (https://github.com/tseemann/prokka, accessed on 8 November 2022) [22]. The assembly was assessed for completeness using BUSCO7 (Version 3) [23], based on the identification of universal single-copy orthologs and the bacteria_odb9.

2.2. Phylogenetic Analysis

A phylogenetic tree was constructed using 1131 core genes identified between the newly sequenced Coxiella PG457 genome and the publicly available genomes of 12 C. burnetii strains (Table S1). All genomes used in the phylogenetic analysis were re-annotated using Prokka3 to ensure consistent gene calling in all genomes. The core genes were identified using Panaroo [24] running in Strict mode with the sequence identity threshold set to 98% and the alignment option set to “core”. The core gene alignment generated by Panaroo was inputted into Iqtree [25] to infer a maximum-likelihood tree using the GTR+I+G model and with 1000 bootstrap replicates. The C. burnetii-only phylogenetic tree was rooted using the novel Coxiella PG457 sequence.
A second phylogenetic tree using the 12 C. burnetii genomes plus Rickettsiella grylli (Accession number: GCF_000168295.1) and the Coxiella endosymbiont of Rhipicephalus microplus (accession number: GCF_002930125.1) was generated using a concatenated alignment of seven housekeeping genes (total length of 32,302 bp) (Table S2). The tree was created with Iqtree (accessed on 31 January 2023) using the GTR+I+G model on the concatenated alignment and rooted using R. grylli.

2.3. Comparative Analysis

The draft genome assembly of the novel Coxiella PG457 was examined for the presence of (i) Coxiella IS1111 transposase sequences, (ii) MLVA gene sequences, and (iii) previously described Coxiella virulence factors. IS1111 sequences were screened for by a combination of read mapping to the C. burnetii Nine Mile RSA439 (NZ_CP018005.1) genome, BLAST searching of the genome assembly, and by use of the IS1111 element search tool available in Coxbase (https://coxbase.q-gaps.de/webapp/analysis/is1111, accessed on 28 January 2023) [22]. MLVA screening utilized the Coxbase MLVA typing tool (https://coxbase.q-gaps.de/webapp/analysis/mlva, accessed on 28 January 2023). BLAST was used to determine the presence of specific Coxiella virulence factors in the Coxiella PG457 draft genome assembly.

3. Results

3.1. Description of Novel Australia Coxiella Draft Genome Assembly

The properties of the assembled draft genome for Coxiella PG457 are presented in Table 1. De novo assembly of the Coxiella-like quality filtered reads resulted in a genome scaffold of 1.9 MB comprising 204 contigs. Automated annotation resulted in 2327 predicted coding sequences (CDS). The assembled Coxiella PG457 draft genome had a G + C content of 42% with three rRNA operons and 43 tRNAs. BUSCO analysis of the C. burnetii PG457 draft genome’s completeness revealed a score of 92.6% [1].
To confirm the presumptive identity of the Coxiella-positive sample sequenced, the full-length 16S rRNA sequence was extracted from the assembled Coxiella draft genome and compared with available sequences in the NCBI database using nucleotide BLAST. This analysis revealed 99.7% identity to the full-length 16S rRNA sequences of a range of C. burnetii strains, including the C. burnetii Nine Mile RSA439 reference genome (CP035112.1), thus identifying this Coxiella strain as belonging to the species C. burnetii. Henceforth, this strain is referred to as C. burnetii PG457. Additionally, PG457 was analyzed with JSpeciesWS (https://jspecies.ribohost.com/jspeciesws/, accessed on 5 May 2023) for a pairwise comparison (ANIbMA and ANImMA) and Tetra correlation search to two reference strains of C. burnetii to ensure the 16s rRNA nucleotide BLAST did not produce erroneous results (Table S3).
An all-versus-all BLAST comparison of C. burnetii PG457 against the completed genome of C. burnetii Nine Mile RSA439 also demonstrates that PG457 is assembled with a high level of completeness and synteny (Supplementary Figure S1). C. burnetii PG457 shares > 90% sequence identity, and limited evidence for genome rearrangement and unique genomic regions, compared with the C. burnetii reference strain.

3.2. Phylogenetic Analysis of C. burnetii PG457

To understand the phylogenetic relationships of the novel C. burnetii strain sequenced in this study to other C. burnetii strains isolated in Australia, Europe, and the North America from human and animal hosts, a phylogenetic tree was constructed using an alignment of 1131 concatenated core genes extracted from 12 publicly available C. burnetii genomes (Figure 1). This analysis revealed that C. burnetii PG457 clustered away from other sequenced C. burnetii strains, forming its own distinct lineage from terrestrial C. burnetii strains isolated in Australia as well as those from the rest of the world.
To gain a broader understanding of these genetic differences, we expanded the phylogenetic analysis to include a Coxiella-like organism, a Coxiella endosymbiont of Rhipicephalus microplus. A phylogenetic tree constructed using a concatenated alignment of seven Coxiella housekeeping genes revealed that, relative to Coxiella-like organisms, the C. burnetii PG457 strain is distinguishable but nevertheless is closely related to other well-characterized C. burnetii strains (Figure 2).

3.3. Identification of Previously Described C. burnetii Features in the Genome of C. burnetii PG457

Having established the identity of the C. burnetii isolate, preliminary genomic analysis was performed to detect the presence of common molecular marker genes and/or markers of C. burnetii pathogenicity.

3.3.1. Coxiella IS1111 Insertion Sequence

The IS1111 transposase sequence is a multicopy genetic element widely targeted for the PCR detection and differentiation of C. burnetii strains [26]. To detect IS1111 sequences in the C. burnetii PG457 draft genome assembly, C. burnetii PG457 reads were mapped to the C. burnetii Nine Mile RSA439 genome (NZ_CP018005.1). No reads were found to align with any of the 20 IS1111 transposase sequences present in the C. burnetii reference genome [1]. BLAST searching of the C. burnetii PG457 genome with IS1111 sequences also failed to detect any of this particular transposase sequence. As a final search strategy, the C. burnetii PG457 genome assembly was imported into the IS1111 element search tool (https://coxbase.q-gaps.de/webapp/analysis/is1111, accessed on 28 January 2023) [27]. No IS1111 sequences were identified. Together, these results suggest that the IS1111 transposase is absent from the C. burnetii PG457 genome.

3.3.2. C. burnetii MLVA Gene Targets

The C. burnetii MLVA typing scheme, consisting of a panel of 17 MLVA loci, is one of many used for fine-detailed genetic differentiation of C. burnetii strains [28]. To identify the presence of MLVA loci, the C. burnetii PG457 draft genome assembly was inputted into the Coxbase MLVA typing tool (https://coxbase.q-gaps.de/webapp/analysis/mlva, accessed on 28 January 2023). Compared with the C. burnetii Nine Mile RSA439 genome, only five loci (ms26, ms27, ms28, ms30, and ms31) could be detected in the C. burnetii PG457 draft genome assembly (Table 2).

3.3.3. Putative C. burnetii Virulence Factors

To gain a preliminary insight into the pathogenesis of this marine C. burnetii strain, we examined the C. burnetii PG457 draft genome assembly for the presence of previously characterized C. burnetii virulence factors. This included searches for components of systems used to interact with the host cell during a C. burnetii infection as well as virulence factors that can be used to differentiate pathogenic and nonpathogenic C. burnetii strains (Table 3). All but one of the genes (adaA) searched for could be found in the C. burnetii PG457 draft genome assembly, including genes associated with the synthesis of Coxiella O-antigen, a component of the C. burnetii lipopolysaccharide, as well as structural components and effectors of the T4SS involved in manipulation and survival in the host cell. This suggests that C. burnetii PG457 is likely to be virulent, both for Australian fur seals and for terrestrial mammals, including humans.

4. Discussion

C. burnetii infections in wildlife have been speculated to be a conservation risk while also posing a threat of spillover infections to humans and domesticated animals [7]. In the current study, we sequenced the genome of a C. burnetii strain detected in an Australian fur seal to gain insight into its relationship with terrestrial C. burnetii strains but also to begin to understand the potential for this pathogen to cause disease in wildlife hosts.
Preliminary phylogenetic analysis of the core genes identified in the whole genome assembly suggests that this previously reported [9,14,35] bacterium is a strain of C. burnetii. However, it is genetically distinct from other pathogenic C. burnetii strains detected in humans and other terrestrial animals both in Australia and elsewhere. While genetically distinct from those strains, the genome of C. burnetii PG457 contains all of the basic genetic hallmarks of previously described C. burnetii genomes, including a similar estimated (i) genome size (1.9–2.1 Mb range) and G + C content (42.4–42.9%) [36] and (ii) number of stable rRNA and tRNA genes. Unfortunately, the sequenced genome of PG457 is not circular. The BUSCO analysis revealed that 7.4% of the sequenced genome is fragmented or missing. It is unknown to what extent this would affect downstream analysis. It is conventionally assumed that sequenced genomes need to have a minimum N50 of 1Mb to ensure no errors occur in the annotation, allowing for conserved genome synteny [37], which the assembled genome of PG457 meets and exceeds.
There are exceptionally few reports of C. burnetii lacking the IS1111 transposase, including only one in humans [35], and sequencing data are not available. Sequencing of C. burnetii strain PG457, which lacks IS1111, illustrates the potential evolutionary relationships between some of the Coxiella-like endosymbionts that lack this insertion sequence and C. burnetii. This is the first sequencing of a C. burnetii that lacks the IS1111 gene. Previous marine mammal studies reporting poor or absent amplification of the IS1111 may very well have been reporting on the same genotype as in this current study [13,14]. This insertion sequence is a multicopy and is often used to differentiate isolates from one another [26]. The genome sequence of this current isolate further supports the hypothesis that the IS1111 transposase is absent from marine mammal strains. The apparent absence of the IS1111 could relate to the missing MLVA loci ms23 and ms33, as these are loci that notoriously contain recognition sites for IS1111 [38,39].
A very distinct MLVA signature appears to be present in this novel isolate. The use of MLVA is considered highly discriminatory between different C. burnetii genotypes [40]. Typical Australian genotypes of C. burnetii have the presence of IS1111, and additionally, three critical MLVA loci, namely ms24, ms28, and ms33 [15]. Previous studies of C. burnetii in Australian fur seals have shown strong evidence that only ms28 is present in these isolates [14,41]. Based on the MLVA-associated genomic grouping, this novel isolate appears to cluster outside of any of the currently accepted genomic groups and any of those represented in Australia [38]. We propose that an MLVA signature for marine mammal isolates of C. burnetii will consist of ms26, ms27, ms28, ms30, and ms31. This assumption, made from the currently available genome sequence data, needs to be verified in samples from other marine mammals and in silico.
Although the exact pathogenic manifestations of C. burnetii in marine mammals are still to be fully illustrated, it is apparent from the initial sequencing data of C. burnetii in Australian fur seals that certain key virulence factors appear to be present in the genome that are shared with pathogenic terrestrial genotypes. Conventionally, genomic groups I and IV contain the most pathogenic genotypes of C. burnetii [38], but the inability to group this novel isolate into any of these previously described genomic groups makes it more difficult to infer pathogenicity. Ideally, the use of a guinea pig model might better illustrate the true pathogenic ability of this marine mammal isolate because it is considered one of the gold standard models to infer pathogenicity [42]. In human cases of Q fever, it appears that certain SNP (single nucleotide polymorphism) genotypes are more likely to cause disease [17], but this has not been determined for PG457. The presence of the tadA, O-antigen, ompA, mceA, dotA, and dotB genes at 94.6–97.6% with the Nine-Mile strain would suggest a strong presence of the LPS and T4SS [18]. This potentially indicates the ability of this novel isolate to induce significant pathology in marine mammals and probably pose a zoonotic risk to persons working with these animals.
Ideally, this novel genome sequence needs to be compared with those of northern-hemisphere marine mammals to determine the evolutionary relatedness of these various strains. Further surveillance is needed to determine if any horizontal transmission can occur between marine mammals and terrestrial mammals. If it is possible to culture C. burnetii from Australian fur seals, potentially a full genome could be sequenced and a viable inoculum made available, to determine its virulence in guinea pigs. If virulent, it is possible that this bacterium has been playing a role in declining marine mammal populations.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pathogens12070893/s1. Figure S1: Whole genome alignment of the Coxiella burnetii PG457 (bottom) and C. burnetii Nine Mile RSA439 reference genome (top). Red shading indicates a nucleotide identity of > 90%; e Phylogenetic tree was constructed using 1131 core genes identified between the newly sequenced Coxiella PG457 genome and the publicly available genomes of 12 C. burnetii strains; e Seven conserved “housekeeping” genes (32,301 bp) detected in the genomes of 13 C. burnetii strains (including C. burnetii PG457) and a Coxiella-like organism (Coxiella of Rhipicephalus microplus); Table S1: Coxiella burnetii sequenced genomes from NCBI; Table S2: Coxiella burnetii conserved house-keeping genes; Table S3: C. burnetii PG457 ANIbMA, ANIMMA and TETRA analysis.

Author Contributions

Conceptualization, B.R.G., M.T. and J.S.; methodology, B.R.G., N.L.B., A.P., M.M. and M.T.; software, N.L.B. and A.P.; validation, B.R.G., N.L.B., A.P. and J.S.; formal analysis, N.L.B., M.M. and A.P.; investigation, B.R.G., M.T., N.L.B. and A.P.; resources, S.G., J.P.Y.A., J.H. and J.S.; data curation, B.R.G., N.L.B. and A.P.; writing—original draft preparation, B.R.G., N.L.B. and A.P.; writing—review and editing, B.R.G., N.L.B., A.P., J.H., M.M., M.M., S.G., J.P.Y.A. and J.S.; visualization, N.L.B.; supervision, J.H., J.P.Y.A. and J.S.; project administration, B.R.G.; funding acquisition, J.S. and S.G.. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Original samples were collected ensuring ethical treatment and complied with the welfare requirements of the University of Melbourne animal ethics committee approval for scavenged animal tissues 19-009, Deakin University animal ethics committee approval B05-2020 under a Department of Environment, Land, Water and Planning wildlife research permit 10009465. This sequencing project did not require ethics approval.

Informed Consent Statement

This study did not involve human participants and therefore informed consent was not applicable.

Data Availability Statement

All data not supplied as Supplementary Files are available on request.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The corresponding author has conducted this research as part of their PhD thesis. Major Mitchell Consulting was paid and contracted to assist with the analysis of the sequencing data.

References

  1. Seshadri, R.; Paulsen, I.T.; Eisen, J.A.; Read, T.D.; Nelson, K.E.; Nelson, W.C.; Ward, N.L.; Tettelin, H.; Davidsen, T.M.; Beanan, M.J. Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc. Natl. Acad. Sci. USA 2003, 100, 5455–5460. [Google Scholar] [CrossRef] [Green Version]
  2. Duron, O.; Noël, V.; McCoy, K.D.; Bonazzi, M.; Sidi-Boumedine, K.; Morel, O.; Vavre, F.; Zenner, L.; Jourdain, E.; Durand, P. The recent evolution of a maternally-inherited endosymbiont of ticks led to the emergence of the Q fever pathogen, Coxiella burnetii. PLoS Pathog. 2015, 11, e1004892. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Eldin, C.; Mélenotte, C.; Mediannikov, O.; Ghigo, E.; Million, M.; Edouard, S.; Mege, J.-L.; Maurin, M.; Raoult, D. From Q fever to Coxiella burnetii infection: A paradigm change. Clin. Microbiol. Rev. 2017, 30, 115–190. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Angelakis, E.; Million, M.; D’amato, F.; Rouli, L.; Richet, H.; Stein, A.; Rolain, J.-M.; Raoult, D. Q fever and pregnancy: Disease, prevention, and strain specificity. Eur. J. Clin. Microbiol. Infect. Dis. 2013, 32, 361–368. [Google Scholar] [CrossRef]
  5. Bond, K.A.; Franklin, L.; Sutton, B.; Stevenson, M.; Firestone, S. Review of 20 years of human acute Q fever notifications in Victoria, 1994–2013. Aust. Vet. J. 2018, 96, 223–230. [Google Scholar] [CrossRef]
  6. Mathews, K.O.; Toribio, J.A.; Norris, J.M.; Phalen, D.; Wood, N.; Graves, S.R.; Sheehy, P.A.; Bosward, K.L. Coxiella burnetii seroprevalence and Q fever in Australian wildlife rehabilitators. One Health 2021, 12, 100197. [Google Scholar] [CrossRef]
  7. González-Barrio, D.; Ruiz-Fons, F. Coxiella burnetii in wild mammals: A systematic review. Transbound. Emer. Dis. 2019, 66, 662–671. [Google Scholar] [CrossRef]
  8. Minor, C.; Kersh, G.J.; Gelatt, T.; Kondas, A.V.; Pabilonia, K.L.; Weller, C.B.; Dickerson, B.R.; Duncan, C.G. Coxiella burnetii in northern fur seals and steller sea lions of Alaska. J. Wildl. Dis. 2013, 49, 441–446. [Google Scholar] [CrossRef] [PubMed]
  9. Gardner, B.R.; Stenos, J.; Hufschmid, J.; Arnould, J.P.; McIntosh, R.R.; Tadepalli, M.; Tolpinrud, A.; Marenda, M.; Lynch, M.; Stent, A. An old pathogen in a new environment–implications of Coxiella burnetii in Australian fur seals (Arctocephalus pusillus doriferos). Front. Mar. Sci. 2022, 9, 6. [Google Scholar] [CrossRef]
  10. González-Barrio, D.; Jado, I.; Fernández-de-Mera, I.G.; del Rocio Fernández-Santos, M.; Rodríguez-Vargas, M.; García-Amil, C.; Beltrán-Beck, B.; Anda, P.; Ruiz-Fons, F. Genotypes of Coxiella burnetii in wildlife: Disentangling the molecular epidemiology of a multi-host pathogen. Environ. Microbiol. Rep. 2016, 8, 708–714. [Google Scholar] [CrossRef]
  11. Cooper, A.; Barnes, T.; Potter, A.; Ketheesan, N.; Govan, B. Determination of Coxiella burnetii seroprevalence in macropods in Australia. Vet. Microbiol. 2012, 155, 317–323. [Google Scholar] [CrossRef] [Green Version]
  12. Cooper, A.; Stephens, J.; Ketheesan, N.; Govan, B. Detection of Coxiella burnetii DNA in wildlife and ticks in northern Queensland, Australia. Vector-Borne Zoonotic Dis. 2013, 13, 12–16. [Google Scholar] [CrossRef] [PubMed]
  13. Duncan, C.; Savage, K.; Williams, M.; Dickerson, B.; Kondas, A.V.; Fitzpatrick, K.A.; Guerrero, J.L.; Spraker, T.; Kersh, G.J. Multiple strains of Coxiella burnetii are present in the environment of St. Paul Island, Alaska. Transbound. Emer. Dis. 2013, 60, 345–350. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Gardner, B.; Arnould, J.; Hufschmid, J.; McIntosh, R.; Fromant, A.; Tadepalli, M.; Stenos, J. Understanding the zoonotic pathogen, Coxiella burnetii in Australian fur seal breeding colonies through environmental DNA and genotyping. Wildl. Res. 2022. [Google Scholar] [CrossRef]
  15. Vincent, G.; Stenos, J.; Latham, J.; Fenwick, S.; Graves, S. Novel genotypes of Coxiella burnetii identified in isolates from Australian Q fever patients. Int. J. Med. Microbiol. 2016, 306, 463–470. [Google Scholar] [CrossRef] [PubMed]
  16. Kersh, G.J.; Lambourn, D.M.; Raverty, S.A.; Fitzpatrick, K.A.; Self, J.S.; Akmajian, A.M.; Jeffries, S.J.; Huggins, J.; Drew, C.P.; Zaki, S.R. Coxiella burnetii infection of marine mammals in the Pacific Northwest, 1997–2010. J. Wildl. Dis. 2012, 48, 201–206. [Google Scholar] [CrossRef]
  17. Larson, C.L.; Martinez, E.; Beare, P.A.; Jeffrey, B.; Heinzen, R.A.; Bonazzi, M. Right on Q: Genetics begin to unravel Coxiella burnetii host cell interactions. Future Microbiol. 2016, 11, 919–939. [Google Scholar] [CrossRef] [Green Version]
  18. Van Schaik, E.J.; Chen, C.; Mertens, K.; Weber, M.M.; Samuel, J.E. Molecular pathogenesis of the obligate intracellular bacterium Coxiella burnetii. Nat. Rev. Microbiol. 2013, 11, 561–573. [Google Scholar] [CrossRef] [Green Version]
  19. Wood, D.E.; Lu, J.; Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019, 20, 257. [Google Scholar] [CrossRef] [Green Version]
  20. Lu, J.; Breitwieser, F.P.; Thielen, P.; Salzberg, S.L. Bracken: Estimating species abundance in metagenomics data. PeerJ Comput. Sci. 2017, 3, e104. [Google Scholar] [CrossRef] [Green Version]
  21. Bankevich, A.; Nurk, S.; Antipov, D.; Gurevich, A.A.; Dvorkin, M.; Kulikov, A.S.; Lesin, V.M.; Nikolenko, S.I.; Pham, S.; Prjibelski, A.D.; et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012, 19, 455–477. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Seemann, T. Prokka: Rapid prokaryotic genome annotation. Bioinformatics 2014, 30, 2068–2069. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Waterhouse, R.M.; Seppey, M.; Simão, F.A.; Manni, M.; Ioannidis, P.; Klioutchnikov, G.; Kriventseva, E.V.; Zdobnov, E.M. BUSCO Applications from Quality Assessments to Gene Prediction and Phylogenomics. Mol. Biol. Evol. 2018, 35, 543–548. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Tonkin-Hill, G.; MacAlasdair, N.; Ruis, C.; Weimann, A.; Horesh, G.; Lees, J.A.; Gladstone, R.A.; Lo, S.; Beaudoin, C.; Floto, R.A. Producing polished prokaryotic pangenomes with the Panaroo pipeline. Genome Biol. 2020, 21, 1–21. [Google Scholar] [CrossRef]
  25. Nguyen, L.-T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [Google Scholar] [CrossRef] [PubMed]
  26. Denison, A.M.; Thompson, H.A.; Massung, R.F. IS1111 insertion sequences of Coxiella burnetii: Characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates. BMC Microbiol. 2007, 7, 1–8. [Google Scholar] [CrossRef] [Green Version]
  27. Fasemore, A.M.; Helbich, A.; Walter, M.C.; Dandekar, T.; Vergnaud, G.; Förstner, K.U.; Frangoulidis, D. CoxBase: An Online Platform for Epidemiological Surveillance, Visualization, Analysis, and Typing of Coxiella burnetii Genomic Sequences. Msystems 2021, 6, e00403–e00421. [Google Scholar] [CrossRef]
  28. Arricau-Bouvery, N.; Hauck, Y.; Bejaoui, A.; Frangoulidis, D.; Bodier, C.C.; Souriau, A.; Meyer, H.; Neubauer, H.; Rodolakis, A.; Vergnaud, G. Molecular characterization of Coxiella burnetii isolates by infrequent restriction site-PCR and MLVA typing. BMC Microbiol. 2006, 6, 1–14. [Google Scholar] [CrossRef] [Green Version]
  29. Planet, P.J.; Kachlany, S.C.; DeSalle, R.; Figurski, D.H. Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification. Proc. Natl. Acad. Sci. USA 2001, 98, 2503–2508. [Google Scholar] [CrossRef] [Green Version]
  30. Lukácová, M.; Barák, I.; Kazár, J. Role of structural variations of polysaccharide antigens in the pathogenicity of Gram-negative bacteria. Clin. Microbiol. Infect. 2008, 14, 200–206. [Google Scholar] [CrossRef] [Green Version]
  31. Martinez, E.; Cantet, F.; Fava, L.; Norville, I.; Bonazzi, M. Identification of OmpA, a Coxiella burnetii Protein Involved in Host Cell Invasion, by Multi-Phenotypic High-Content Screening. PLoS Pathog. 2014, 10, e1004013. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Fielden, L.F.; Moffatt, J.H.; Kang, Y.; Baker, M.J.; Khoo, C.A.; Roy, C.R.; Stojanovski, D.; Newton, H.J. A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection. Infect. Immun. 2017, 85, e01046-16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Beare, P.A.; Larson, C.L.; Gilk, S.D.; Heinzen, R.A. Two systems for targeted gene deletion in Coxiella burnetii. Appl. Env. Microbiol. 2012, 78, 4580–4589. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Zhang, G.; To, H.; Russell, K.E.; Hendrix, L.R.; Yamaguchi, T.; Fukushi, H.; Hirai, K.; Samuel, J.E. Identification and characterization of an immunodominant 28-kilodalton Coxiella burnetii outer membrane protein specific to isolates associated with acute disease. Infect. Immun. 2005, 73, 1561–1567. [Google Scholar] [CrossRef] [Green Version]
  35. Marmion, B.P.; Storm, P.A.; Ayres, J.G.; Semendric, L.; Mathews, L.; Winslow, W.; Turra, M.; Harris, R.J. Long-term persistence of Coxiella burnetii after acute primary Q fever. QJM 2005, 98, 7–20. [Google Scholar] [CrossRef] [Green Version]
  36. Abou Abdallah, R.; Million, M.; Delerce, J.; Anani, H.; Diop, A.; Caputo, A.; Zgheib, R.; Rousset, E.; Sidi Boumedine, K.; Raoult, D.; et al. Pangenomic analysis of Coxiella burnetii unveils new traits in genome architecture. Front. Microbiol. 2022, 13, 4258. [Google Scholar] [CrossRef]
  37. Liu, D.; Hunt, M.; Tsai, I.J. Inferring synteny between genome assemblies: A systematic evaluation. BMC Bioinform. 2018, 19, 1–13. [Google Scholar] [CrossRef] [Green Version]
  38. Hemsley, C.M.; Essex-Lopresti, A.; Norville, I.H.; Titball, R.W. Correlating Genotyping Data of Coxiella burnetii with Genomic Groups. Pathogens 2021, 10, 604. [Google Scholar] [CrossRef]
  39. Sidi-Boumedine, K.; Duquesne, V.; Prigent, M.; Yang, E.; Joulié, A.; Thiéry, R.; Rousset, E. Impact of IS1111 insertion on the MLVA genotyping of Coxiella burnetii. Microbes Infect. 2015, 17, 789–794. [Google Scholar] [CrossRef]
  40. Svraka, S.; Toman, R.; Skultety, L.; Slaba, K.; Homan, W.L. Establishment of a genotyping scheme for Coxiella burnetii. FEMS Microbiol. Lett. 2006, 254, 268–274. [Google Scholar] [CrossRef] [Green Version]
  41. Gardner, B.R.; Hufschmid, J.; Stenos, J.; Tadepalli, M.; Sutton, G.; Fromant, A.; Eizenberg, Y.; Geeson, J.J.; Arnould, J.P. Pacific Gulls (Larus pacificus) as Potential Vectors of Coxiella burnetii in an Australian Fur Seal Breeding Colony. Pathogens 2023, 12, 122. [Google Scholar] [CrossRef] [PubMed]
  42. Long, C.M.; Beare, P.A.; Cockrell, D.C.; Larson, C.L.; Heinzen, R.A. Comparative virulence of diverse Coxiella burnetii strains. Virulence 2019, 10, 133–150. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Pathogens 12 00893 g001 550
Figure 1. Rooted phylogenetic tree of 13 C. burnetii isolates, including C. burnetii PG457, overlaid with host and isolate locations. The phylogenetic tree was constructed based on alignment of core 1131 genes identified using Panaroo [24], and bootstrap values are shown as percentages of 1000 replicants. Scale indicates number of substitutions per site. There is a possibility that C. burnetii strains RSA425 and RSA297, while originally Australian isolates, may have been contaminated with, and overgrown by, North American strains during passaging in laboratories many decades ago (Personal communication S. Graves).
Figure 1. Rooted phylogenetic tree of 13 C. burnetii isolates, including C. burnetii PG457, overlaid with host and isolate locations. The phylogenetic tree was constructed based on alignment of core 1131 genes identified using Panaroo [24], and bootstrap values are shown as percentages of 1000 replicants. Scale indicates number of substitutions per site. There is a possibility that C. burnetii strains RSA425 and RSA297, while originally Australian isolates, may have been contaminated with, and overgrown by, North American strains during passaging in laboratories many decades ago (Personal communication S. Graves).
Pathogens 12 00893 g001
Pathogens 12 00893 g002 550
Figure 2. Maximum likelihood phylogenetic tree based on the concatenated alignment of 7 conserved genes (32,301 bp) detected in the genomes of 13 C. burnetii strains (including C. burnetii PG457) and a Coxiella-like endosymbiont/bacterium (Coxiella of Rhipicephalus microplus). The tree was rooted to Ricketsiella grylli. Scale indicates number of substitutions per site.
Figure 2. Maximum likelihood phylogenetic tree based on the concatenated alignment of 7 conserved genes (32,301 bp) detected in the genomes of 13 C. burnetii strains (including C. burnetii PG457) and a Coxiella-like endosymbiont/bacterium (Coxiella of Rhipicephalus microplus). The tree was rooted to Ricketsiella grylli. Scale indicates number of substitutions per site.
Pathogens 12 00893 g002
Table
Table 1. Coxiella PG457 draft genome assembly statistics.
Table 1. Coxiella PG457 draft genome assembly statistics.
CriteriaStatistic
Number of scaffolds201
Number of contigs204
Scaffold sequence length1.907 MB
Maximum scaffold length115,883 KB
Maximum contig length105,116 KB
Percentage of sequence in scaffold > 5060.15%
Table
Table 2. Detection of C. burnetii MLVA loci in the C. burnetii PG457 and C. burnetii Nine Mile RSA439 genome using the Coxbase MLVA search tool (https://coxbase.q-gaps.de/webapp/analysis/mlva).
Table 2. Detection of C. burnetii MLVA loci in the C. burnetii PG457 and C. burnetii Nine Mile RSA439 genome using the Coxbase MLVA search tool (https://coxbase.q-gaps.de/webapp/analysis/mlva).
StrainLocus
Characteristic		MS01MS03MS20MS21MS22MS23MS24MS26MS27MS28MS30MS31MS33MS34
C. burnetii PG457Product lengthN.D. aN.D.N.D.N.D.N.D.N.D.N.D.132263132306142N.D.N.D.
Flank lengthN.D.N.D.N.D.N.D.N.D.N.D.N.D.104249112205106N.D.N.D.
Repeat sizeN.D.N.D.N.D.N.D.N.D.N.D.N.D.966187N.D.N.D.
Repeat numberN.D.N.D.N.D.N.D.N.D.N.D.N.D.32355N.D.N.D.
C. burnetii RSA439Product length248227402210246157344150276150306150262210
Flank length1761429613617490135104249112205106193175
Repeat size16123312117796618776
Repeat number479669295465695
a N.D. = Not detected.
Table
Table 3. Preliminary analysis of the previously described Coxiella virulence factors in the C. burnetii PG457 draft genome assembly.
Table 3. Preliminary analysis of the previously described Coxiella virulence factors in the C. burnetii PG457 draft genome assembly.
Gene/Gene ProductProduct Description and FunctionDetected in
C. burnetii PG457?		Nucleotide
Identity to C. burnetii RSA439 Gene		Percentage Gene
Coverage		Nucleotide Identity to C. burnetii Cb175 GenePercentage Gene
Coverage
tadAType 2 and Type 4 family secretion system ATPase, TadA; predicted involvement in Coxiella adherence [29]Yes96.2%99%96.2%100%
O-antigen/LPSO-antigen ligase family protein, membrane transport (lptA,B,C); synthesize components of the Coxiella lipopolysaccharide unique to virulent strains [30]Yes95.4%100%95.43%100%
ompAOuter Membrane Protein A;
Coxiella invasin [31]		Yes96.8%100%96.8%100%
mceAMitochondrial Coxiella effector protein A;
effector targeting host cell mitochondria during Coxiella infection [32]		Yes97.6%100%97.6%100%
dotAType IV secretion system protein, DotA; structural component of the Dot/Icm Type IV secretion system complex [33]Yes94.6%100%94.6%100%
dotBType IV secretion system protein, DotB; structural component of the Dot/Icm Type IV secretion system complex [33]Yes96.6%99%96.57%99%
adaACoxiella burnetii-specific acute disease antigen, adaA; outer membrane protein, originally thought to differentiate strains causing acute and chronic Q fever [34]No−−−−
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Gardner, B.R.; Bachmann, N.L.; Polkinghorne, A.; Hufschmid, J.; Tadepalli, M.; Marenda, M.; Graves, S.; Arnould, J.P.Y.; Stenos, J. A Novel Marine Mammal Coxiella burnetii—Genome Sequencing Identifies a New Genotype with Potential Virulence. Pathogens 2023, 12, 893. https://doi.org/10.3390/pathogens12070893

AMA Style

Gardner BR, Bachmann NL, Polkinghorne A, Hufschmid J, Tadepalli M, Marenda M, Graves S, Arnould JPY, Stenos J. A Novel Marine Mammal Coxiella burnetii—Genome Sequencing Identifies a New Genotype with Potential Virulence. Pathogens. 2023; 12(7):893. https://doi.org/10.3390/pathogens12070893

Chicago/Turabian Style

Gardner, Brett R., Nathan L. Bachmann, Adam Polkinghorne, Jasmin Hufschmid, Mythili Tadepalli, Marc Marenda, Stephen Graves, John P. Y. Arnould, and John Stenos. 2023. "A Novel Marine Mammal Coxiella burnetii—Genome Sequencing Identifies a New Genotype with Potential Virulence" Pathogens 12, no. 7: 893. https://doi.org/10.3390/pathogens12070893

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop