How a new species of Lyme disease bacteria was discovered

A new agent of the tick-borne illness known as Lyme disease has emerged in the upper Midwest.  The bacterium is genetically related to Borrelia burgdorferi, until now believed to be the only cause of Lyme disease in the United States.  The name proposed for the bacterium is Borrelia mayonii because the work was conducted at the Mayo Clinic.  B. mayonii has not been detected in patients outside of the Midwest (so far).  The findings are described in The Lancet Infectious Diseases.

The new species was discovered at the Mayo Clinic during routine testing of specimens (blood, cerebral spinal fluid, and joint fluid) received from all regions of the U.S.  Over 100,000 specimens collected from 2003 through 2014 were tested for Lyme disease bacteria by real-time PCR . The PCR probes were designed to detect the oppA1 gene from Borrelia species belonging to the Lyme disease group, known in the scientific literature as "B. burgdorferi sensu lato."  The Lyme disease group comprises 18 species that fall into the same genetic cluster within the genus Borrelia.  They include species known or suspected to cause Lyme disease (B. burgdorferi, B. garinii, B. afzelii, B. spielmanii, B. valaisiana, B bissettii, B. bavariensis, and B. lusitaniae) and another ten species that do not cause illness.  The PCR probes do not react with DNA from species belonging to the other cluster of Borrelia, the relapsing fever group.

The key to the discovery of the new species was the melting temperature analysis routinely programmed onto the end of real-time PCR runs.  The oppA1 PCR products amplified from B. burgdorferi strains have melting temperatures of 63.6 through 64.9°C.  For other Lyme disease species, the melting temperature ranges from 52.3°C (B. valaisiana) to 59.2°C (B. californiensis).  Therefore, the melting temperature of the oppA1 PCR product was used to distinguish B. burgdorferi from other Lyme disease Borrelia.

Over 9,000 specimens were collected from the states of Minnesota, Wisconsin, and North Dakota from January 2012 through September 2014.  102 were PCR positive, and most of the PCR products had the melting temperature profile of B. burgdorferi.  However, six had melting temperatures ranging from 60.4°C to 61.2°C, too low to be B. burgdorferi but too high to be any other member of the Lyme disease group.  The novel spirochetes were cultured from the blood of two of the patients.  The DNA sequence of several "housekeeping" genes of the new isolates differed enough from those of other Borrelia species to signify that a new Borrelia species has been found.  The investigators named the new spirochete Borrelia mayonii.  No specimen collected from other regions of the U.S. exhibited the atypical melting temperatures, and neither did any collected earlier than 2012 from the Midwest.  These findings led the authors to conclude that B. mayonii has recently emerged in the upper Midwest and that the six patients are the first known cases of Lyme disease to be caused by the new species.

The investigators also collected Ixodes scapularis ticks in Wisconsin.  PCR and melting temperature analysis showed that 19 of 658 ticks (2.9%) were positive for B. mayonii, 195 (29.6%) positive for B. burgdorferi, and two positive for both.

One striking feature of B. mayonii infections is the large number of spirochetes circulating within the patients.  The densities ranged from 420,000 to 6,400,000 bacterial cells per milliliter, at least a hundred times higher than observed in the blood of patients with B. burgdorferi infections.  The numbers were high enough that spirochetes could be seen in blood collected from one of the patients.

Fig. 1b from Pritt et al., 2016

The six patients had many of the typical Lyme disease symptoms:  headache, neck pain, muscle aches, joint pain, and fatigue.  Although mild fever is also common in Lyme disease, two of the six patients had severe fevers with temperature readings approaching 40°C (104°F).  Four had nausea or were vomiting, which are also uncommon Lyme disease symptoms.  Two patients were hospitalized because of the severity of their illness.  Lyme disease may be missed in those infected with B. mayonii because of the unusual symptoms.

The standard two-tier antibody test, which uses B. burgdorferi antigens to detect reactive antibody, may help with the diagnosis.  Blood specimens from five of the six patients were tested.  Four patients either tested positive or, if negative initially, tested positive with blood drawn weeks later.  The one patient who tested negative had blood drawn only on the first day of illness, so it's likely that the antibody response hadn't kicked in fully.  The test appears to help with the diagnosis of Lyme disease caused by B. mayonii, but the number of patients tested was too small to draw firm conclusions.

The authors conclude:

In view of the differing clinical manifestations for patients infected with the novel B burgdorferi sensu lato genospecies, it is likely that Lyme borreliosis is not being considered—and therefore not diagnosed—in some patients with this infection. The clinical range of illness must be better defined in additional patients to ensure that physicians can recognise the infection and distinguish it from other tick-borne infections. Many tick-borne pathogens have global distribution, therefore studies are needed to establish the geographic distribution of human beings and ticks infected with the novel B. burgdorferi sensu lato genopecies. Finally, clinicians should be aware of the potential role of oppA1 PCR for diagnosing infection with this novel pathogen.

Reference

Pritt BS, Mead PS, Johnson DK, Neitzel DF, Respicio-Kingry LB, Davis JP, Schiffman E, Sloan LM, Schriefer ME, Replogle AJ, Paskewitz SM, Ray JA, Bjork J, Steward CR, Deedon A, Lee X, Kingry LC, Miller TK, Feist MA, Theel ES, Patel R, Irish CL, & Petersen JM (2016). Identification of a novel pathogenic Borrelia species causing Lyme borreliosis with unusually high spirochaetaemia: a descriptive study. The Lancet. Infectious diseases. PMID: 26856777

Do nonspiral spirochetes help clean our environment?

Members of the spirochete phylum Spirochaetes are recognized easily by their long spiral shape, which allows their periplasmic flagella to power them through viscous environments.  But scientists are discovering that not all spirochetes share this peculiar shape.  Two bacterial isolates recovered from freshwater sediments in Michigan were spherical and lacked flagella, yet phylogenetic analysis of their 16S rRNA and other genes placed them firmly within the Spirochaetes.  The genus Sphaerochaeta was created to accommodate the new isolates, which were designated Sphaerochaeta globosa and Sphaerochaeta pleomorpha.

Sphaerochaeta pleomorpha viewed by phase contrast microscopy.  Arrowheads point to protrusions.  Panel B shows the round spirochetes organized as "strings of pearls."  Figure 1a and 1b from Ritalahti et al., 2012.

Sphaerochaeta globosa viewed by phase contrast microscopy.  Figure 2a from Ritalahti et al., 2012.

The disease-causing spirochetes such as Borrelia burgdorferi and Leptospira species are shape changers.  Although they are often observed with the familiar spiral morphology, they sometimes morph into nonmotile round bodies when stressed, only to revert to the spiral form when conditions improve (see images below).  Could the Sphaerochaeta strains sprout flagella and morph into the spiral form under the right conditions?  It doesn't appear likely.  Sphaerochaeta retain their round shape under a variety of growth conditions, and their genomes lack motility and chemotaxis genes, including those encoding the components of the flagellum.

The Lyme disease spirochete B. burgdorferi viewed by electron microscopy.  Panel A:  B. burgdorferi in its standard growth medium BSKII, which contains serum.  Panel B:  Most of the spirochetes appear as round bodies after being starved for serum for 48 hours.  Bar, 2 µm.   Figure 1A and 1B from Alban et al., 2000.

Views of B. burgdorferi by phase contrast microscopy.  Panel A: B. burgdorferi starved for serum for 48 hours.  Panel B:  Less than one minute after the culture is replenished with serum, the round bodies convert back to the spiral form.  Bar, 5 µm.  Figure 2A and 2B from Alban et al., 2000.

Sphaerochaeta spirochetes have another unusual property.  Electron microscopy revealed what could be a peptidoglycan-layered cell wall (see image below), yet they grow fine even when high concentrations of ampicillin are dumped into the growth meduim.  The genome sequence revealed the reason for their resistance to the antibiotic.  Although the two Sphaerochaeta strains had the genes necessary to make peptidoglycan, they were missing the genes encoding the enzymes that strengthen the cell wall by cross-linking the peptidoglycan.  These missing enzymes are the targets of β-lactams, the penicillin class of antibiotics that includes ampicillin.  Without the cross-linking enzymes, one may expect the cell wall to be fragile, but it isn't.  The strains grow fine in hypotonic medium, which would have caused the bacteria to burst if they had a weak cell wall.  What strengthens the Sphaerochaeta cell wall to keep it intact under physical strain remains a mystery.

Cell wall architecture of Sphaerochaeta pleomorpha viewed by electron microscopy.  OM, outer membrane; PS, periplasmic space; CW, cell wall.  Figure 1d from Ritalahti et al., 2012.

Even though Sphaerochaeta reside in oxygen-poor environments, they don't live alone.  They are members of a close-knit microbial community that includes bacteria of the genus Dehalococcoides, which respire by reducing organic chlorides instead of oxygen.  Dehalococcoides have attracted attention because of their potential for cleaning up groundwater and other sensitive environments contaminated with chlorinated organic compounds, pollutants that originated mainly from past industrial and agricultural activities.  Although the production of these toxic compounds has ceased in many countries, the pollutants persist in the environment and must be detoxified.  This is where Dehalococcoides bacteria may be beneficial.  They obtain energy by anaerobic respiration of chlorinated organic molecules, which strips off the chloride atoms, rendering the compounds nontoxic.

Dehaloccoides bacteria do not grow well on their own unless other members of the microbial community are also present.  This indicates that the other microbes provide something that the Dehalococcoides need for optimal growth.  Sphaerochaeta bacteria extract energy from sugars by fermentation, generating a mixture of waste products that include acetate and H₂.  Dehalococcoides have a strict requirement for acetate as a carbon source, and they must use hydrogen as the electron donor for anaerobic respiration of organic chlorides.  Members of Sphaerochaeta may provide these critical substrates to Dehalococcoides.

S. globosa and S. pleomorpha are the best-characterized nonspiral spirochetes, but they were not the first round spirochetes to be found.  A report from 1992 described a round, cold-loving spirochete recovered from Ace Lake in Antarctica.  This spirochete is a member of the genus Spirochaeta, the closest relative of Sphaerochaeta.  More recently, another round nonmotile spirochete, Spirochaeta coccoides, was isolated from the hindgut of a termite.  Based on its genome sequence, reclassification of Spirochaeta coccoides into the genus Sphaerochaeta was proposed recently.  The residence of nonspiral spirochetes in such diverse environments could mean that they are more widespread than we think.


References

Caro-Quintero, A., Ritalahti, K.M., Cusick, K.D., Loffler, F.E., & Konstantinidis, K.T. (2012). The chimeric genome of Sphaerochaeta: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology mBio, 3 (3) DOI: 10.1128/mBio.00025-12

Ritalahti, K.M., Justicia-Leon, S.D., Cusick, K.D., Ramos-Hernandez, N., Rubin, M., Dornbush, J., & Loffler, F.E. (2011). Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochaetes INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 62 (1), 210-216 DOI: 10.1099/ijs.0.023986-0

Alban P.S., Johnson P.W., & Nelson D.R. (2000). Serum-starvation-induced changes in protein synthesis and morphology of Borrelia burgdorferi. Microbiology (Reading, England), 146 ( Pt 1), 119-127 PMID: 10658658

Franzmann P.D., & Dobson S.J. (1992). Cell wall-less, free-living spirochetes in Antarctica. FEMS microbiology letters, 76 (3), 289-292 PMID: 1385265

Dröge S., Fröhlich J., Radek R., & König H. (2006). Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus. Applied and environmental microbiology, 72 (1), 392-397 PMID: 16391069

Abt, B., Han, C., Scheuner, C., Lu, M., Lapidus, A., Nolan, M., Lucas, S., Hammon, N., Deshpande, S., Cheng, J., Tapia, R., Goodwin, L., Pitluck, S., Liolios, K., Pagani, I., Ivanova, N., Mavromatis, K., Mikhailova, N., Huntemann, M., Pati, A., Chen, A., Palaniappan, K., Land, M., Hauser, L., Brambilla, E., Rohde, M., Spring, S., Gronow, S., Göker, M., Woyke, T., Bristow, J., Eisen, J.A., Markowitz, V., Hugenholtz, P., Kyrpides, N.C., Klenk, H.-P., & Detter, J.C. (2012). Complete genome sequence of the termite hindgut bacterium Spirochaeta coccoides type strain (SPN1T), reclassification in the genus Sphaerochaeta as Sphaerochaeta coccoides comb. nov. and emendations of the family Spirochaetaceae and the genus Sphaerochaet Standards in Genomic Sciences, 6 (2), 194-209 DOI: 10.4056/sigs.2796069

Taş, N., van Eekert, M.H.A., de Vos, W.M., & Smidt, H. (2009). The little bacteria that can - diversity, genomics and ecophysiology of ‘Dehalococcoides’ spp. in contaminated environments Microbial Biotechnology, 3 (4), 389-402 DOI: 10.1111/j.1751-7915.2009.00147.x

The 14th species of the Lyme disease group of Borrelia

The cluster of genetically related Borrelia species that includes the Lyme disease spirochete B. burgdorferi is known in the scientific community as Borrelia burgdorferi sensu lato ("in the wider sense"). Three members of B. burgdorferi sensu lato account for most cases of Lyme disease worldwide. They are B. burgdorferi sensu stricto ("in the stricter sense"), B. garinii, and B. afzelii. Several other species of the cluster are suspected of causing a small number of Lyme disease cases in Europe and Asia.

The United States is home to at least four species of B. burgdorferi sensu lato. They are B. burgdorferi sensu stricto (the only species known to cause Lyme disease in the U.S.), B. bissettii, B. andersonii, and B. californiensis. The discovery of a fifth named U.S. species, christened Borrelia carolinensis, was published in the Journal of Clinical Microbiology earlier this year. The new species hails from South Carolina. Most of the isolates were cultured from cotton mice and eastern wood rats, but one isolate was obtained from an Ixodes minor tick feeding on an eastern wood rat. Whether B. carolinensis is capable of inducing Lyme disease is unknown.

The number of named species of B. burgdorferi sensu lato found worldwide now stands at 14:

• B. burgdorferi sensu stricto
• B. garinii
• B. afzelii
• B. andersonii
• B. bissettii
• B. californiensis
• B. carolinensis
• B. japonica
• B. lusitaniae
• B. sinica
• B. spielmanii
• B. tanukii
• B. turdi
• B. valaisiana

If you read my last post, you will notice that Borrelia lonestari, detected in one case of the Lyme-like illness STARI, is missing from the list. Although STARI clinically resembles a mild form of Lyme disease, genetically B. lonestari is more closely related to the set of Borrelia that causes relapsing fever.

Featured article

Rudenko, N., Golovchenko, M., Grubhoffer, L., and Oliver, J.H. (2009). Borrelia carolinensis sp. nov., a new (14th) member of the Borrelia burgdorferi sensu lato complex from the southeastern region of the United States. Journal of Clinical Microbiology 47(1):134-141. DOI: 10.1128/JCM.01183-08

The twentieth species of Leptospira

A new species of Leptospira was isolated from soil in Johor, Malaysia by researchers at the Universiti Putra Malaysia. The spirochete was dubbed Leptospira kmetyi to honor Emil Kmety, a Slovak bacteriologist who had made numerous contributions to the understanding of the genus Leptospira. L. kmetyi is the twentieth species of Leptospira to be validly published. The sequence of its 16S rRNA gene places L. kmetyi among the "pathogenic" species of Leptospira, as shown in the phylogenetic tree below (Figure 1 from Slack et al., 2009). (Click on the image for a larger version.) Further studies are needed to prove that L. kmetyi is truly pathogenic.

The tree shows that 19 of the species cluster into four major groupings or "clades" within the genus Leptospira as follows:

Pathogenic

• L. borgpetersenii
• L. weilii
• L. alexanderi
• L. santarosai
• L. noguchii
• L. interrogans
• L. kirschneri
• Leptospira genomospecies 1
• L. kmetyi

Novel

• L. wolffii
  

Intermediate

• L. fainei
• L. broomii
• L. inadai

Saprophytic

• Leptospira genomospecies 3
• L. biflexa
• L. wolbachii
• Leptospira genomospecies 4
• Leptospira genomospecies 5
• L. meyeri

The "novel" clade, which currently has L. wolffii as its only member, was first proposed last year in a paper by Slack and colleagues.

The species that was excluded from the phylogenetic analysis is L. licerasiae, which is a recently described member of the intermediate clade (Matthias et al., 2008).

By the way, I have never liked the designation "intermediate" because readers may assume an intermediate pathogenic potential between the pathogenic and saprophytic clades. At least one intermediate member, L. fainei, can cause severe disease, including Weil's syndrome and pulmonary hemorrhage (bleeding of the lungs).

Featured paper

Slack, A.T., Khairani-Bejo, S., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Bahaman, A.R., Craig, S., Harrower, B.J., and Smythe, L.D. (2009). Leptospira kmetyi sp. nov. isolated from an environmental source in Malaysia. International Journal of Systematic and Evolutionary Microbiology 59(4):705-708. DOI: 10.1099/ijs.0.002766-0

Other references

Matthias, M.A., Ricaldi, J.N., Cespedes, M., Diaz, M.M., Galloway, R.L., Saito, M., Steigerwalt, A.G., Patra, K.P., Vidal Ore, C., Gotuzzo, E., Gilman, R.H., Levett, P.N., and Vinetz, J.M. (2008). Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon. PLOS Neglected Tropical Diseases 2(4):e213. DOI: 10.1371/journal.pntd.0000213

Slack, A.T., Kalambaheti, T., Symonds, M.L., Dohnt, M.F., Galloway, R.L., Steigerwalt, A.G., Chaicumpa, W., Bunyaraksyotin, G., Craig, S., Harrower, B.J., and Smythe, L.D. (2008). Leptospira wolffii sp nov., isolated from a human with suspected leptospirosis in Thailand. International Journal of Systematic and Evolutionary Microbiology 58(10):2305-2308. DOI: 10.1099/ijs.0.64947-0