Category Archives: genetics

Microbial Forensics of a Natural Pneumonic Plague Outbreak

For bioterrorism agents like Yersinia pestis it is necessary to identify the strain and its source specifically enough for forensic use. Categorizing an epidemic isolate and tracing its source is always important for public health measures, but the level of precision is far higher for legal uses. Developing forensic techniques to characterize and parse very similar strains of a species and trace it to a specific location robs terrorists (and states) of the ability to deny responsibility for an attack (Koblentz & Tucker, 2010). The ability to launch a secret and deniable attack on an enemy has been viewed as one of attractive advantages of biological warfare.

A Chinese group led by Ruifu Yang and Yujun Cui recognized that only whole genome sequencing could adequately parse the strains of the monomorphic species Yersinia pestis but that the computing power necessary to compare entire genomic sequences as the database enlarges is impractical (Yan et al, 2014). Unlike most pathogens, typing only specific regions of the genome are just not enough to get a unique genetic fingerprint for low genetic variability pathogens like Yersinia pestis. This is yet another indication of the genomic similarity of all Yersinia pestis strains.

The Chinese group developed a two stage method of classification detailed enough for forensic work.  They took a twelve person outbreak of pneumonic plague contracted from a dog in 2009 in the Qinghai area of Tibet / western China, specifically at Xinghai as their test case (Wang et al, 2010). In the first step they took six cases including the two dogs who died in the outbreak and compared them to 24 strains representing the 23 phylogroups of the phylogenetic tree. This comparison selected which branch of the phylogenetic tree the outbreak belonged. There were no SNP (single nucleotide polymorphisms) different between the seven isolates confirming a common source, one of the dogs based on outbreak narratives. The seven isolates were all the same strain belonging to branch 1.IN2 of the tree. The second step was to then compare the isolates to all known strains of 1.IN2 shown below. Since these strains all come from the Qinghai-Tibetan plateau, they were able to add other strains historically isolated from this region.

Distribution of 1.IN in Qinghai  (site source)
Distribution of 1.IN2 in Qinghai (Yan et al, 2014, click to enlarge)

The results localized the new isolates (r) as being from the same focus as strains g, r, s, t. u plus, interestingly, the 0.PE7 strain (green b) that is over 300 SNPs different from the 1.IN2 strains. All of these other strains from this branch are scattered around the Qinghai region near Lake Qinghai. The polysomy (branch point) that produced all of the 1.IN2 in Xinghai (g,r,s,t,u) is located closer to the eastern end of Lake Qinghai, where the Chinese team hypothesizes this these strains began. The new outbreak isolates did not match any previous isolates from Xinghai which is testimony to the degree of movement of these strains around the region. Without the case narrative, they would not have been able to identify the specific foci at Xinghai, but would have got it to the region of east Qinghai lake. This illustrates how important sampling all of these foci are because a biological attack is likely to be far from its site of environmental isolation. Characterization of all laboratory strains, obviously, needs to happen as well for forensic tracing.

Reconstructing the historical epidemiology of this region will be an area of continuing research. The location of 0.PE7, the most genetically ancestral strain ever found — the closest the common ancestor of all Yersinia pestis, plus the likelihood that the ‘big bang’ epidemic (or epizootic), that produced the third pandemic, represented by node 12, was also in this region. (Each of the nodes represents a bang of evolutionary diversity, with all major branch points in the lineage probably representing large epidemics or epizootics.) The full diversity of strains in this region (unrelated to the outbreak isolates) are not shown in the figure above. This same group lead by Ruifu Yang  produced the primary phylogenetic tree of Yersinia pestis in China that noted that the molecular clock is not constant (Cui et al, 2012), here calculates that N12 is about 212 years old (95% confidence being 116 to 336 years ago) (Yan et al, 2014).  They note that in the history of Qinghai, there was a major human outbreak in the year 1754 CE linked to a Buddhist missionary working in Qinghai and Gansu provinces (Yan et al, 2014). Its is unclear if we can trust this narrative at all; scapegoats are common in plague narratives. Linking the 1.IN2 strains from Qinghai to four of the five o.IN2 isolates from Tibet suggest that the epidemic moved from Qinghai to Tibet in one ancient epidemic, though remaining isolate from Tibet looks like a more recent transmission from Qinghai. Regardless of the movements of 1.IN2, this area is believed to have been a site of long-term survival of Yersinia pestis, potentially over a thousand years, so that it has a lot to teach us about enduring foci.

Microbial forensics has already been used in criminal investigations, court cases and intelligence operations, such as the ‘Amerithrax’ (anthrax) attacks of 2001, anthrax spores sprayed over Japan by a cult, and suspicious plague cases in New York City (Yan et al, 2014). Phylogenetic microbial forensics was successfully used to show the intentional transmission of HIV from Dr Richard Schmidt to his girlfriend in his 1998 trial. This was the first successful use of microbial forensics in a court case (Koblentz & Tucker, 2010). In these cases, isolates are taken from the accused, the victim, other sexual partners, and the local population so show phylogenetic linkage between the accused and victim in the context of the local epidemiology.  The United States, United Kingdom, Sweden, the Netherlands, Japan, Canada, Germany, Australia, Singapore, and now China are involved in the development of microbial forensics (Koblentz & Tucker, 2010; Yan et al, 2014).

Reference

Koblentz, G. D., & Tucker, J. B. (2010). Tracing an Attack: The Promise and Pitfalls of Microbial Forensics. Survival, 52(1), 159–186. doi:10.1080/00396331003612521

Yan Y, Wang H, Li D, Yang X, Wang Z, et al. (2014) Two-Step Source Tracing Strategy of Yersinia pestis and Its Historical Epidemiology in a Specific Region. PLoS ONE 9(1): e85374. doi:10.1371/journal.pone.0085374

Wang, H., Cui, Y., Wang, Z., Wang, X., Guo, Z., Yan, Y., et al. (2010). A Dog-Associated Primary Pneumonic Plague in Qinghai Province, China. Clinical Infectious Diseases, 52(2), 185–190. doi:10.1093/cid/ciq107

Cui, Y., Yu, C., Yan, Y., Li, D., Li, Y., Jombart, T., et al. (2012). Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proceedings of the National Academy of Sciences, 110(2), 577–582. doi:10.1073/pnas.1205750110/-/DCSupplemental/sd01.xls

Historians Chronicling Plague Genetic Discoveries

After my last post critiquing Cohn’s scientific interpretations, I think its only fair to write about all the historians who are actively engaging and incorporating scientific findings in their work. I’ve communicated with a lot of historians who are following the scientific work on the plague and I know there will be some articles and books coming out over the next year or so that incorporate some of new genetics in historical analysis.

So for science folks, these two articles give us some insight into how historians see plague genetics unfolding. Little concentrates on the early drama over plague genetics. Bolton covers that material also, but also looks at newer information on transmission dynamics too.

Little, L. K. (2011). Plague Historians in Lab Coats. Past & Present, 213(1), 267–290. doi:10.1093/pastj/gtr014

Bolton, J.L. ‘Looking for Yersinia pestis: scientists, historians and the Black Death’ in L. Clark and C. Rawcliffe (eds.), Society in an Age of Plague, The Fifteenth Century XII (Woodbridge: Boydell, 2013), publication date 15 August 2013, ISBN 9781843838753. (In the same book/issue as Cohn’s paper discussed in the last post.)

Overall, I am really optimistic about the interdisciplinary work that can be done on the plague.

Antibiotic Resistance, Agriculture, and the Plague

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Antibiotics have ended the uncontrollable outbreaks of plague in humans that stalked our ancestors. Today, outbreaks are usually snuffed out after a couple of cases with antibiotic treatment of patients, prophylactic treatment of contacts and vector control. Our greatest risks from plague today are a pneumonic plague outbreak/attack and the emergence of antibiotic resistance.

Beginning in 1995 several antibiotic resistant strains of Yersinia pestis emerged in Madagascar. Two strains isolated from different districts of Madagascar in 1995 were resistant to multiple antibiotics (1). Genetic analysis shows that not only are the resistance plasmids of different derivation but the ribotypes of the Yersina pestis are also different. There is no discernible connection between the development of these two strains. Strain 17/95 is resistant to multiple drugs including all of the antibiotics used to treat plague and for prophylaxis (1). The second strain isolated that year, strain 16/95, is resistant to Streptomycin the primary drug of choice but vulnerable to other drugs commonly used to treat plague. From 1996 to 1998, several more Y. pestis strains each resistant to a single antibiotic were also isolated in Madagascar in humans, rats and fleas (1).

Like the majority of microbes with antibiotic resistance, Y. pestis gained these plasmids by conjugation, a type of lateral gene transfer.  Experiments have shown that Y. pestis readily exchanges antibiotic resistance plasmids with E. coli and other species in the flea gut (1). This is believed to be the primary site where Y. pestis interacts with other plasmid bearing bacterial species. The plasmid isolated from Strain 17/95 (IP275) in 1995 has been identified as “nearly identical” to multi-drug resistant (MDR) plasmids isolated from Salmonella enterica (ser. Newport) and Yersinia ruckeri, a fish pathogen and with a similar core to MDR plasmids found in E. coli, Klebsiella species, and multiple serotypes of Salmonella isolated from agricultural products (2). These plasmids have a common plasmid backbone categorized as IncA/C.

Past interaction between Salmonella enterica and Yersinia pestis has been known for a long time. The Yersinia pestis plasmid pFra is 97% identical to the exclusively human pathogen Salmonella enterica ser. typhi’s cryptic pHMC2 plasmid (2). The pFra plasmid contains the F1 capsule protein and the murine toxin, important virulence factors. The location of transfer between Yersinia pestis and the human only Salmonella enterica ser. typhi has not been determined (2, 4).

Antibiotic resistant plasmids linked to IP275 have been found in many agricultural products — in livestock, grocery stores, and ill humans (2). It is not hard to imagine flea mediated transfer of an antibiotic resistant pathogen from livestock to rats and Yersinia pestis picking up the resistance plasmid where-ever co-infection is occurring.  Antibiotic resistance in livestock enteric pathogens must now be considered a risk for not only Y. pestis but also other similar zoonotic pathogens (2). Rats feeding on livestock feed containing antibiotics would set up a situation that favors the retention of the antibiotic resistance plasmids.

In an effort to see how wide-spread the problem is, an antibiotic resistance survey was conducted on 392 isolates Y. pestis from 17 countries (3). The survey was conducted for all eight antimicrobials commonly used for treatment or prophylaxis. The good news is that they found no resistance in human, animal or flea isolates (3). However, the survey was not as wide-spread as it sounds on the surface. The United States, Uganda and Madagascar contributed 84% of the 229 human cases, and the United States alone contributed 81.7% of animal cases and 92% of the flea isolates (3).  Known antibiotic resistant Y. pestis strains could not be included due to restricted access (3). Isolates from central Asia are meager: four each from China and India, three from Nepal, one from Iran, and eight from Kazakhstan; most collected in the 1960s or earlier (3).  Of the 229 human cases, 32 died and 10 of these had been treated with antibiotics that the strains were shown to be susceptible (3). This gives a 14% fatality rate overall with approximately a third of the deaths occurring despite antibiotic treatment. These survey results make it all the more important that we understand what happened in Madagascar in the 1990s.

Surveillance for antibiotic resistance should now be standard on all Y. pestis isolates.  Once we could begin with the belief that antibiotic resistant plague was an act of biological warfare but no longer. Active monitoring is, as always, the key, now with special attention to areas where livestock and plague foci overlap.

 

References

  1. Galimand, M. ,  Carniel, E.  and Courvalin, P. Resistance of Yersinia pestis to Antimicrobial Agents. Antimicrob. Agents Chemother. 2006, 50(10):3233. DOI:10.1128/AAC.00306-06 [Mini-review]
  2. Welch TJ, Fricke WF, McDermott PF, White DG, Rosso ML, Rasko DA, Mammel MK, Eppinger M, Rosovitz MJ, Wagner D, Rahalison L, Leclerc JE, Hinshaw JM, Lindler LE, Cebula TA, Carniel E, & Ravel J (2007). Multiple antimicrobial resistance in plague: an emerging public health risk. PloS one, 2 (3) PMID: 17375195
  3. Urich SK, Chalcraft L, Schriefer ME, Yockey BM, & Petersen JM (2012). Lack of antimicrobial resistance in Yersinia pestis isolates from 17 countries in the Americas, Africa, and Asia. Antimicrobial agents and chemotherapy, 56 (1), 555-8 PMID: 22024826
  4. Prentice MB, James KD, Parkhill J, Baker SG, Stevens K, Simmonds MN, Mungall KL, Churcher C, Oyston PC, Titball RW, Wren BW, Wain J, Pickard D, Hien TT, Farrar JJ, & Dougan G (2001). Yersinia pestis pFra shows biovar-specific differences and recent common ancestry with a Salmonella enterica serovar Typhi plasmid. Journal of bacteriology, 183 (8), 2586-94 PMID: 11274119