1. Transcriptional Profiling of the Yersinia pestis Life Cycle

B. Joseph Hinnebusch, Florent Sebbane, and Viveka Vadyvaloo

DNA microarray technology enables a comprehensive, systems biology approach to investigate the microbial gene expression program associated with adaptation to different environments. Monitoring the whole-genome transcriptional response of pathogens within infected tissues has rarely been achieved, but has been possible with Yersinia pestis. The transcriptional profiles of Y. pestis in infective fleas and in the lymph node of rats during bubonic plague were compared to identify important adaptational responses associated with successful colonization of the flea, transmission, and the establishment of disease in the mammal. The differential patterns of gene expression indicate metabolic reprogramming, response to different stresses, and specific induction of virulence and transmission factors as Y. pestis alternates between its two hosts.

2. Yersinia pseudotuberculosis Gene Expression in Plasma

Michael Marceau and Michel Simonet

Yersinia pseudotuberculosis is a septicemic pathogen for rodents and many other animals. However, in certain immunosuppressive conditions, it may also invade the human bloodstream and little is known about the physiological events that take place once the microorganism has entered this compartment. DNA arrays are powerful tools for comparing wide and complex RNA population samples and, therefore, are appropriate to gain insight into the metabolic pathways and virulence factors expressed by the bacterium in this situation. Using such a technology, we compared the overall gene transcription patterns (the transcriptome) of Y. pseudotuberculosis cultured in either human plasma or Luria-Bertani medium. In this chapter, we will try to decipher, and sometimes speculate a little bit, on the physiological events behind the most salient transcriptional regulatory events detected in our experiments, with the intention of making the story as less descriptive as possible. Discordances between our data and those obtained in identical conditions with Y. pestis will be discussed when necessary.

3. Host Transcriptome Responses to Yersinia pestis Infection

Zongmin Du and Ruifu Yang

Yersinia pestis, the etiological agent of plague, is highly virulent and has acquired an ability to transmit among hosts via fleabite. The high bacterial load in the blood meal from terminal septicemic rodents makes the fleabite transmission route feasible. Molecular mechanisms underlying the high virulence and the unique transmission strategy of Y. pestis have not been clearly elucidated till now. Host transcriptomic responses to Y. pestis in host immune cells or in infected tissues have provided valuable insights into the pathogenic mechanisms adopted by this pathogen. It is now established by detailed histopathology analysis and transcriptional profiling studies on infected animal tissues that the immune response is delayed in both bubonic and pneumonic plague animal models, and neutrophil infiltration and proinflammatory cytokine production occur only after the infection progresses into a systemic septicemic stage. This chapter reviews the recent progress in studies on the host transcriptional responses to Y. pestis infection and prospects the future trends that will lead us to go further in probing into the pathogenicity of this pathogen.

4 . Transcriptional Profiling of Yersinia enterocolitica-host Cell Interactions

Reinhard Hoffmann, Ekaterina Lenk, and Jürgen Heesemann

The infection of the host is a complex biological process which prompted infection biologists to pursue reductionist approaches to unravel molecular events of pathogen-host interaction. DNA microarray provides us with a systems biology approach to gain a more holistic picture. Transcriptional profiling of host cell-pathogen interactions results in vast data sets which have to be carefully analyzed by considering the pathogen on a clonal level, the particular cell type (cell lines or primary cells) and the infection model. Here we summarize and discuss the available transcriptional profiling data obtained from Yersinia enterocolitica infection models in relation to the general gene expression program of host cells to microbial infection.

5. Proteome Analysis of Yersinia pestis and the Mammalian Host Response to Y. pestis Infection

Rembert Pieper and Scott N. Peterson

In the last few years, proteomic analyses of Y. pestis have been focused on the identification of proteins derived from in vitro cultures that sought to mimic important aspects of in vivo environments and have provided valuable insight into dynamic abundance profiles of numerous proteins and their subcellular localizations, comparing growth conditions relevant in the context of the flea vector/mammalian host life cycle. A proteomic analysis of Y. pestis under iron starvation conditions revealed marked changes in the utilization of pathways related to energy metabolism, stress response and Fe-S cluster assembly. Virulence-associated factors were often found to be highly expressed only under specific environmental conditions. This included T3SS subunits (such as LcrV and YopD), Pla protease, the phospholipase D2 Ymt, the outer membrane (OM) receptor for iron/yersiniabactin, the β-barrel OM adhesion protein Ail and the capsular F1 antigen. Some of these proteins were also major antigens (Caf1, LcrV, YopD and YopE) although the human immune response appears to target a larger variety of Y. pestis proteins than the aforementioned virulence factors.

6. Yersinia pestis Metabolic Network

Ali Navid and Eivind Almaas

Bubonic plague is one of the deadliest diseases known to man. Unfortunately, despite all of our medical advances, we still do not have a working vaccine against this disease. Worse yet, discovery of anti-microbial resistant strains of Yersinia pestis, the causative agent of plague, could soon render our current therapeutic means ineffective. Unique characteristics of bacterial metabolism constitute one of the primary sets of targets for drug design. Accordingly, metabolism of Y. pestis has been one of the most studied aspects of its physiology.

7. Plague Treatment and Resistance to Antimicrobial Agents

Marc Galimand and Patrice Courvalin

For many years, Yersinia pestis was considered as uniformly susceptible to antimicrobial agents that are active against Gram-negative bacteria. In 1995, the first two multidrug-resistant strains of Y. pestis were identified in Madagascar and shown to contain self-transmissible plasmids, pIP1202 conferring resistance to all antimicrobial agents recommended for plague treatment and prophylaxis and pIP1203 to a smaller array of drugs. Both plasmids could be transferred by conjugation from the source Y. pestis strains to other Y. pestis and Escherichia coli. Plasmid pIP1203 could be transferred from E. coli to Y. pestis in the midgut of co-infected rat fleas, common vectors of plague. Comparative analysis of the DNA sequence of pIP1202 revealed a shared IncA/C plasmid backbone with multidrug resistant plasmids from Salmonella enterica and Yersinia ruckeri suggesting recent acquisition from a common ancestor. In addition, similar IncA/C backbones were detected in numerous multidrug resistant enterobacterial pathogens isolated from retail meat products in the United States as well as in multidrug resistant plasmids from aquatic environments. This bacterial reservoir of mobile resistance determinants, probably widespread globally, has the potential to disseminate to bacterial pathogens, including Y. pestis, and therefore represents a significant public health concern.

8. Enteropathogenic Yersinia: Antibiotic Resistance and Susceptibility of Yersinia enterocolitica and Yersinia pseudotuberculosis

Jeanette N. Pham

Yersinia enterocolitica and Yersinia pseudotuberculosis, the two enteropathogenic species of the genus Yersinia, are poles apart in their natural resistance to β-lactam antibiotics. While Yersinia pseudotuberculosis, as a species, is susceptible to all antibiotics used in the treatment of Gram-negative infections, Y. enterocolitica susceptibility to β-lactam antibiotics varies. Due to the presence of chromosomal β-lactamases, Y. enterocolitica are inherently resistant to ampicillin and cephalothin. The consistent pattern of β-lactam antibiotic susceptibility of each bioserotype and subgroup within a bioserotype is explained by the production or the lack of production of two chromosomally encoded β-lactamases. One is the non inducible broad spectrum enzyme A, a β-lactamase of molecular class A, and the other one is enzyme B, an inducible cephalosporinase of molecular class C. Fluoroquinolones alone or in combination with a third generation cephalosporin are very effective at treating severe infections caused by Y. pseudotuberculosis or Y. enteterocolitica.

9. Acellular Vaccines Against Plague

E. Diane Williamson and Petra C.F. Oyston

In this chapter we review the progress to date on the development of acellular vaccines to protect against plague; we examine the pathogenicity of the causative organism and how an understanding of this has led to the identification of these new vaccine candidates. The approaches available to demonstrate the potential benefit arising from the use of such candidate vaccines in a human population, where vaccine efficacy in man cannot be determined directly, are discussed. The concept of deriving immune correlates of protective efficacy in authentic animal models of the human disease and then identifying surrogate markers of protection for application to clinical trials is discussed, together with an assessment of the prospects ultimately for eventual vaccine licensure. We explore the potential impact of candidate vaccines on disease occurrence, particularly the likelihood of achieving defined sub-unit vaccines which provide prophylaxis against pneumonic plague, since this represents a new capability against the most hazardous form of the disease.

10. Live Vaccines Against Plague and Pseudotuberculosis

Christian E. Demeure

The reemergence of plague in the world, the appearance of antibiotic-resistant strains and the risk that genetically modified Y. pestis could serve as a bioterrorist weapon have fostered a renewed interest for vaccination. Currently, researchers mainly follow two distinct vaccinal strategies: one is the development of acellular sub-unit vaccines based on two recombinant targets (F1 and V), and the other is the development of improved live vaccines. Live plague vaccines have been previously largely used in humans and their efficiency against bubonic plague is not disputed. Rather, critics pointed to a limited duration of protection, an unverified protection against pneumonic plague, instability of vaccine seed strain characteristics and the fact that vaccination could induce severe local and systemic reactions. New live vaccine candidates should combine both the known advantages of replicating vaccines: humoral and cell-mediated immune responses, robustness against mutant microorganisms, easiness of mass production and use, limited cost, etc. whilst providing guarantees in terms of security, stability and efficiency against pneumonic plague. They are based not only on attenuated Y. pestis strains, but also on other Yersiniae and live vectors (Salmonella, viruses) expressing Y. pestis antigens. Vaccines against enteropathogenic Yersiniae are also developed.

11. North American Plague Models of Enzootic Maintenance, Epizootic Spread, and Spatial and Temporal Distributions

Rebecca J. Eisen, Kenneth L. Gage and

Plague is a severe, primarily flea-borne, rodent-associated zoonosis caused by Yersinia pestis. The majority of human infections are associated with epizootic periods that are defined by rapid transmission in rodent populations. In this chapter, we highlight how modeling has been used to integrate field- and laboratory-derived data to provide insights into identifying when and where epizootics are most likely to occur, how Y. pestis is maintained during periods of apparent quiescence, what triggers a transition to the epizootic phase, and the forces that drive plague epizootics. Understanding these dynamics is critically important for targeting limited public health resources for surveillance, prevention and control, informing wildlife management decisions, and anticipating future disease trends in relation to changing climatic and land use patterns.

12 . Surveillance and Control of Plague

Jean-Marc Duplantier

Surveillance focuses primarily on human cases, then on vectors and reservoirs, but it is important not to neglect the bacteria (virulence and resistance to antimicrobial agents). Absence of human cases does not mean that plague does not continue to circulate in rodents. Recent re-emergences show the importance of monitoring historic foci considered as extinct. There is a need for surveillance in shantytowns, mines, refugee camps, etc., even far from known foci. It is essential to improve the declaration of human cases. Specific identification of reservoirs is necessary as their sensitivity or resistance to plague varies. Rodent surveillance should be based mainly by estimating their abundance, flea indexes and Yersinia pestis prevalence. Specific identification of fleas is also essential as the different species have not the same transmission efficiency and the same level of resistance to insecticides. Pest control must focus as a priority on the fleas, but it is imperative to combine rodent and flea control. It is preferable to use anticoagulant rodenticides and dust formulations of the insecticides and to focus their application in burrows or bait boxes. As surveillance is costly, determining indicators easy to collect is essential and modeling of long-time series proved that it is possible.

13. Tracing of Enteropathogenic Yersinia

Maria Fredriksson-Ahomaa

Yersiniosis caused by Y. enterocolitica and Y. pseudotuberculosis is primarily acquired via contaminated food or water. However, several difficulties have been associated with isolating pathogenic Yersinia strains from food and environmental samples. The detection rates of enteropathogenic yersiniae in animal reservoirs, foods and in the environment have been shown to be clearly higher by PCR than by culturing. In epidemiological studies, subtyping of Y. enterocolitica and Y. pseudotuberculosis strains is necessary to identify infection sources and transmission routes. A number of different DNA-based methods have been used in molecular typing of enteropathogenic Yersinia spp. Pulsed-field gel electrophoresis (PFGE) is so far the most frequently used typing method for Y. enterocolitica and Y. pseudotuberculosis because it is one of the most powerful tools to subtype these two pathogens. Pigs have shown to be the main reservoir of human pathogenic Y. enterocolitica strains because of the high prevalence of these strains in the tonsils and the high similarity of pig strains to human strains. However, the transmission routes from pigs to humans, especially to young children, are still unknown. Contaminated fresh produce and drinking water have been demonstrated as important sources of human Y. pseudotuberculosis infections but yet the transmission routes have remained unclear.

14. Surveillance and Control of Enteric Yersinioses

Truls Nesbakken

Yersinia enterocolitica is a zoonotic bacterium that has its main reservoir within the domestic pig population. Accordingly, interventions in the meat chain are essential for protection of consumers against infection with pathogenic Y. enterocolitica. Occurrence of Y. enterocoliticaY. enterocolitica is less frequent in mixed breeding-finishing herds than in fattening herds. Accordingly, purchase of animals from other herds, with an unknown carrier state of Y. enterocolitica, should be avoided. Slaughter hygiene, including enclosure of the anus into a plastic bag after rectum-loosening, and hygienic handling of the head and the plucks during slaughter and dressing is crucial to avoid carcass contamination. Modified atmosphere packaging of meat emphasizes the importance of temperature control during storage at refrigeration temperature. Avoidance of cross-contamination in the kitchen is particularly necessary because Y. enterocolitica is able to propagate at refrigeration temperature. It is important not to drink from raw water supplies that are liable to contamination by animals, and to ensure that drinking water supplies are treated effectively so that Y. enterocolitica are inactivated or eliminated. Some of the preventive measures listed for Y. enterocolitica are also valid for Y. pseudotuberculosis, in particular measures listed regarding vegetables, water, personal and kitchen hygiene and animal contact.