EVOLUTION OF PATHOGENS

by Acad. Vladimir SERGIEV, Director of the Ye. I. Martsinovsky Institute of Medical Parasitology and Tropical Medicine, I. M. Sechenov Moscow Medical Academy, Russian Academy of Medical Sciences (RAMS)

Last year, in 2006, our magazine told you about the selective effect of microorganisms on the evolution of the human genome, and about the impact of pathogens - viruses and bacteria - on the destinies of nations and civilizations. But what about changes in the pathogenic agents themselves? How do they survive in spite of the constant struggle waged against them?

ORIGINATION OF CLONES

Today we have clearly traced the formative course of certain pathogenic agents responsible for mass diseases of man. In many instances the causes of such diseases can be blamed on his social activities. Like, for example, the renaissance of the ТВ bacterium Mycobacterium tuberculosis.

Primarily soil mycobacteria became obligate parasites in the intestine of animals and birds and, consequently, came to be transmitted by the fecal-oral mode among populations of warm-blooded animals and fowl. Man, the Homo sapiens, came in touch with this pathogen about 5 thousand years ago in the process of domestication of cattle, sheep and goats. Consuming milk, Caucasoids developed an ability of lactolysis - that is to digest milk and milk products by means of corresponding enzymes - not only in childhood but also in adult years (unlike representatives of the other races losing this ability in puberty). Indirectly this factor contributed to the emergence of a human-adapted pathogenic species, as seen in the high affinity of DNA contained in the human and bovine types as well as in the fact that from among other ТВ mycobacteria (not counting in M. tuberculosis), man is susceptible only to the bovine pathogen, M. bovis.

Parasitizing in man, the originally animal pathogen mutated and acquired specific characteristics to become what is now known as M. tuberculosis. It is characterized by high virulence and pathogenicity for the new host organism, man, while it has become much less dangerous to animals. Furthermore, it has a unique ability of being transmitted through air (an air-borne and droplet infection spread by infected droplets coughed up by victims of the active disease). This bacterium, however, does not survive in soil - unlike the M. avium (yet another kind of ТВ bacillus), which can live on in the hen-house litter for no less than ten years.

Still another example of this kind: the importation of the rodent musk-rat from Canada to Siberia in the 20th century caused a mutation of the Russian tick-borne encephalitis virus through its adaptation to the hitherto unknown host. This produced the pathogen of Omsk hemorrhagic fever.

Several hypotheses have been suggested on the origin of HIV-1, the human immunodeficiency virus. Epidemi-

Articles in this rubric reflect the authors' opinion. - Ed.

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Trichinella (Trichinella spiralis), caustative agent of trichinosis (trichinelliasis): a - intestinal form (fragment); b - larvae (experimental infestation); c - larvae in mice.

cally this disease is related to Africa's regions where, judging by genomes, similar viruses occur widely in primates. There have been at least three independent cases provoked by this causative agent brought from the chimpanzee to the human population in the Republic of Congo. As to HIV-2, it is a "gift" of the African smoke-colored monkeys mangabeys (Cerocebus) that have passed the pathogen to humans on several occasions.

This list could be continued. But there are also other formative mechanisms for pathogenic agents infecting man. Drug resistance (tolerance) is one, which is largely a result of the targeted selection of microorganisms by irrationally used antibiotics. There is an explicit correlation between the mix of antibiotics on sale and the level of resistance of streptococci (Streptococcus pneumoniae). In fact, the same preparations are used in livestock and poultry farming: worldwide as much as 170 million tons of meat is produced each year.

Pathogens' fitness is also increased by regular immunoprophylaxis. The pressing of mass vaccinations touches off antigenic changes in the populations of causative agents whereby clones not contained in the vaccines start proliferating by and large. Thus, in the 1990s the mass immunization of children with a combination vaccine against pertussis (whooping cough), diphtheria and tetanus sent the pertussis incidence rate up in Australia, the Low Countries, Canada and the United States. A study of the circulating strains showed substantial changes in their characteristics compared with the pre-inoculation period. Here in Russia the causative agent of whooping cough acquired new properties: its "wild" clone of the serotype 1.2.3 - of wide occurrence half a century ago - came to be supplanted in most cases with the serotype 1.0.3, which whipped up the incidence rate since both domestic and foreign vaccines could not handle it.

Also, the appearance of new viral and bacterial clones-unrelated to vaccination - may be responsible for outbursts of epidemics. Such was the case of the first and second pandemic of memingococcic meningitis of the serogroup A, which did not bypass the Soviet Union in 1967 and 1968, and in the early 1980s. In 1995 the clone ST-7 entered the stage to cause much trouble in Chad in 1998 and, a year after, in Sudan as well. This strain stems from the meningococcus, the "culprit" responsible for the pandemics of the 1960s, 1970s and 1980s.

In Brazil more than 40,000 measles cases were registered in 1997, and 42 deaths caused thereby. A B6 genotype virus was isolated from the victims. Since it was also traced in other countries of South America, the epidemic was thought to be of clonal nature.

HIV affecting drug addicts, who inject mind-expanding drugs intravenously, staged its appearance in our country in 1996 and spread apace subsequently. In just five years the number of HIV-infected cases soared dozens of times over; nearly all the victims contracted only one variant of the HIV-1 virus. The level of its genetic variability was a mere 5 percent, which proves that this viral clone got into this country but recently. Incidentally, this clone does not occur among drug addicts elsewhere. Probably it made a beeline for this country right from Africa.

Advance scientific technologies may generate novel problems. Unfortunately. Of late due to the intensive work in the field of transplantology it is feared that the transplantation of cells, organs and tissues of animals (from pigs, primates) to human recipients, combined with the use of immunosuppressive preparations, may pose a risk of contagion with infectious agents - both known and unknown ones - the xenopathogens (foreign pathogens). Even a more dramatic scenario is not ruled out either-an unpredictable epidemic of "xenozoonoses".

Presumably the formation of pathogenic agents infecting man has taken place with the resettlement of obligate parasites from animals to man. The same is true of free-living microorganisms. But the evolution of pathogens does not end there. It is proceeding apace today, what with

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Ankylostoma (Ancylostoma duodenalej, intestinal parasite in man and animals responsible for ankylostomiasis (trismus, lockjaw).

Flatworms of the cestodes class (Cestoidea): a - beef tapeworm (Taeniarhynchus saginatus), a pathogen responsible for taeniarhynchosis (infestation with Taeniorhynchus); b - broad tapeworm (Diphyllobotrium latum), pathogen of diphyllobothriasis.

the attempts to suppress the parasites with chemotherapeutic preparations and vaccines.

Diminished virulence is one of the mechanisms enabling a parasite to beat a host's immune defenses. A pathogen varies and modifies its virulence, and seeks to build up its strength in a host macroorganism and thus ensure its transmission from host to host (for with a low immune barrier there can develop a lethal disease that could kill the pathogen's survival chances, too, and its further proliferation consequently).

The milder clinical picture in course of an epidemic is observed during outbreaks of meningococcal meningitis. In the beginning of an epidemic a large number of acute forms of the disease is registered, with a lethal outcome just a few hours after contamination. Afterwards, especially at the end of the epidemic, such lethalities are few, if any. Supposedly the mollification of clinical symptoms is connected with a lower pathogenicity of circulating viral strains in consequence of their passage through the immune organisms of the victims.

The lower level of pathogenicity is also observed in the poliomyelitis virus. According to molecular data, the propagation of this pathogen under natural conditions and its replication in a host organism results in the accumulation of mutations because of errors in the transcription of RNA molecules. Such defects undermine the viability of the poliovirus. But as a result of natural hybridization, the viability and virulence of this causative agent may go up spasmodically and lead to epidemic outbreaks.

Research workers of our Institute have observed a drop in the virulence of the pathogen responsible for zoonotic cutaneous leishmaniasis (Leishmania major), an infectious skin diseases, when cultivated in nutrient media; in this case the pathogen loses its ability to infect man. None the less its passage through highly susceptible laboratory animals causes an abrupt jump in its pathogenicity when it regains the ability to trigger a pathology in humans. Its virulence is also up in cases of diminished immunity (quite often intestinal worms, or helminths, are responsible for that). Even opportunistic pathogens can invade such an organism. In a similar way, opisthorchiasis (infecting cats and humans consuming river fish; this disease is caused by the Opistorchis, or liver fluke) spurs the incidence of grave forms of tuberculosis badly amenable to medical treatment. In yet another instance, trichinelliasis (or trichinosis, a disease affecting man and animals, and provoked by the roundworm Trichinella) is capable of suppressing transplantational immunity.

Unfavorable factors of the habitation medium may also operate as immunosuppressors. Thus, in the 1990s Dr. Oleg Bukharin (Corresponding Member of the Russian Academy of Medical Sciences, member of the Russian Academy of Sciences), who heads the Institute of Cell and Intracell Symbiosis (RAS Ural Branch, Ohrenburg) and his colleagues obtained a unique result on technogenic hazards. They demonstrated that people living in industrial districts contaminated heavily by chemical pollutants are sitting ducks for pathogenic and toxigenic microorganisms, while such invasions go down farther away from industrial objects.

"NEW" PROPERTIES OF PATHOGENS

In 1985 Dr. Robert Calwell of the United States discovered uncultivable bacterial forms retaining their viability and virulence. It became clear that the current notions concerning the life of pathogens in the ambient medium - notions predicated on bacteriological studies only - are not fully watertight. A seemingly dying parasite actually adapts itself to changed conditions in what is known as adaptive variability. Lapsing into a quiescent state, microorganisms slow down their rate of metabolism and discontinue reproduction. Such quiescent, dormant bacterial forms, including the causative agents of cholera and plague, have been found in bodies of water and soil on endemic (peculiar to a particular people or locality) and enzootic (relative to diseases confined to a definite local-

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ity) territories. Under changed conditions such bacteria revert to their former state and, regaining their pathogenicity, are capable of triggering an epidemic again. Such conservative mechanisms ensure the survival of pathogens in unfavorable interim (interepidemic and interepizootic) periods.

The circulation of an infectious agent in blood is a characteristic feature of many diseases. An organism contracting classical infections (typhoid, dysentery) forms morphologically modified agents that cannot be identified by conventional bacteriological methods. It was formerly believed that L-forms (microbes devoid of the cell wall) and uncultivable forms of bacteria (not detected in standard laboratory cultivation) proliferate in the external medium only. But, as it has been proved now, this process takes place in the host organism; excreted into an ambient environment, the pathogens convert to vegetative forms outside the human body and thus keep the epidemic process alive.

It is common knowledge that microorganisms can associate into communities. For a long time, however, this was believed to be an ultraprimitive form of their anonymous conglomeration without any inner structure - just a huge mass of identical elements. But bacteria can communicate actively. Microbiologists have achieved a breakthrough by discovering the mechanisms implicated in such communication. These are not "dumb weights" at all - they are capable of vigorous interaction. As it has been proved recently, they, like human beings, make the best of the advantages of collective behavior so as to ensure their survival. They do that via low-molecular-weight chemical compounds, as diversified as man's every-day vocabulary.

The phenomenon of such interaction has been dubbed "quorum sensing".* Getting into the extracellular medium, signal molecules activate concrete genes upon reaching a critical concentration. This process of autoinduction is involved in mechanisms responsible for neutralization of antibiotics, formation of biofilms and production of pathogenicity factors in many bacteria. As shown by the latest data, such bacteria also produce signal molecules capable of stimulating, inhibiting or inactivating the pathogenic potential of other microbes. The key factors implicated in the progress of infection are the controlled numerical strength of "quorum sensing" and the aggregate virulence of microorganisms.

Such a peculiar function as the formation of special structures, the "biofilms", is also a sequel to the social behavior of pathogenic bacteria. These films built by pathogens are a major survival strategy in the hostile environment, for enclosed within such films they are protected against antibacterial preparations - antibiotics, bacteriophages and phagocytes. Many human diseases - such as lung mucoviscidosis, tuberculosis, chronic otitis and dental caries - are blamed on the above structures.

A solitary bacterial cell excreting polysaccharides first attaches itself to a solid substrate surface. Dividing, it begets a bacterial colony within a polysaccharide matrix thus formed and gives rise to an orderly community that may take in other bacterial species as well. Tightly attached to the substrate and to one another, the bacteria are also protected by an extracellular polymer matrix they produce. A viable biofilm is composed of cells (15 percent) surrounded by the matrix (85 percent), and this boosts manifold the resistance of the bacterial community, compared with "free lances" outside, to antimicrobial medication.

As shown by German research scientists but recently: by sending signals bacteria can modulate certain physiological processes in a host organism - these interactive mechanisms are termed "cross-talk" (communication border crossings). The "quorum sensing" type not only regulates a broad spectrum of bacterial physiological processes. Autoinductors of bacterial origin produced by the human microflora are capable of setting off a pathophysiological restructuring of some functional systems of a macroorganism.

* See: V. Bondarenko, "Human Microflora: Norm and Pathology". Science in Russia, No. I, 2007. - Ed.

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Causative agents of perilous diseases: a - strain of the bird flu virus (H5N1); b - HIV-1 virus.

Novel data on the inherent organizational complexity of microorganisms have made it possible to decipher certain heretofore unknown phases in the pathogenesis of infectious and ostensibly somatic diseases of man. Further studies into the social behavior of bacteria also pave the way for new antimicrobial preparations.

UNKNOWN PATHOGENS OF KNOWN DISEASES

The author of the present article shares fully the opinion of many infectionists: "All human diseases are of two categories: infectious and absolutely infectious." There have been many conjectures about the infectious character of the overwhelming majority of presumably somatic (bodily) diseases. Way back in the early 20th century the outstanding biologist and pathologist Elie Metchnikoff (Nobel Prize, 1908) wrote this: "There is every reason to suppose that... diabetes mellitus develops under the effect of microbes..." And he continued "... with the further progress of scientific methods it will be possible to detect parasites causing malignant tumors..." Metchnikoff likewise referred to cardiovascular and psychic diseases, among others.

Today ever new facts are coming to light to corroborate the infectious nature of cardiovascular diseases. For one, Chlamydiae are thought to be one of the causative agents, in particular, Chlamydiae pneumoniae. The role of certain helminths (worms) has been proved in the etiology of these diseases. Such pathologies as myocarditis and chronic cardiac insufficiency result from the hematogenic transport (through blood) of ova (eggs) of intestinal flat-worms - trematodes. Thus, the 15 percent death-rate of heart failures in the Philippines is put down to trematode-caused myocarditis. These helminths, also occurring in Russia's Far East, have not been yet studied for their effect on heart pathology.

Allergic myocarditis is one of the most frequent organic pathologies attending trichinelliasis (trichinosis) and most often is a terminal case (the same complication also attends opisthorchiasis). Episodes of trichinosis-caused myocarditis concur with the death of trichinous larvae in the myocardium, and accompany destruction of muscular fibers; simultaneously, specific antigens of the parasite infiltrate tissues. Researchers of our Institute have detected focal infiltrates with an admixture of eosinophils (leukocytes implicated in allergic reactions of the organism) and expiring larvae in the cardiac tissues of lethal trichinous cases; the same result is obtained under experimental conditions as well.

The infectious nature of yet another terrible pathology, the oncological diseases, leaves no doubt either. For instance, the viruses of hepatitis В and С cause liver cancer, and the pathogen of opisthorchiasis also provokes cholangiocarcinoma (malignant cholangioma). Type 16 and 18 papillomaviruses are the frequent cause of cervical and vesical carcinomas. The Epstein-Barr herpes virus is capable of inducing malignancies of the nasopharynx and stomach as well as lymphogranulomatosis; the causative agent of gastric ulcer is a risk factor of adenocarcinoma of the stomach. Certain helminths (worms) are found to be cancerogenic. By the data of the World Health Organization (WHO), as much as 84 percent of some malignancies are of infectious origin.

But there is a paradox in that - what with the heightened "layman's" interest in new infections, something quite important escapes one's notice: that amongst so-called "new infections (listed in periodical WHO bulletins) a great many diseases, formerly thought to be somatic, are actually caused by pathogens identified in recent years.

Conceptually the identification of an infectious pathology depends on the detection of a causative agent, its components or toxins in patients. The pathogen may be in there, in the locus of the pathological process, it may travel throughout the organism to get to a site anatomically remote from the afflicted organ. In the later case toxins or metabolic products excreted by pathogenic microorganisms into the microorganism's liquid media - mostly blood - produce a specific pathogenic effect.

It was the German microbiologist Robert Koch (Nobel Prize, 1905) who, in 1890, first articulated standard requirements for determining the etiological role of an infectious agent responsible for a particular disease. A parasite could be regarded as a causative agent under these conditions: it should show up in each event of a disease and under circumstances leading to pathological changes; this agent should not come up in any other diseases as a random or nonpathogenic one; its pure culture, when administered to man or animals, should cause a malady.

The limitations of these postulates became obvious soon afterwards. Many pathogens were found to be asymptomatic in disease carriers; and many agents identified by direct microscopy could not be cultivated in vitro. For

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ТВ pathogens

Plasmodia (Plasmodia vivaces), malaria pathogens.

example, today a mere 7-to-50 percent from among genuine anaerobes (microorganisms that can exist without atmospheric oxygen) are cultivated this way.

A novel approach is offered by the identification of nucleic acid genomic sequences related to a putative pathogen. Such sequences, present in most infectious pathologies, are detected predominantly in affected organs, and not elsewhere. Upon recovery the number of nucleic acid copies goes down. This is how the hepatitis С virus, responsible for the incidence of this perilous form of hepatitis, was discovered; and this is how the TT virus, likewise causing infectious hepatitis (also known as Botkin's disease) was detected, too.

Many diseases, however, break out when a macroorganism is infested with several pathogens simultaneously. This factor poses problems for correct diagnostication and adequate therapy. Diseases caused by seven and even eight agents at once have been described. Microbial communities thus identified not only point to a grave pathological process, they may also indicate the inadequacy of a treatment strategy. Different species of microorganisms can act in concert (synergy).

Thus, the clinical picture of AIDS (acquired immunodeficiency syndrome) combines several viral infections [hepatitis С (68 percent), hepatitis В (48 percent)], mycotic (66 percent) and bacterial (46 percent)] infections. The presence of HIV stimulates cirrhosis of the liver in chronic hepatitis С cases.

Clinicians are still inadequately informed about the effects of mixed and complementary parasitocenoses. Hence diagnostic errors. Inflammatory diseases of the genital organs of the small pelvis (pelvis minor) should be in most cases diagnosed as monoinfections. If microorganisms - not parasitosis agents (pathogenic protozoa) - are detected in the pathological material, the very fact is blown out of all proportion, so much so that any identified microbe is considered the main causative agent of a disease, while other "accomplices" are overlooked.

Conversely, it may happen when the discovery of antibodies to any microorganism is regarded as proof positive of its etiological role for a particular illness. Both approaches, if carried too far, hamper correct diagnostication.

What with the problems related to identification of even known pathogens isolated from patients down with classical infectious diseases, we can well understand the difficulties in the isolation of an unknown pathogen in a somatic pathology. Such attempts are continued nonetheless. An active search is going on to spot an infectious agent responsible for atherosclerosis. Evidence has been obtained on the probable pathogenic role of Chlamidiae, of the herpes simplex virus and the cytomegalovirus. The etiological role of the 5 В type papillomavirus as a causative agent of psoriasis is likewise postulated. Well-founded suppositions have been made putting down certain psychic disorders to the group of Born's viruses (pathogens of an infectious encephalomyelitis). A method of electron transmission microscopy has made it possible to prove the active implication of pathogenic microorganisms in the development of certain illnesses formerly regarded as noninfectious ones (like neurocirculatory dystonia and juvenile arterial hypertension). Special antibacterial therapy in such cases allows to arrest the illness.

Concluding, may I stress it once again: only identification of a real pathogen will make it possible to put prevention of all diseases on a sound footing and do away with empirical trial and error practices in this important matter.

Illustrations supplied by the author