PhD-student in Medical Microbiology, Lund University, Sweden
When the bacteriologist and "accidental" discoverer of penicillin Alexander Fleming held his Nobel Lecture on December 11th, 1945 (17 years after his revolutionary discovery and only a few years removed from the commercialization of penicillin for clinical use), he warned:
But I would like to sound one note of warning /…/ It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them, and the same thing has occasionally happened in the body. The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant.
Antibiotics, which are substances that are anti-bacterial, have existed in nature long before man discovered them. Most clinically used antibiotics are originally isolated from bacteria and fungi that secrete these substances in their environment to kill microbial competitors in order to gain a fitness advantage (they are themselves resistant to the compounds). Naturally, these processes have historically occurred outside of the human host (we are not the center of the universe, no matter what journalists think). Primarily, soil bacteria such as Streptomyces are extremely proficient at producing a plethora of antibacterial substances. Competitors in the soil that are unable to cope with the antibiotics die quickly, whereas other microorganisms that develop protection against them, or resistance, survive. Antibiotics and antibiotic resistance have thus historically been components of microbial territorial wars, long before they played a role in the human world.
Evolutionary, resistance mechanisms have their roots in other cellular processes. For example, a transportation pump originally involved in the export of unwanted material out of the bacterial cell can mutate to also pump out antibiotics. An enzyme that is normally involved in the degradation of carbohydrates may mutate so that it can break down antibiotics. In an evolutionary cat-and-mouse game, the winner is whomever manages to generate the most advantageous properties, such as antibiotic resistance where there are antibiotics.
The human bacterial flora, i.e. the bacterial population that inhabits our body, is massive. It consists of over 10 000 different species, and for each human cell in our body, we carry ten bacterial cells. Today, almost all pathogenic bacteria, including the opportunists that belong to the normal flora, harbor one or more antibiotic resistance genes, giving rise to clinically increasingly difficult-to-treat infections. Resistance genes spread among different species, especially when you expose the flora to unnecessary selection pressure by using antibiotics when not needed. Several scientifically relevant questions arise regarding resistance mechanisms: how did these genes end up in human pathogens (which basically were not subjected to antibiotic pressure before the 1900s)?
Three different hypotheses are plausible. When human pathogens began to be exposed to the man-made antibiotics sometime around 80 years ago, they either 1) developed resistance mechanisms from scratch (in analogy with soil bacteria) or 2) managed to acquire antibiotic resistance genes from environmental bacteria through different processes. From there, pathogens spread the resistance genes among themselves inside the human host. The third option is that 3) human pathogens have had antibiotic resistance genes for a long time but they were first noticed when antibiotic selection pressure was applied upon them.
The answers to these questions, however, have eluded researchers due to: how does one examine genetic material from our since long dead bacterial flora? It is not possible to investigate bacteria from 100 years, 200 years, 500 years or 1000 years ago, right? Surely, you cannot? You cannot!
Well, you can. Enter: ancient DNA (aDNA) technology. Using highly sensitive methods, rigorous contamination avoidance measures and powerful calculation software, researchers can reconstruct parts of the genetic content found on ancient archaeological findings. Especially dental plaque has proven to be a rich source of reasonably intact DNA suitable for aDNA extraction. In addition, investigators have recently discovered that viral DNA can be detected in up to 1500-year-old samples.
Parenthesis: it is completely imperative to avoid contamination from "current" environmental bacteria when performing these analyses, which is much more difficult than you would think as germs are everywhere. Just read the Materials & Methods from one aDNA study showing the type of precautions necessary for these studies:
In Munich the pre-PCR DNA analyses, including the decontamination procedure, DNA extraction, and assembly of the reactions for PCR amplification; were carried out in the new aDNA laboratories at the ArchaeoBioCenter (Ludwig-Maximillians-University, Munich). This aDNA laboratory is located several kilometers from the laboratory used for the post PCR analyses, which included the actual amplification process and sequencing; the post PCR laboratory is situated at the Bundeswehr Institute of Microbiology in Munich. Movement of samples between the laboratories was always unidirectional: from the aDNA laboratories to the post PCR laboratory. The pre-PCR laboratories are dedicated solely to aDNA analysis and have specialized equipment, such as airlocks, HEPA filtered air, positive air pressure, and UV air flow cleaner. In addition, extensive cleaning protocols using bleach and UV irradiation are in place. All possible further methodological precautions were also taken, such as mock extractions, PCR blanks, and independent replications of extractions and amplifications.
So, what have investigators found in their aDNA laboratories?
A recent study published in Nature Genetics examined dental plaque in four skeletons dated to 950-1200 AD from a monastery graveyard in Germany. In addition to mapping the oral flora and the medieval human's diet (cabbage, pork and bread, FYI, i.e. German), the researchers examined the prevalence of different pathogens in the oral cavity, the presence of bacterial virulence proteins and human immune proteins, AND made a total genomic reconstruction of the periodontal pathogen Tannerella forsythia. Furthermore, they investigated the prevalence of antibiotic resistance genes (read those sentences again to form an estimation of the amount of work that went into this article -> 35 authors and supplementary material of 102 pages). The authors could detect resistance genes to aminoglycosides, β-lactams, bacitracin, bacteriocin and macrolides in the fossilized calculus, showing that antibiotic resistance genes were present in the bacterial flora of humans 1000 years before we began using antibiotics clinically! Moreover, precursors to many efflux pumps currently involved in antibiotic resistance were found. But since the human oral flora was rarely, if ever, exposed to antibiotics during the Middle Ages, the researchers state that the purpose and function of these genes in the ancient bacteria remains unclear.
But microbiologists are not only fond of medieval dental plaque. Recently French scientists threw themselves over a fossilized turd that was found in a sealed vessel from the 14th century, and therefore had escaped contamination from the outside world for about 700 years. Using aDNA, they managed to determine the total viral DNA in the coprolite (as no one wants to openly admit that they work with dried turds, they rebrand the fecal matter to coprolite). The interesting findings are that they observed a resistance gene for chloramphenicol as well as viral DNA from many bacteriophages (i.e., viruses that infect bacteria and not humans) that usually infect environmental bacteria Streptomyces. Chloramphenicol is a potent antibiotic produced by Streptomyces and to which it is resistant against. Sometimes when phages infect bacteria, they accidentally bring some of the bacterial DNA with them when they kill the cell and spread on. These genes can then be transferred to other bacteria that in turn are infected by the same virus, that is; phages can act as vectors for the exchange of genetic material between different bacteria. Now, if the 1300-century creator of the microbiologically-valuable coprolite managed to ingest some bacteriophages (while eating or drinking), which first infected Streptomyces and accidentally incorporated the bacterial chloramphenicol resistance gene in its own DNA, this virus may have acted as a reservoir for the spread of this gene inside the human flora. This could serve as one reason as to why the medieval human bacterial flora harbored resistance genes without being subjected to selection pressure. These genes may have been expressed at very low levels or perhaps not expressed at all.
aDNA can also be used for other purposes than to study the origin of antibiotic resistance. It can for instance be used to settle disputes, such as, let's say, two schools of researchers arguing about the aetiological agent of the 6th century Justinianic plague. Then it might so happen that one of them gets a hold of skeletons with dental plaque belonging to plague victims from the ancient Roman Empire's heyday, analyze the genetic content via aDNA and present strong evidence of excessive presence of the bacteria Yersinia pestis in these skeletons. To really twist the knife around, they might even make a phylogenetic tree and determine the bacterial origin to Asia. So thanks to aDNA technology, and researchers' persistent efforts to prove who the best is, nana nana na na, we now know much more about the 6th century Justinianic plague's origin and epidemiology.
When Alexander Fleming thus warned of antibiotic resistance in human pathogens in 1945 he probably did not know that he was dealing with forces that had bubbled beneath the surface for centuries, perhaps millennia. Nevertheless, it must be said that however academically interesting the origin of antibiotic resistance is, there is no doubt the current clinical manifestation of antibiotic resistant pathogens are a direct result of man's overuse of antibiotics in medicine and agriculture. For perhaps the basis for these mechanisms have existed in our normal flora for a long time, but they never provided any fitness advantage to the bacteria until we applied a colossally massive selection pressure on them. And should we fail to effectively deal with the alarming rise of antibiotic resistant pathogens, we will soon find ourselves back in the times of plagues, and perhaps it will be our dental plaques that scientists analyze for resistance genes 1400 years from now.
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