Doi:10.1016/j.tim.2004.07.004

Human intestinal bacteria as reservoirsfor antibiotic resistance genes Abigail A. Salyers, Anamika Gupta and Yanping Wang Department of Microbiology, University of Illinois, Urbana, IL 61801, USA Human intestinal bacteria have many roles in human through the human colon on a regular basis are pathogens health, most of which are beneficial or neutral for the such as Streptococcus pneumoniae and Staphylococcus host. In this review, we explore a more sinister side of aureus; these bacteria are normally found in the nose or intestinal bacteria; their role as traffickers in antibiotic throat but can pass through the colon if swallowed resistance genes. Evidence is accumulating to support Until recently, such bacteria were thought to be transients the hypothesis that intestinal bacteria not only that spent little time in the human colon, but some recent exchange resistance genes among themselves but reports suggest that S. aureus might transiently or might also interact with bacteria that are passing persistently colonize the human colon in low numbers, through the colon, causing these bacteria to acquire especially in hospitalized patients In addition to and transmit antibiotic resistance genes.
such pathogenic transients, there are potentially patho-genic members of the intestinal microflora itself, such as Until recently, bacterial pathogens were the primary focus Escherichia coli, Enterococcus species, Clostridium of studies of antibiotic resistance genes and their spread.
species and Bacteroides species . Is it possible Now, scientists are starting to wonder whether this focus that such diverse bacteria can and do regularly exchange is too narrow. Could the microflora of the human colon, DNA under conditions found in the human colon? normally considered innocuous or beneficial, be playing amore sinister role in human health as reservoirs for Assessing the extent to which resistance gene transfer antibiotic resistance genes? The reservoir hypothesis is depicted in According to this view, human How can the actual extent of resistance gene transfer in intestinal bacteria not only share resistance genes among the human colon be assessed? One approach would be to themselves but can also acquire from or donate resistance feed people resistant animal bacteria, then determine genes to bacteria that are just passing through the whether genes carried by these bacteria enter human intestine . The possibility that resistance gene spread colonic bacteria. This approach has not been taken for two in the human colon might be a serious threat to human obvious reasons. First, such an experiment would be health was first raised in connection with post-surgical considered unethical in most countries. Second, such a infections, which are usually caused by the normal study would be prohibitively expensive, especially in view microflora of the patient or the patient’s caretakers .
of the fact that it is not clear how long the duration of the Recently, concern about resistance gene transfers in the sample collection period should be. Such a study could human colon has expanded to include agriculture alternatively be done in laboratory animals. Notably, There is no question that feeding antibiotics to livestock to enhance an animal’s growth selects for antibiotic resistantbacteria in the animal’s intestine but to what extent are such bacteria a threat to human health? Afterall, farms are located at a considerable distance fromplaces, such as cities, where high concentrations of people are found. Nonetheless, there is a very significant link between farm and city: the food supply. It is now wellestablished that antibiotic resistant bacteria from chick- ens, pigs and cattle enter the food supply and can be foundin meat offered for sale in supermarkets Figure 1. The resistance gene reservoir hypothesis. Bacteria that normally reside inthe human colon, most of which are normally benign, transfer resistance genes If these foods are not properly cooked, the resistant among themselves. This type of transfer becomes a problem if the commensals, bacteria will enter the intestinal tracts of consumers and many of which are opportunistic pathogens, go on to cause post-surgicalinfections. Bacteria that are merely passing through the human colon will be in will have the opportunity to commingle with members of transit through the colon long enough to transfer or acquire genes by conjugation.
the resident human microflora . Also passing These bacteria might return to the sites where they are usually found (e.g. themouth and skin) by contamination of these sites with excreted bacteria. Gene Corresponding author: Abigail A. Salyers ([email protected]).
transfer could be occurring in the mouth, where thick biofilms are found, but here we focus on the colon for simplicity.
0966-842X/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2004.07.004 there have been a few reports of resistance gene transfer subgroup of these strains revealed that all the tetQ genes in laboratory animals. For example, transfer of antibiotic were at least 94% identical and the ermF genes had even resistance genes among enterococci has been demon- fewer sequence differences. The increased carriage of strated in germfree rodents In another study, a these genes over the past three decades indicated that species of Lactococcus was eliminated from the normal horizontal transfer was occurring frequently enough to microflora of rodents and replaced by an antibiotic have spread these genes widely over a time period that is resistant strain of the same species . Transfer of a small in evolutionary terms . In addition, the fact that plasmid carrying an antibiotic resistance gene was the genes were found in people with no recent history of demonstrated in this model. A problem with interpreting antibiotic use (community isolates) indicates that these the results of studies that use laboratory rodents is that genes are maintained stably once they are acquired. This the normal microflora of rodents is different from that of is bad news for those who have accepted as an article of humans. Also, the use of germfree animals eliminates the faith that susceptible strains are inherently more fit than bulk of the microflora that is normally present and focuses resistant ones and that stopping use of an antibiotic would attention on normally minor populations that in the inevitably result in the disappearance of the resistant germfree animal become the predominant bacteria.
strain. Importantly, stably maintained antibiotic resist- Given the problems inherent in such prospective ance genes are fairly common and in many cases resistant studies, a second, retrospective approach has been bacteria hold their own quite well when faced with taken, where DNA sequences were determined for competition with resistant strains .
resistance genes found in different bacteria in the To determine what type of element was transferring human colon or in other sites and were compared. The tetQ, DNA from some of the strains carrying tetQ was assumption is that if genes found in two different bacterial probed with DNA from a conjugative transposon known to species are at least 95% identical, then this gene must carry tetQ, known as CTnDOT. The digest pattern on a have been transferred horizontally by one means or Southern blot was consistent with tetQ being part of a another. The 95% cutoff is somewhat arbitrary, but finding conjugative transposon in the CTnDOT family nearly identical sequences in different species rules out CTnDOT and related conjugative transposons have an the possibility of convergent evolution, where selective unusual feature. Their transfer is stimulated 100–1000 pressures on different bacteria produce proteins that are fold by tetracycline . Therefore, it is possible that virtually identical even though they evolved separately.
the extensive transfer of tetQ, which has occurred during Because selection is for a specific amino acid sequence and the past three decades, was triggered by the use of not for a specific DNA sequence, third base differences enable the DNA sequences that encode identical proteins However, there could be other stimulatory conditions. A surprising finding of this survey was that even during thepre-1970 years, when tetracycline had not yet been widely Getting the goods on intestinal anaerobesIn our studies, we chose to focus on Bacteroides species.
used, the carriage of tetQ was as high as 20–30% ().
Similar to the strains found in the 1990 s, tetQ was carried the human colon Because of their high concen- on a CTnDOT type element in the earlier isolates.
tration, they appear most likely to be involved in Consequently, horizontal gene transfer was occurring horizontal gene transfer events. Bacteroides species even before heavy use of tetracycline was begun .
harbor two types of conjugative elements: conjugative Another surprising outcome of this survey was the plasmids and conjugative transposons .
finding that two other erm genes in addition to ermF – Initially, two resistance genes were monitored in natural ermB and ermG – had moved into Bacteroides isolates of Bacteroides species: tetQ and ermF To species during the period between 1970 and the 1990 s date, tetQ has only been found on conjugative transposons (). These erm genes had previously been associated in Bacteroides spp. The ermF gene has been found on both almost exclusively with Gram-positive bacteria, not with conjugative transposons and conjugative plasmids the Gram-negative Bacteroides species Sub- . A survey of 289 strains, representing more than sequent studies have shown that ermB and ermG are 10 different Bacteroides species, revealed that in the carried on conjugative transposons that are unrelated at period before 1970, 20–30% of isolates carried tetQ, the DNA sequence level to any conjugative transposons whereas ermF was found only rarely. By contrast, in the found in Bacteroides species to date . This raises 1990 s, over 80% of strains carried tetQ and 15% carried the possibility that ermB and ermG have entered ermF () . DNA sequence analysis of a selected Bacteroides species from Gram-positive bacteria.
Table 1. Prevalence of tetQ, ermF, ermG and ermB genes in colonic Bacteroides spp.
aBacteroides isolates from the colon of people who were healthy and did not have a history of recent antibiotic use.
bIsolates from people with Bacteroides infections.
Transactions between major populations of intestinal Research of the sort described in this review has been As already mentioned, Bacteroides species account for controversial because it can be interpreted as support for w20–30% of bacteria isolated from the human colon. Most concerns about possible effects of agricultural use of of the remaining 70–80% of colonic isolates consists of antibiotics on bacteria that cause human infections.
poorly characterized Gram-positive anaerobes. The well- People in the animal agricultural field are quick to point studied facultative species, such as E. coli and the out that currently there is no ‘smoking gun’ linking the enterococci, are numerically minor, constituting less use of antibiotics on the farm with the appearance of than 1% of colonic isolates A question that needs to resistance genes in human pathogens and subsequent be answered is what types of conjugative elements are treatment failures resulting from agriculture-associated found in the Gram-positive anaerobes, and are these resistance gene transfer. It is important to remember, bacteria participating in a significant way in the exchange however, that absence of evidence is not the same as of antibiotic resistance genes among bacteria in the evidence of absence. The diversity of bacteria found in the various microfloras of the human and animal body is The original question raised in this article was whether staggering. Most of these bacteria and the resistance members of the normal flora of the human intestine could genes they carry have not been studied a great deal. The exchange genes with bacterial pathogens that might be fact that conjugative transposons, which were unknown present in low numbers or just passing through the until recently, appear to be a driving force in the transfer intestine. Evidence that such transfers can and do happen of antibiotic resistance genes in the human body is is summarized in where several genes are shown unexpected and illustrates the principle that scientists along with the genera in which they have been found. In know a lot less than they think they do about mechanisms the case of the erm genes, the sequence identity of the of horizontal gene transfer in nature . It is genes found in different species is usually 99% or higher.
important that we establish what selective or stimulatorypressures are driving the spread of antibiotic resistance The ermB gene has been found in a variety of pathogenic genes so that we can assess the relative contributions of Gram-positive bacteria, including Streptococcus pneumo- different types of antibiotic use or other human activities niae and Clostridium perfringens The ermB and to this spread. Only then will it be possible to design ermG genes were found in more than one species, there- effective strategies for preventing further increases in the fore these genes appear to have entered Bacteroides incidence of antibiotic-resistant bacteria.
species more than once in the past One of thefew reports that implicates the human colonic Gram-positive anaerobes in resistance gene transfer is a recent report of vancomycin resistance genes, such as those This article has focused on the transfer of antibiotic found in pathogenic enterococci, in the colonic anaerobe resistance genes in nature, but gene transfer among It is also worth noting that bacteria from different sites appear to be exchanging genes. For example, Porphyro-monas gingivalis is an oral anaerobe and Prevotella ruminicola is normally found in the rumen of cattle and the intestines of pigs. It is easy to imagine howP. gingivalis might be involved in gene transfers because it is constantly being swallowed, and strains from the colon could be reintroduced into the mouth by the fecal- Streptococcus spp.
Clostridia spp.
oral route. How a rumen anaerobe, which is very sensitiveto oxygen, made contact with human colonic bacteria (if that is where P. ruminicola picked up tetQ) and then made it back into the animal is harder to imagine.
A caveat is in order. The strategy of using virtually identical genes found in different genera and species to deduce that there is some genetic conduit open betweenthose species has a couple of limitations. First, it is usually not possible to ascertain the direction of the transfer. The only reason we feel comfortable about saying that ermG and ermB appear to have entered Bacteroides species from some other species of bacteria is that the pre-1970 strainsdid not carry these genes. A second limitation is that there Figure 2. Evidence that transfer of resistance genes occurs between Gram-positive is no way to ascertain how many transfers it took for a and Gram-negative bacteria in the mammalian colon and in other environmentalsites. This evidence consists of finding virtually identical resistance genes in natural gene to move between two of the species shown in .
isolates representing different bacterial species. The resistance gene is contained in Moreover, there are almost certainly unknown players, the oval that connects the Gram-positive (left boxes) and Gram-negative (right such as the Gram positive colonic anaerobes and even soil boxes) bacterial species, in which virtually identical copies of the resistance genehave been found. Examples of possible transfer events are shown here, however, this is not meant as a complete listing of all cases found in the literature.
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