Clustered, Regularly Interspaced Short Palindromic Repeats (CRISPR) abound in the genomes of almost all archaebacteria and nearly half the eubacteria sequenced. Through a genetic interference mechanism, bacteria with CRISPR regions carrying copies of the DNA of previously encountered phage and plasmids abort the replication of phage and plasmids with these sequences. Thus it would seem that protection against infecting phage and plasmids is the selection pressure responsible for establishing and maintaining CRISPR in bacterial populations. But is it? To address this question and provide a framework and hypotheses for the experimental study of the ecology and evolution of CRISPR, I use mathematical models of the population dynamics of CRISPR-encoding bacteria with lytic phage and conjugative plasmids. The results of the numerical (computer simulation) analysis of the properties of these models with parameters in the ranges estimated for Escherichia coli and its phage and conjugative plasmids indicate: (1) In the presence of lytic phage there are broad conditions where bacteria with CRISPR-mediated immunity will have an advantage in competition with non-CRISPR bacteria with otherwise higher Malthusian fitness. (2) These conditions for the existence of CRISPR are narrower when there is envelope resistance to the phage. (3) While there are situations where CRISPR-mediated immunity can provide bacteria an advantage in competition with higher Malthusian fitness bacteria bearing deleterious conjugative plasmids, the conditions for this to obtain are relatively narrow and the intensity of selection favoring CRISPR weak. The parameters of these models can be independently estimated, the assumption behind their construction validated, and the hypotheses generated from the analysis of their properties tested in experimental populations of bacteria with lytic phage and conjugative plasmids. I suggest protocols for estimating these parameters and outline the design of experiments to evaluate the validity of these models and test these hypotheses.

In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination—broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT)—plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR) to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1) the contribution of HGR to the rate of adaptive evolution in these populations and (2) the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1) HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2) once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent invasion of non-recombining populations, even when recombination engenders a modest fitness cost; and (3) because of the density- and frequency-dependent nature of HGR in bacteria, this capacity to increase rates of adaptive evolution is not sufficient as a selective force to provide a recombining population a selective advantage when it is rare. Under realistic conditions, homologous gene recombination will increase the rate of adaptive evolution in bacterial populations and, once established, selection for higher rates of evolution will promote the maintenance of bacteria-encoded mechanisms for HGR. On the other hand, increasing rates of adaptive evolution by HGR is unlikely to be the sole or even a dominant selective pressure responsible for the original evolution of transformation.

Background: Using mathematical deterministic models of the epidemiology of hospital-acquired infections and antibiotic resistance, it has been shown that the rates of hospital-acquired bacterial infection and frequency of antibiotic infections can be reduced by (i) restricting the admission of patients colonized with resistant bacteria, (ii) increasing the rate of turnover of patients, (iii) reducing transmission by infection control measures, and (iv) the use of second-line drugs for which there is no resistance. In an effort to explore the generality and robustness of the predictions of these deterministic models to the real world of hospitals, where there is variation in all of the factors contributing to the incidence of infection, we developed and used a stochastic model of the epidemiology of hospital-acquired infections and resistance. In our analysis of the properties of this model we give particular consideration different regimes of using second-line drugs in this process. Methods: We developed a simple model that describes the transmission of drug-sensitive and drug-resistant bacteria in a small hospital. Colonized patients may be treated with a standard drug, for which there is some resistance, and with a second-line drug, for which there is no resistance. We then ran deterministic and stochastic simulation programs, based on this model, to predict the effectiveness of various treatment strategies. Results: The results of the analysis using our stochastic model support the predictions of the deterministic models; not only will the implementation of any of the above listed measures substantially reduce the incidences of hospital-acquired infections and the frequency of resistance, the effects of their implementation should be seen in months rather than the years or decades anticipated to control resistance in open communities. How effectively and how rapidly the application of second-line drugs will contribute to the decline in the frequency of resistance to the first-line drugs depends on how these drugs are administered. The earlier the switch to second-line drugs, the more effective this protocol will be. Switching to second-line drugs at random is more effective than switching after a defined period or only after there is direct evidence that the patient is colonized with bacteria resistant to the first antibiotic. Conclusions: The incidence of hospital-acquired bacterial infections and frequencies of antibiotic resistant bacteria can be markedly and rapidly reduced by different readily implemented procedures. The efficacy using second line drugs to achieve these ends depends on the protocol used for their administration.

Background In 1982 Smith and Huggins showed that bacteriophages could be at least as effective as antibiotics in preventing mortality from experimental infections with a capsulated E. coli (K1) in mice. Phages that required the K1 capsule for infection were more effective than phages that did not require this capsule, but the efficacies of phages and antibiotics in preventing mortality both declined with time between infection and treatment, becoming virtually ineffective within 16 hours. Results We develop quantitative microbiological procedures that (1) explore the in vivo processes responsible for the efficacy of phage and antibiotic treatment protocols in experimental infections (the Resistance Competition Assay, or RCA), and (2) survey the therapeutic potential of phages in vitro (the Phage Replication Assay or PRA). We illustrate the application and utility of these methods in a repetition of Smith and Huggins' experiments, using the E. coli K1 mouse thigh infection model, and applying treatments of phages or streptomycin. Conclusions 1) The Smith and Huggins phage and antibiotic therapy results are quantitatively and qualitatively robust. (2) Our RCA values reflect the microbiological efficacies of the different phages and of streptomycin in preventing mortality, and reflect the decline in their efficacy with a delay in treatment. These results show specifically that bacteria become refractory to treatment over the term of infection. (3) The K1-specific and non-specific phages had similar replication rates on bacteria grown in broth (based on the PRA), but the K1-specific phage had markedly greater replication rates in mouse serum.

Background: The first step in invasive disease caused by the normally commensal bacteria Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae is their colonization of the nasal passages. For any population to colonize a new habitat it is necessary for it to be able to compete with the existing organisms and evade predation. In the case of colonization of these species the competition is between strains of the same and different species of bacteria and the predation is mediated by the host's immune response. Here, we use a neonatal rat model to explore these elements of the ecology of nasal colonization by these occasionally invasive bacteria. Results: When neonatal rats are colonized by any one of these species the density of bacteria in the nasal passage rapidly reaches a steady-state density that is species-specific but independent of inoculum size. When novel populations of H. influenzae and S. pneumoniae are introduced into the nasal passages of neonatal rats with established populations of the same species, residents and invaders coexisted. However, this was not the case for S. aureus - the established population inhibited invasion of new S. aureus populations. In mixed-species introductions, S. aureus or S. pneumoniae facilitated the invasion of another H. influenzae population; for other pairs the interaction was antagonistic and immune-mediated. For example, under some conditions H. influenzae promoted an immune response which limited the invasion of S. pneumoniae. Conclusions: Nasal colonization is a dynamic process with turnover of new strains and new species. These results suggest that multiple strains of either H. influenzae or S. pneumoniae can coexist; in contrast, S. aureus strains require a host to have no other S. aureus present to colonize. Levels of colonization (and hence the possible risk of invasive disease) by H. influenzae are increased in hosts pre-colonized with either S. aureus or S. pneumoniae.

Agents that kill or induce suicide in the organisms that produce them or other individuals of the same genotype are intriguing puzzles for ecologists and evolutionary biologists. When those organisms are pathogenic bacteria, these suicidal toxins have the added appeal as candidates for the development of narrow spectrum antibiotics to kill the pathogens that produce them. We show that when clinical as well as laboratory strains of Streptococcus pneumoniae are maintained in continuous culture (chemostats), their densities oscillate by as much as five orders of magnitude with an apparently constant period. This dynamic, which is unanticipated for single clones of bacteria in chemostats, can be attributed to population-wide die-offs and recoveries. Using a combination of mathematical models and experiments with S. pneumoniae, we present evidence that these die-offs can be attributed to the autocatalytic production of a toxin that lyses or induces autolysis in members of the clone that produces it. This toxin, which our evidence indicates is a protein, appears to be novel; S. pneumoniae genetic constructs knocked out for lytA and other genes coding for known candidates for this agent oscillate in chemostat culture. Since this toxin lyses different strains of S. pneumoniae as well as other closely related species of Streptococcus, we propose that its ecological role is as an allelopathic agent. Using a mathematical model, we explore the conditions under which toxins that kill members of the same clone that produces them can prevent established populations from invasion by different strains of the same or other species. We postulate that the production of the toxin observed here as well as other bacteria-produced toxins that kill members of the same genotype, ‘clonal suicide’, evolved and are maintained to prevent colonization of established populations by different strains of the same and closely related species.

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIRis maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIRin these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility—a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria—their “existence conditions.”.

The immune systems that protect organisms from infectious agents invariably have a cost for the host. In bacteria and archaea CRISPR-Cas loci can serve as adaptive immune systems that protect these microbes from infectiously transmitted DNAs. When those DNAs are borne by lytic viruses (phages), this protection can provide a considerable advantage. CRISPR-Cas immunity can also prevent cells from acquiring plasmids and free DNA bearing genes that increase their fitness. Here, we use a combination of experiments and mathematical-computer simulation models to explore this downside of CRISPR-Cas immunity and its implications for the maintenance of CRISPR-Cas loci in microbial populations. We analyzed the conjugational transfer of the staphylococcal plasmid pG0400 into Staphylococcus epidermidis RP62a recipients that bear a CRISPR-Cas locus targeting this plasmid. Contrary to what is anticipated for lytic phages, which evade CRISPR by mutations in the target region, the evasion of CRISPR immunity by plasmids occurs at the level of the host through loss of functional CRISPR-Cas immunity. The results of our experiments and models indicate that more than 10-4of the cells in CRISPR-Cas positive populations are defective or deleted for the CRISPR-Cas region and thereby able to receive and carry the plasmid. Most intriguingly, the loss of CRISPR function even by large deletions can have little or no fitness cost in vitro. These theoretical and experimental results can account for the considerable variation in the existence, number and function of CRISPR-Cas loci within and between bacterial species. We postulate that as a consequence of the opposing positive and negative selection for immunity, CRISPR-Cas systems are in a continuous state of flux. They are lost when they bear immunity to laterally transferred beneficial genes, re-acquired by horizontal gene transfer, and ascend in environments where phage are a major source of mortality.

Increasing concerns about the rising rates of antibiotic therapy failure and advances in single-cell analyses have inspired a surge of research into antibiotic persistence. Bacterial persister cells represent a subpopulation of cells that can survive intensive antibiotic treatment without being resistant. Several approaches have emerged to define and measure persistence, and it is now time to agree on the basic definition of persistence and its relation to the other mechanisms by which bacteria survive exposure to bactericidal antibiotic treatments, such as antibiotic resistance, heteroresistance or tolerance. In this Consensus Statement, we provide definitions of persistence phenomena, distinguish between triggered and spontaneous persistence and provide a guide to measuring persistence. Antibiotic persistence is not only an interesting example of non-genetic single-cell heterogeneity, it may also have a role in the failure of antibiotic treatments. Therefore, it is our hope that the guidelines outlined in this article will pave the way for better characterization of antibiotic persistence and for understanding its relevance to clinical outcomes.

The canonical view of phage - bacterial interactions in dense, liquid cultures is that the phage will eliminate most of the sensitive cells; genetic resistance will then ascend to restore high bacterial densities. Yet there are various mechanisms by which bacteria may remain sensitive to phages but still attain high densities in their presence – because bacteria enter a transient state of reduced adsorption. Importantly, these mechanisms may be cryptic and inapparent prior to the addition of phage yet result in a rapid rebound of bacterial density after phage are introduced. We describe mathematical models of these processes and suggest how different types of this ‘phenotypic’ resistance may be elucidated. We offer preliminary in vitro studies of a previously characterized E. coli model system and Campylobacter jejuni illustrating apparent phenotypic resistance. As phenotypic resistance may be specific to the receptors used by phages, awareness of its mechanisms may identify ways of improving the choice of phages for therapy. Phenotypic resistance can also explain several enigmas in the ecology of phage-bacterial dynamics. Phenotypic resistance does not preclude the evolution of genetic resistance and may often be an intermediate step to genetic resistance.