Biofilms are surface-attached groups of microbial cells encased in an extracellular matrix that are significantly less susceptible to antimicrobial agents than non-adherent, planktonic cells. Biofilm-based infections are, as a result, extremely difficult to cure. A wide range of molecular mechanisms contribute to the high degree of recalcitrance that is characteristic of biofilm communities. These mechanisms include, among others, interaction of antimicrobials with biofilm matrix components, reduced growth rates and the various actions of specific genetic determinants of antibiotic resistance and tolerance. Alone, each of these mechanisms only partially accounts for the increased antimicrobial recalcitrance observed in biofilms. Acting in concert, however, these defences help to ensure the survival of biofilm cells in the face of even the most aggressive antimicrobial treatment regimens. This review summarises both historical and recent scientific data in support of the known biofilm resistance and tolerance mechanisms. Additionally, suggestions for future work in the field are provided.

Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria

Many bacteria can infect and persist inside their hosts for long periods of time In this Review, Fisher, Gollan and Helaine discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections Many bacteria can infect and persist inside their hosts for long periods of time This can be due to immunosuppression of the host, immune evasion by the pathogen and/or ineffective killing by antibiotics Bacteria can survive antibiotic treatment if they are resistant or tolerant to a drug Persisters are a subpopulation of transiently antibiotic-tolerant bacterial cells that are often slow-growing or growth-arrested, and are able to resume growth after a lethal stress The formation of persister cells establishes phenotypic heterogeneity within a bacterial population and has been hypothesized to be important for increasing the chances of successfully adapting to environmental change The presence of persister cells can result in the recalcitrance and relapse of persistent bacterial infections, and it has been linked to an increase in the risk of the emergence of antibiotic resistance during treatment If the mechanisms of the formation and regrowth of these antibiotic-tolerant cells were better understood, it could lead to the development of new approaches for the eradication of persistent bacterial infections In this Review, we discuss recent developments in our understanding of bacterial persisters and their potential implications for the treatment of persistent infections

Persistent bacterial infections and persister cells

BACKGROUND The escalating crisis of multidrug resistance is raising fears of untreatable infections caused by bacterial “superbugs.” However, many patients already suffer from infections that are effectively untreatable due to innate bacterial mechanisms for persistence. This phenomenon is caused by the formation of specialized persister cells that evade antibiotic killing and other stresses by entering a physiologically dormant state, irrespective of whether they possess genes enabling antibiotic resistance. The recalcitrance of persister cells is a major cause of prolonged and recurrent courses of infection that can eventually lead to complete antibiotic treatment failure. Regularly growing bacteria differentiate into persister cells stochastically at a basal rate, but this phenotypic conversion can also be induced by environmental cues indicative of imminent threats for the bacteria. Size and composition of the persister subpopulation in bacterial communities are largely controlled by stress signaling pathways, such as the general stress response or the SOS response, in conjunction with the second messenger (p)ppGpp that is almost always involved in persister formation. Consequently, persister formation is stimulated under conditions that favor the activation of these signaling pathways. Such conditions include bacterial biofilms and hostile host environments, as well as response to damage caused by sublethal concentrations of antibiotics. ADVANCES The limited comprehensive understanding of persister formation and survival is a critical issue in controlling persistent infections. However, recent work in the field has uncovered the molecular architecture of several cellular pathways underlying bacterial persistence, as well as the functional interactions that generate heterogeneous populations of persister cells. These results confirm the long-standing notion that persistence is intimately connected to slow growth or dormancy in the sense that a certain level of physiological quiescence is attained. Most prominently, the central role of toxin-antitoxin (TA) modules has been explained in considerable detail. In the model organism Escherichia coli K-12, two major pathways of persister formation via TA modules are both controlled by (p)ppGpp and involve toxin HokB and a panel of mRNA endonuclease toxins, respectively. Whereas activation of the membrane-associated toxin HokB depends on the enigmatic (GTPase) guanosine triphosphatase Obg and causes persister formation by abolishing the proton-motive force, mRNA endonuclease toxins are activated through antitoxin degradation by protease Lon and globally inhibit translation. In addition to these two pathways, toxin TisB is activated in response to DNA damage by the SOS response and promotes persister formation in a manner similar to HokB. Beyond TA modules, many additional factors (such as cellular energy metabolism or drug efflux) have been found to contribute to persister formation and survival, but their position in particular molecular pathways is often unclear. Altogether, this diversity of mechanisms drives the formation of a highly heterogeneous ensemble of persister cells that displays multistress and multidrug tolerance as the root of the recalcitrance of persistent infections. OUTLOOK Though recent advances in the field have greatly expanded our understanding of the molecular mechanisms underlying persister formation, important facets have remained elusive and should be addressed in future studies. One example is the upstream signaling input into the pathways mediating bacterial persister formation (e.g., the nature of the pacemaker driving stochastic persister formation). Similarly, it is often not well understood how—beyond the general idea of dormancy—persister cells can survive the action of lethal antibiotics. Finally, one curious aspect of the persister field is recurrent inconsistency between the results obtained by different groups. We speculate that these variations may be linked to subtle differences in experimental procedures inducing separate yet partially redundant pathways of persister formation. It is evident that the elucidation of this phenomenon may not only consolidate progress in the field but also offer the chance to gain insights into the molecular basis and control of bacterial persistence.

http://science.sciencemag.org/content/sci/354/6318/aaf4268.full.pdf

Mechanisms of bacterial persistence during stress and antibiotic exposure.

Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing dormant cells-activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.

Activated ClpP kills persisters and eradicates a chronic biofilm infection

Bacterial persister cells constitute a subpopulation of genetically identical, metabolically slow-growing cells that are highly tolerant of antibiotics and other environmental stresses. Recent studies have demonstrated that gene loci known as toxin-antitoxin (TA) modules play a central role in the persister state. Under normal growth conditions, antitoxins potently inhibit the activities of the toxins. In contrast, under conditions of stress, the antitoxins are selectively degraded, freeing the toxins to inhibit essential cellular processes, such as DNA replication and protein translation. This inhibition results in rapid growth arrest. In this Review, we highlight recent discoveries of these multifaceted TA systems with a focus on the newly uncovered mechanisms, especially conditional cooperativity, that are used to regulate cell growth and persistence. We also discuss the potential for targeting TA systems for antimicrobial drug discovery.

Toxin-antitoxin systems in bacterial growth arrest and persistence

Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a Delta yafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-type E. coli, the Delta yafQ strain had up to a approximately 2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression of yafQ from a high-copy-number plasmid resulted in up to a approximately 10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.

The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm

Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms.

Entamoeba histolytica infections of the gastrointestinal tract are common in the developing world but rare in North America. The authors present two cases: one involving an individual who had not travelled to an endemic area and another involving an individual who was born in Bulgaria. Both presented with severe abdominal pain and diarrhea. Endoscopic assessment revealed scattered colonic ulcerations and one patient was found to have a liver abscess on imaging. Stool ova and parasite studies were negative in both cases and both were diagnosed on review of colonic biopsies. On review of all Entamoeba cases in the Calgary Health Zone (Alberta), ova and parasite analysis found an average of 63.7 Entamoeba cases per year and a pathology database review revealed a total of seven cases of invasive E histolytica (2001 to 2011). Both patients responded well to antibiotic therapy. E histolytica should be considered in new-onset colitis, especially in individuals from endemic areas.

/pdf/invasive-amoebiasis-a-review-of-entamoeba-infections-394rwejps7.pdf

Invasive Amoebiasis: A Review of Entamoeba Infections Highlighted with Case Reports

We report on a patient who initially presented with delayed puberty and an absent uterus on imaging with ultrasound and MRI. She was subsequently diagnosed with Turner Syndrome. Turner Syndrome typically presents with early loss of ovarian function and should be considered when primary ovarian insufficiency is present with apparent absent uterus on imaging. Follow-up imaging of the apparent absent uterus post-estrogen replacement therapy is important to confirm a normal uterus. A diagnosis of an absent uterus can be psychologically traumatic for patients and families, and can have significant implications for future fertility options.

Sarah Akierman

Papers

The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm

Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms.

Invasive Amoebiasis: A Review of Entamoeba Infections Highlighted with Case Reports

Turner Syndrome and apparent absent uterus: a case report and review of the literature.

Gut microbiota biofilm disruptions by Giardia: Pathology in human enterocytes and germ-free mice