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1.  Significant Reduction of Antibiotic Use in the Community after a Nationwide Campaign in France, 2002–2007 
PLoS Medicine  2009;6(6):e1000084.
Didier Guillemot and colleagues describe the evaluation of a nationwide programme in France aimed at decreasing unnecessary outpatient prescriptions for antibiotics. The campaign was successful, particularly in reducing prescriptions for children.
Background
Overuse of antibiotics is the main force driving the emergence and dissemination of bacterial resistance in the community. France consumes more antibiotics and has the highest rate of beta-lactam resistance in Streptococcus pneumoniae than any other European country. In 2001, the government initiated “Keep Antibiotics Working”; the program's main component was a campaign entitled “Les antibiotiques c'est pas automatique” (“Antibiotics are not automatic”) launched in 2002. We report the evaluation of this campaign by analyzing the evolution of outpatient antibiotic use in France 2000–2007, according to therapeutic class and geographic and age-group patterns.
Methods and Findings
This evaluation is based on 2000–2007 data, including 453,407,458 individual reimbursement data records and incidence of flu-like syndromes (FLSs). Data were obtained from the computerized French National Health Insurance database and provided by the French Sentinel Network. As compared to the preintervention period (2000–2002), the total number of antibiotic prescriptions per 100 inhabitants, adjusted for FLS frequency during the winter season, changed by −26.5% (95% confidence interval [CI] −33.5% to −19.6%) over 5 years. The decline occurred in all 22 regions of France and affected all antibiotic therapeutic classes except quinolones. The greatest decrease, −35.8% (95% CI −48.3% to −23.2%), was observed among young children aged 6–15 years. A significant change of −45% in the relationship between the incidence of flu-like syndromes and antibiotic prescriptions was observed.
Conclusions
The French national campaign was associated with a marked reduction of unnecessary antibiotic prescriptions, particularly in children. This study provides a useful method for assessing public-health strategies designed to reduce antibiotic use.
Editors' Summary
Background
In 1928, Alexander Fleming discovered penicillin, the first antibiotic (a drug that kills bacteria). By the early 1940s, large amounts of penicillin could be made and, in the following decades, several other classes of powerful antibiotics were discovered. For a time, it looked like bacteria and the diseases that they cause had been defeated. But bacteria rapidly became resistant to these wonder drugs and nowadays, antibiotic resistance is a pressing public-health concern. Almost every type of disease-causing bacteria has developed resistance to one or more antibiotic in clinical use, and multidrug-resistant bacteria are causing outbreaks of potentially fatal diseases in hospitals and in the community. For example, multidrug-resistant Streptococcus pneumoniae (multidrug-resistant pneumococci or MRP) is now very common. S. pneumoniae colonize the nose and throat (the upper respiratory tract) and can cause diseases that range from mild ear infections to life-threatening pneumonia, particularly in young children and elderly people.
Why Was This Study Done?
For years, doctors have been prescribing (and patients have been demanding) antibiotics for viral respiratory infections (VRIs) such as colds and flu even though antibiotics do not cure viral infections. This overuse of antibiotics has been the main driving force in the spread of MRP. Thus, the highest rate of S. pneumoniae antibiotic resistance in Europe occurs in France, which has one of the highest rates of antibiotic consumption in the world. In 2001 France initiated “le plan national pour préserver l'efficacité des antibiotiques” to reduce the inappropriate use of antibiotics, particularly for the treatment of VRIs among children. The main component of the program was the “Antibiotiques c'est pas automatique” (“Antibiotics are not automatic”) campaign, which ran from 2002 to 2007 during the winter months when VRIs mainly occur. The campaign included an educational campaign for health care workers, the promotion of rapid tests for diagnosis of streptococcal infections, and a public information campaign about VRIs and about antibiotic resistance. In this study, the researchers evaluate the campaign by analyzing outpatient antibiotic use throughout France from 2000 to 2007.
What Did the Researchers Do and Find?
The researchers obtained information about antibiotic prescriptions and about the occurrence of flu-like illnesses during the study period from the French National Health Insurance database and national disease surveillance system, respectively. After adjusting for variations in the frequency of flu-like illnesses, compared to the preintervention period (2000–2002), the number of antibiotic prescriptions per 100 inhabitants decreased by a quarter over the five winters of the “Antibiotics are not automatic” campaign. The use of all major antibiotic classes except quinolones decreased in all 22 regions of France. Thus, whereas in 2000, more than 70 prescriptions per 100 inhabitants were issued during the winter in 15 regions, by 2006/7, no regions exceeded this prescription rate. The greatest decrease in prescription rate (a decrease of more than a third by 2006/7) was among children aged 6–15 years. Finally, although the rates of antibiotic prescriptions reflected the rates of flu-like illness throughout the campaign, by 2006/7 this relationship was much weaker, which suggests that fewer antibiotics were being prescribed for VRIs.
What Do These Findings Mean?
These findings indicate that the “Antibiotics are not automatic” campaign was associated with a reduction in antibiotic prescriptions, particularly in children. Because the whole French population was exposed to the campaign, these findings do not prove that the campaign actually caused the reduction in antibiotic prescriptions. The observed decrease might have been caused by other initiatives in France or elsewhere or by the introduction of a S. pneumoniae vaccine during the study period, for example. However, an independent survey indicated that fewer members of the public expected an antibiotic prescription for a VRI at the end of the campaign than at the start, that more people knew that antibiotics only kill bacteria, and that doctors were more confident about not prescribing antibiotics for VRIs. Thus, campaigns like “Antibiotics are not automatic” may be a promising way to reduce the overuse of antibiotics and to slow the spread of antibiotic resistance until new classes of effective antibiotics are developed.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000084.
This study is further discussed in a PLoS Medicine Perspective by Stephen Harbarth and Benedikt Huttner
The Bugs and Drugs Web site from the UK National electronic Library of Infection provides information about antibiotic resistance and links to other resources
The US National Institute of Allergy and Infectious Diseases provides information on antimicrobial drug resistance and on pneumococcal pneumonia
The US Centers for Disease Control and Prevention also have information on antibiotic resistance (in English and Spanish)
The European Surveillance of Antimicrobial Consumption Web site provides information on antibiotic consumption in European countries
Les antibiotiques c'est pas automatique provides information about the “Antibiotics are not automatic” campaign (in French)
Information on the Plan National pour Pérserver l'efficacité des antibiotiques is also available (in French)
doi:10.1371/journal.pmed.1000084
PMCID: PMC2683932  PMID: 19492093
2.  The Pharmaco –, Population and Evolutionary Dynamics of Multi-drug Therapy: Experiments with S. aureus and E. coli and Computer Simulations 
PLoS Pathogens  2013;9(4):e1003300.
There are both pharmacodynamic and evolutionary reasons to use multiple rather than single antibiotics to treat bacterial infections; in combination antibiotics can be more effective in killing target bacteria as well as in preventing the emergence of resistance. Nevertheless, with few exceptions like tuberculosis, combination therapy is rarely used for bacterial infections. One reason for this is a relative dearth of the pharmaco-, population- and evolutionary dynamic information needed for the rational design of multi-drug treatment protocols. Here, we use in vitro pharmacodynamic experiments, mathematical models and computer simulations to explore the relative efficacies of different two-drug regimens in clearing bacterial infections and the conditions under which multi-drug therapy will prevent the ascent of resistance. We estimate the parameters and explore the fit of Hill functions to compare the pharmacodynamics of antibiotics of four different classes individually and in pairs during cidal experiments with pathogenic strains of Staphylococcus aureus and Escherichia coli. We also consider the relative efficacy of these antibiotics and antibiotic pairs in reducing the level of phenotypically resistant but genetically susceptible, persister, subpopulations. Our results provide compelling support for the proposition that the nature and form of the interactions between drugs of different classes, synergy, antagonism, suppression and additivity, has to be determined empirically and cannot be inferred from what is known about the pharmacodynamics or mode of action of these drugs individually. Monte Carlo simulations of within-host treatment incorporating these pharmacodynamic results and clinically relevant refuge subpopulations of bacteria indicate that: (i) the form of drug-drug interactions can profoundly affect the rate at which infections are cleared, (ii) two-drug therapy can prevent treatment failure even when bacteria resistant to single drugs are present at the onset of therapy, and (iii) this evolutionary virtue of two-drug therapy is manifest even when the antibiotics suppress each other's activity.
Author Summary
In this study, we combine pharmacodynamic experiments using pathogenic strains of E. coli and S. aureus with mathematical and computer simulation models to explore the relative efficacies of two-drug antibiotic combinations in clearing infections and preventing the emergence of resistance. We develop a pharmacodynamic method that provides a convenient way to determine whether drug combinations will interact synergistically, antagonistically, additively or suppressively. We find that it is not possible to predict the nature and form of drug interactions based on what is known about the mode of action of individual drugs, thus illustrating the necessity of assessing the efficacy of drug combinations empirically. Our simulations of the within-host population and evolutionary dynamics of bacteria undergoing multi-drug treatment indicate that the form of the interaction between drugs observed experimentally can substantially affect the rate of clearance of the infection. On the other hand, the form of these interactions plays a minimal role in the emergence of resistance. Even when antibiotics are suppressive, two-drug therapy can prevent the ascent of bacteria resistant to single drugs that are present at the start of therapy and/or generated during the course of the infection.
doi:10.1371/journal.ppat.1003300
PMCID: PMC3617031  PMID: 23593006
3.  Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids 
mBio  2013;4(4):e00281-13.
ABSTRACT
Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.
IMPORTANCE  The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.
IMPORTANCE 
The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of “repelling” the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and “traps” it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.
doi:10.1128/mBio.00281-13
PMCID: PMC3735187  PMID: 23882013
4.  Elevated Mutagenesis Does Not Explain the Increased Frequency of Antibiotic Resistant Mutants in Starved Aging Colonies 
PLoS Genetics  2013;9(11):e1003968.
The frequency of mutants resistant to the antibiotic rifampicin has been shown to increase in aging (starved), compared to young colonies of Eschierchia coli. These increases in resistance frequency occur in the absence of any antibiotic exposure, and similar increases have also been observed in response to additional growth limiting conditions. Understanding the causes of such increases in the frequency of resistance is important for understanding the dynamics of antibiotic resistance emergence and spread. Increased frequency of rifampicin resistant mutants in aging colonies is cited widely as evidence of stress-induced mutagenesis (SIM), a mechanism thought to allow bacteria to increase mutation rates upon exposure to growth-limiting stresses. At the same time it has been demonstrated that some rifampicin resistant mutants are relatively fitter in aging compared to young colonies, indicating that natural selection may also contribute to increased frequency of rifampicin resistance in aging colonies. Here, we demonstrate that the frequency of mutants resistant to both rifampicin and an additional antibiotic (nalidixic-acid) significantly increases in aging compared to young colonies of a lab strain of Escherichia coli. We then use whole genome sequencing to demonstrate conclusively that SIM cannot explain the observed magnitude of increased frequency of resistance to these two antibiotics. We further demonstrate that, as was previously shown for rifampicin resistance mutations, mutations conferring nalidixic acid resistance can also increase fitness in aging compared to young colonies. Our results show that increases in the frequency of antibiotic resistant mutants in aging colonies cannot be seen as evidence of SIM. Furthermore, they demonstrate that natural selection likely contributes to increases in the frequency of certain antibiotic resistance mutations, even when no selection is exerted due to the presence of antibiotics.
Author Summary
Antibiotic resistance is one of the most pressing threats on human health worldwide. Such resistance has been increasing largely due to widespread antibiotic usage. However, it has also been noticed that under certain growth limiting conditions, there is an increase in resistance frequency that is independent of the presence of antibiotics. Such increases in antibiotic resistance frequency can greatly affect the dynamics of antibiotic resistance emergence and spread. Yet currently their causes are far from understood. Many assume that we observe more resistance mutations when growth is limited, because more mutations occur under such conditions. Here we use whole genome sequencing to show that increases in resistance frequency to two different antibiotics under starvation cannot be explained by increased mutagenesis. We further show that at least some of the increase in resistance frequency is likely to be explained by natural selection that favors certain resistance mutations conferring increased fitness under starvation. These results are intriguing as they demonstrate that positive selection may contribute to increases in the frequency of certain antibiotic resistance mutations, even in the absence of selection exerted by the presence of antibiotics.
doi:10.1371/journal.pgen.1003968
PMCID: PMC3828146  PMID: 24244205
5.  When the Most Potent Combination of Antibiotics Selects for the Greatest Bacterial Load: The Smile-Frown Transition 
PLoS Biology  2013;11(4):e1001540.
Finding the most potent combinations of antibiotics in the lab can be a challenge if antibiotic interactions are not robust to evolutionary adaptation.
Conventional wisdom holds that the best way to treat infection with antibiotics is to ‘hit early and hit hard’. A favoured strategy is to deploy two antibiotics that produce a stronger effect in combination than if either drug were used alone. But are such synergistic combinations necessarily optimal? We combine mathematical modelling, evolution experiments, whole genome sequencing and genetic manipulation of a resistance mechanism to demonstrate that deploying synergistic antibiotics can, in practice, be the worst strategy if bacterial clearance is not achieved after the first treatment phase. As treatment proceeds, it is only to be expected that the strength of antibiotic synergy will diminish as the frequency of drug-resistant bacteria increases. Indeed, antibiotic efficacy decays exponentially in our five-day evolution experiments. However, as the theory of competitive release predicts, drug-resistant bacteria replicate fastest when their drug-susceptible competitors are eliminated by overly-aggressive treatment. Here, synergy exerts such strong selection for resistance that an antagonism consistently emerges by day 1 and the initially most aggressive treatment produces the greatest bacterial load, a fortiori greater than if just one drug were given. Whole genome sequencing reveals that such rapid evolution is the result of the amplification of a genomic region containing four drug-resistance mechanisms, including the acrAB efflux operon. When this operon is deleted in genetically manipulated mutants and the evolution experiment repeated, antagonism fails to emerge in five days and antibiotic synergy is maintained for longer. We therefore conclude that unless super-inhibitory doses are achieved and maintained until the pathogen is successfully cleared, synergistic antibiotics can have the opposite effect to that intended by helping to increase pathogen load where, and when, the drugs are found at sub-inhibitory concentrations.
Author Summary
We take an evolutionary approach to a problem from the medical sciences in seeking to understand how our knowledge of rapid bacterial evolution should shape the way we treat pathogens with antibiotic drugs. We pay particular attention to combinations of different drugs that are purposefully used to produce potent therapies. Textbook orthodoxy in medicine and pharmacology states one should hit the pathogen hard with the drug and then prolong the treatment to be certain of clearing it from the host; how effective this approach is remains the subject of discussion. If the textbooks are correct, a combination of two antibiotics that prevents bacterial growth more than if just one drug were used should provide a better treatment strategy. Testing alternatives like these, however, is difficult to do in vivo or in the clinic, so we examined these ideas in laboratory conditions where treatments can be carefully controlled and the optimal combination therapy easily determined by measuring bacterial densities at every moment for each treatment trialled. Studying drug concentrations where antibiotic synergy can be guaranteed, we found that treatment duration was crucial. The most potent combination therapy on day 1 turned out to be the worst of all the therapies we tested by the middle of day 2, and by day 5 it barely inhibited bacterial growth; by contrast, the drugs did continue to impair growth if administered individually.
doi:10.1371/journal.pbio.1001540
PMCID: PMC3635860  PMID: 23630452
6.  Prospects for Advancing Tuberculosis Control Efforts through Novel Therapies 
PLoS Medicine  2006;3(8):e273.
Background
Development of new, effective, and affordable tuberculosis (TB) therapies has been identified as a critical priority for global TB control. As new candidates emerge from the global TB drug pipeline, the potential impacts of novel, shorter regimens on TB incidence and mortality have not yet been examined.
Methods and Findings
We used a mathematical model of TB to evaluate the expected benefits of shortening the duration of effective chemotherapy for active pulmonary TB. First, we considered general relationships between treatment duration and TB dynamics. Next, as a specific example, we calibrated the model to reflect the current situation in the South-East Asia region. We found that even with continued and rapid progress in scaling up the World Health Organization's DOTS strategy of directly observed, short-course chemotherapy, the benefits of reducing treatment duration would be substantial. Compared to a baseline of continuing DOTS coverage at current levels, and with currently available tools, a 2-mo regimen introduced by 2012 could prevent around 20% (range 13%–28%) of new cases and 25% (range 19%–29%) of TB deaths in South-East Asia between 2012 and 2030. If effective treatment with existing drugs expands rapidly, overall incremental benefits of shorter regimens would be lower, but would remain considerable (13% [range 8%–19%] and 19% [range 15%–23%] reductions in incidence and mortality, respectively, between 2012 and 2030). A ten-year delay in the introduction of new drugs would erase nearly three-fourths of the total expected benefits in this region through 2030.
Conclusions
The introduction of new, shorter treatment regimens could dramatically accelerate the reductions in TB incidence and mortality that are expected under current regimens—with up to 2- or 3-fold increases in rates of decline if shorter regimens are accompanied by enhanced case detection. Continued progress in reducing the global TB burden will require a balanced approach to pursuing new technologies while promoting wider implementation of proven strategies.
Mathematical modeling suggests that new tuberculosis treatments that are shorter than the current 6-month standard regimen would lead to considerable reductions in incidence and mortality of TB, which remains the leading cause of global deaths.
Editors' Summary
Background.
One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that is the main cause of tuberculosis (TB). In most people, the infection remains dormant, but every year eight million people develop active TB, usually in their lungs, and two million people die from the disease. For most of the second half of the 20th century, TB was in decline, particularly in developed countries, due to the availability of powerful antibiotics. Recently, however, global efforts to control TB have been set back by the HIV/AIDS epidemic—people with damaged immune systems are very susceptible to TB—and the emergence of antibiotic-resistant bacteria. In the 1990s, the World Health Organization (WHO) introduced DOTS as the recommended strategy for global TB control. Central to DOTS is “directly observed short-course chemotherapy.” To cure TB, several antibiotics have to be taken daily for 6 months. Patients must complete this treatment, even if they feel better sooner, to prevent relapse and the emergence of drug-resistant bacteria. The DOTS approach ensures that patients do this by having trained observers watch them swallow each dose of their medication for the entire 6-month period.
Why Was This Study Done?
This year, WHO and partners launched a renewed Global Plan to Stop TB, which aims to reduce, by 2015, the number of people who are sick with TB (disease prevalence) and the number of people who die each year from the disease (mortality) to half the 1990 levels. Because sick people often infect others, reducing disease prevalence will also reduce the number of new infections each year (disease incidence). The Global Plan to Stop TB includes a commitment to expand and intensify the DOTS strategy (in the year 2004, only around half of new active, infectious TB cases were detected under DOTS). The drug combinations currently used for DOTS consist of four or more different antibiotics, which have all been around and in use for many years. Recently, renewed effort has gone into the search for new TB treatments. Several candidate drugs have been identified and are now being tested, and scientists expect that some of them will be able to cure patients quicker than the current 6-month regimen. In this study, the researchers wanted to understand the potential benefits of such shorter treatments.
What Did the Researchers Do and Find?
The researchers developed a mathematical model that considers the acquisition, progression, diagnosis, and treatment of M. tuberculosis infection and its clinical consequences. They used the model to predict how changes in treatment duration might affect TB incidence, prevalence, and mortality. In the model, shorter treatment durations are connected to higher cure rates, as each additional month of treatment means more time to ensure that patients keep taking their medications, and more doses that might be missed. Since patients who drop out of treatment early can continue to infect other people, the model shows how a 2-month course of antibiotics will produce a quicker decline in the incidence of TB than a 6-month course by reducing these opportunities to infect others.
  The researchers then refined their model to include current conditions in South-East Asia, an area where DOTS is being scaled up and where one-third of all new TB cases occur. This “calibrated” model indicates that even if DOTS is scaled up as planned, shorter drug courses would still reduce TB incidence and deaths much quicker than a 6-month course. If, for example, a 2-month drug treatment were introduced by 2012, it might prevent 13% of the new cases and 19% of the TB deaths that would otherwise occur in South-East Asia between 2012 and 2030. These benefits might be even greater if the new drug regimen freed up resources to improve the systematic effort to detect new TB cases. On the other hand, delaying its implementation until 2022 would erase three-quarters of the predicted benefits.
What Do These Findings Mean?
Like all mathematical models, this one makes many assumptions that, if incorrect, will change the predictions. With this caveat, the study confirms that the planned scale-up of DOTS will greatly reduce TB incidence and mortality over the next few years. However, it also suggests that the impact of DOTS could be substantially improved by introducing shorter drug regimens. The earlier such shorter treatments become available, the greater their benefits would be. By reducing the opportunities for patients to default on their treatment and by shortening the period during which they can infect others, the researchers predict that the rate of decline in TB incidence and mortality could be up to three times higher for antibiotics that only have to be taken for 2 months compared with those that need to be taken for 6 months. But, they stress, strategies for reducing the global TB burden must strike a balance between pursuing new treatment and detection strategies and promoting wider implementation of proven strategies. And while there is hope that new shorter treatments will prove effective over the next few years, this will only become clear as candidates are rigorously tested.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030273.
• US National Institute of Allergy and Infectious Diseases patient fact sheet on TB
• US Centers for Disease Control and Prevention information for patients and professionals on TB
• MedlinePlus encyclopedia entry on TB
• NHS Direct Online patient information on TB from the UK National Health Service
• World Health Organization information on global TB control
• Global Alliance for TB Drug Development information on current initiatives to develop new TB drugs
• Wikipedia page on TB treatment (note that Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030273
PMCID: PMC1523376  PMID: 16866578
7.  Antibiotic Selection Pressure and Macrolide Resistance in Nasopharyngeal Streptococcus pneumoniae: A Cluster-Randomized Clinical Trial 
PLoS Medicine  2010;7(12):e1000377.
Jeremy Keenan and colleagues report that during a cluster-randomized clinical trial in Ethiopia, nasopharyngeal pneumococcal resistance to macrolides was significantly higher in communities randomized to receive azithromycin compared with untreated control communities.
Background
It is widely thought that widespread antibiotic use selects for community antibiotic resistance, though this has been difficult to prove in the setting of a community-randomized clinical trial. In this study, we used a randomized clinical trial design to assess whether macrolide resistance was higher in communities treated with mass azithromycin for trachoma, compared to untreated control communities.
Methods and Findings
In a cluster-randomized trial for trachoma control in Ethiopia, 12 communities were randomized to receive mass azithromycin treatment of children aged 1–10 years at months 0, 3, 6, and 9. Twelve control communities were randomized to receive no antibiotic treatments until the conclusion of the study. Nasopharyngeal swabs were collected from randomly selected children in the treated group at baseline and month 12, and in the control group at month 12. Antibiotic susceptibility testing was performed on Streptococcus pneumoniae isolated from the swabs using Etest strips. In the treated group, the mean prevalence of azithromycin resistance among all monitored children increased from 3.6% (95% confidence interval [CI] 0.8%–8.9%) at baseline, to 46.9% (37.5%–57.5%) at month 12 (p = 0.003). In control communities, azithromycin resistance was 9.2% (95% CI 6.7%–13.3%) at month 12, significantly lower than the treated group (p<0.0001). Penicillin resistance was identified in 0.8% (95% CI 0%–4.2%) of isolates in the control group at 1 year, and in no isolates in the children-treated group at baseline or 1 year.
Conclusions
This cluster-randomized clinical trial demonstrated that compared to untreated control communities, nasopharyngeal pneumococcal resistance to macrolides was significantly higher in communities randomized to intensive azithromycin treatment. Mass azithromycin distributions were given more frequently than currently recommended by the World Health Organization's trachoma program. Azithromycin use in this setting did not select for resistance to penicillins, which remain the drug of choice for pneumococcal infections.
Trial registration
www.ClinicalTrials.gov NCT00322972
Please see later in the article for the Editors' Summary
Editors' Summary
Background
In 1928, Alexander Fleming discovered penicillin, the first antibiotic (a drug that kills bacteria). By the early 1940s, scientists were able to make large quantities of penicillin and, in the following decades, several other classes of powerful antibiotics were discovered. For example, erythromycin—the first macrolide antibiotic—was developed in the early 1950s. For a time, it looked like bacteria and the diseases that they cause had been defeated. But bacteria rapidly become resistant to antibiotics. Under the “selective pressure” of an antibiotic, bacteria that have acquired a random change in their DNA that allows them to survive in the antibiotic's presence outgrow nonresistant bacteria. What's more, bacteria can transfer antibiotic resistance genes between themselves. Nowadays, antibiotic resistance is a major public health concern. Almost every type of disease-causing bacteria has developed resistance to one or more antibiotic in clinical use and multi-drug resistant bacteria are causing outbreaks of potentially fatal diseases in hospitals and in the community.
Why Was This Study Done?
Although epidemiological studies (investigations of the causes, distribution, and control of disease in population) show a correlation between antibiotic use and antibiotic resistance in populations, such studies cannot prove that antibiotic use actually causes antibiotic resistance. It could be that the people who use more antibiotics share other characteristics that increase their chance of developing antibiotic resistance (so-called “confounding”). A causal link between antibiotic use and the development of antibiotic resistance can only be established by doing a randomized controlled trial. In such trials, groups of individuals are chosen at random to avoid confounding, given different treatments, and outcomes in the different groups compared. Here, the researchers undertake a randomized clinical trial to assess whether macrolide resistance is higher in communities treated with azithromycin for trachoma than in untreated communities. Azithromycin—an erythromycin derivative—is used to treat common bacterial infections such as middle ear infections caused by Streptococcus pneumoniae. Trachoma—the world's leading infectious cause of blindness—is caused by Chlamydia trachomatis. The World Health Organization's trachoma elimination strategy includes annual azithromycin treatment of at-risk communities.
What Did the Researchers Do and Find?
In this cluster-randomized trial (a study that randomly assigns groups of people rather than individuals to different treatments), 12 Ethiopian communities received mass azithromycin treatment of children aged 1–10 years old at 0, 3, 6, and 9 months, and 12 control communities received the antibiotic only at 12 months. The researchers took nasopharyngeal (nose and throat) swabs from randomly selected treated children at 0 and 12 months and from randomly selected control children at 12 months. They isolated S. pneumoniae from the swabs and tested the isolates for antibiotic susceptibility. 70%–80% of the children tested had S. pneumoniae in their nose or throat. In the treated group, 3.6% of monitored children were carrying azithromycin-resistant S. pneumoniae at 0 months, whereas 46.9% were doing so at 12 months—a statistically significant increase. Only 9.2% of the monitored children in the untreated group were carrying azithromycin-resistant S. pneumoniae at 12 months, a significantly lower prevalence than in the treated group. Importantly, there was no resistance to penicillin in any S. pneumoniae isolates obtained from the treated children at 0 or 12 months; one penicillin-resistant isolate was obtained from the control children.
What Do These Findings Mean?
These findings indicate that macrolide resistance is higher in nasopharyngeal S. pneumoniae in communities receiving intensive azithromycin treatment than in untreated communities. Thus, they support the idea that frequent antibiotic use selects for antibiotic resistance in populations. Although the study was undertaken in Ethiopian communities with high rates of nasopharyngeal S. pneumoniae carriage, this finding is likely to be generalizable to other settings. Importantly, these findings have no bearing on current trachoma control activities, which use less frequent antibiotic treatments and are less likely to select for azithromycin resistance. The lack of any increase in penicillin resistance, which is usually the first-line therapy for S. pneumoniae infections, is also reassuring. However, although these findings suggest that the benefits of mass azithromycin treatment for trachoma outweigh any potential adverse affects, they nonetheless highlight the importance of continued monitoring for the secondary effects of mass antibiotic distributions.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000377.
The Bugs and Drugs website provides information about antibiotic resistance and links to other resources
The US National Institute of Allergy and Infectious Diseases provides information on antimicrobial drug resistance and on diseases caused by S. pneumoniae (pneumococcal diseases)
The US Centers for Disease Control and Prevention also have information on antibiotic resistance (in English and Spanish)
The World Health Organization has information about the global threat of antimicrobial resistance and about trachoma (in several languages)
More information about the trial described in this paper is available on ClinicalTrials.gov
doi:10.1371/journal.pmed.1000377
PMCID: PMC3001893  PMID: 21179434
8.  Optimal Drug Synergy in Antimicrobial Treatments 
PLoS Computational Biology  2010;6(6):e1000796.
The rapid proliferation of antibiotic-resistant pathogens has spurred the use of drug combinations to maintain clinical efficacy and combat the evolution of resistance. Drug pairs can interact synergistically or antagonistically, yielding inhibitory effects larger or smaller than expected from the drugs' individual potencies. Clinical strategies often favor synergistic interactions because they maximize the rate at which the infection is cleared from an individual, but it is unclear how such interactions affect the evolution of multi-drug resistance. We used a mathematical model of in vivo infection dynamics to determine the optimal treatment strategy for preventing the evolution of multi-drug resistance. We found that synergy has two conflicting effects: it clears the infection faster and thereby decreases the time during which resistant mutants can arise, but increases the selective advantage of these mutants over wild-type cells. When competition for resources is weak, the former effect is dominant and greater synergy more effectively prevents multi-drug resistance. However, under conditions of strong resource competition, a tradeoff emerges in which greater synergy increases the rate of infection clearance, but also increases the risk of multi-drug resistance. This tradeoff breaks down at a critical level of drug interaction, above which greater synergy has no effect on infection clearance, but still increases the risk of multi-drug resistance. These results suggest that the optimal strategy for suppressing multi-drug resistance is not always to maximize synergy, and that in some cases drug antagonism, despite its weaker efficacy, may better suppress the evolution of multi-drug resistance.
Author Summary
The use of antibiotics against bacterial infections has led to the emergence of multi-drug resistant pathogens such as tuberculosis and MRSA. In order to control resistance, clinicians have increasingly turned to multi-antibiotic therapies. The common wisdom is to use combinations of drugs that act synergistically to kill the infection, but the impact of drug synergy on the evolution of resistance is unclear. Using mathematical simulations of an in vivo infection model, we asked what level of drug synergy would minimize the risk of multi-drug resistance while preserving the efficacy of treatment. We found that synergy may increase or decrease the risk of multi-drug resistance in a given treatment, depending on infection properties such as mutation rate and the availability of resources. Surprisingly, under conditions of strong competition for resources within the host, we found that maximal synergy—currently favored in clinical settings—can actually increase the risk of multi-drug resistance. Our results identify conditions under which drug synergy exacerbates the problem of multi-drug resistance, and offer guidelines for the selection of drug pairs that suppress it.
doi:10.1371/journal.pcbi.1000796
PMCID: PMC2880566  PMID: 20532210
9.  Hedging against Antiviral Resistance during the Next Influenza Pandemic Using Small Stockpiles of an Alternative Chemotherapy 
PLoS Medicine  2009;6(5):e1000085.
Mathematically simulating an influenza pandemic, Joseph Wu and colleagues predict that using a secondary antiviral drug early in local epidemics would reduce global emergence of resistance to the primary stockpiled drug.
Background
The effectiveness of single-drug antiviral interventions to reduce morbidity and mortality during the next influenza pandemic will be substantially weakened if transmissible strains emerge which are resistant to the stockpiled antiviral drugs. We developed a mathematical model to test the hypothesis that a small stockpile of a secondary antiviral drug could be used to mitigate the adverse consequences of the emergence of resistant strains.
Methods and Findings
We used a multistrain stochastic transmission model of influenza to show that the spread of antiviral resistance can be significantly reduced by deploying a small stockpile (1% population coverage) of a secondary drug during the early phase of local epidemics. We considered two strategies for the use of the secondary stockpile: early combination chemotherapy (ECC; individuals are treated with both drugs in combination while both are available); and sequential multidrug chemotherapy (SMC; individuals are treated only with the secondary drug until it is exhausted, then treated with the primary drug). We investigated all potentially important regions of unknown parameter space and found that both ECC and SMC reduced the cumulative attack rate (AR) and the resistant attack rate (RAR) unless the probability of emergence of resistance to the primary drug pA was so low (less than 1 in 10,000) that resistance was unlikely to be a problem or so high (more than 1 in 20) that resistance emerged as soon as primary drug monotherapy began. For example, when the basic reproductive number was 1.8 and 40% of symptomatic individuals were treated with antivirals, AR and RAR were 67% and 38% under monotherapy if pA = 0.01. If the probability of resistance emergence for the secondary drug was also 0.01, then SMC reduced AR and RAR to 57% and 2%. The effectiveness of ECC was similar if combination chemotherapy reduced the probabilities of resistance emergence by at least ten times. We extended our model using travel data between 105 large cities to investigate the robustness of these resistance-limiting strategies at a global scale. We found that as long as populations that were the main source of resistant strains employed these strategies (SMC or ECC), then those same strategies were also effective for populations far from the source even when some intermediate populations failed to control resistance. In essence, through the existence of many wild-type epidemics, the interconnectedness of the global network dampened the international spread of resistant strains.
Conclusions
Our results indicate that the augmentation of existing stockpiles of a single anti-influenza drug with smaller stockpiles of a second drug could be an effective and inexpensive epidemiological hedge against antiviral resistance if either SMC or ECC were used. Choosing between these strategies will require additional empirical studies. Specifically, the choice will depend on the safety of combination therapy and the synergistic effect of one antiviral in suppressing the emergence of resistance to the other antiviral when both are taken in combination.
Editors' Summary
Background
Every winter, millions of people catch influenza—a viral infection of the airways—and about half a million people die as a result. These seasonal “epidemics” occur because small but frequent changes in the viral proteins (antigens) to which the human immune system responds mean that an immune response produced one year provides only partial protection against influenza the next year. Influenza viruses also occasionally appear that contain major antigenic changes. Human populations have little or no immunity to such viruses so they can start deadly pandemics (global epidemics). The 1918–19 influenza pandemic, for example, killed 40–50 million people. The last influenza pandemic was in 1968 and many experts fear the next pandemic might strike soon. To prepare for such an eventuality, scientists are trying to develop vaccines that might work against an emerging pandemic influenza virus. In addition, many governments are stockpiling antiviral drugs for the large-scale treatment of influenza and for targeted prophylaxis (prevention). Antiviral drugs prevent the replication of the influenza virus, thereby shortening the length of time that an infected person is ill and protecting uninfected people against infection. Their widespread use should, therefore, slow the spread of pandemic influenza.
Why Was This Study Done?
Although some countries are stockpiling more than one antiviral drug in preparation for an influenza pandemic, many countries are investing in large stockpiles of a single drug, oseltamivir (Tamiflu). But influenza viruses can become resistant to antiviral drugs and the widespread use of a single drug (the primary antiviral) is likely to increase the risk that a resistant strain will emerge. If this did happen, the ability of antiviral drugs to slow the spread of a pandemic would be greatly reduced. In this study, the researchers use a mathematical model of influenza transmission to investigate whether a small stockpile of a secondary antiviral drug could be used to prevent the adverse consequences of the emergence of antiviral-resistant pandemic influenza viruses.
What Did the Researchers Do and Find?
The researchers used their model of influenza transmission to predict how two strategies for the use of a small stockpile of a secondary antiviral might affect the cumulative attack rate (AR; the final proportion of the population infected) and the resistant attack rate (RAR; the proportion of the population infected with an influenza virus strain resistant to the primary drug, a measure that may reflect the impact of antiviral resistance on death rates during a pandemic). In a large, closed population, the model predicted that both “early combination chemotherapy” (treatment with both drugs together while both are available) and “sequential multi-drug chemotherapy” (treatment with the secondary drug until it is exhausted, then treatment with the primary drug) would reduce the AR and the RAR compared with monotherapy unless the probability of emergence of resistance to the primary drug was very low (resistance rarely occurred) or very high (resistance emerged as soon as the primary drug was used). The researchers then introduced international travel data into their model to investigate whether these two strategies could limit the development of antiviral resistance at a global scale. This analysis predicted that, provided the population that was the main source of resistant strains used one of the strategies, both strategies in distant, subsequently affected populations would be able to reduce the AR and RAR even if some intermediate populations failed to control resistance.
What Do These Findings Mean?
As with all mathematical models, the accuracy of these predictions depends on the assumptions used to build the model and the data fed into it. Nevertheless, these findings suggest that both of the proposed strategies for the use of small stockpiles of secondary antiviral drugs should limit the spread of drug-resistant influenza virus more effectively than monotherapy with the primary antiviral drug. Thus, small stockpiles of secondary antivirals could provide a hedge against the development of antiviral resistance during the early phases of an influenza pandemic and are predicted to be a worthwhile public-health investment. However, note the researchers, experimental studies—including determinations of which drugs are safe to use together, and how effectively a given combination prevents resistance compared with each drug used alone—are now needed to decide which of the strategies to recommend in real-life situations. In the context of the 2009 global spread of swine flu, these findings suggest that public health officials might consider zanamivir (Relenza) as the secondary antiviral drug for resistance-limiting strategies in countries that have stockpiled oseltamivir.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000085.
The US Centers for Disease Control and Prevention provides information about influenza for patients and professionals, including specific information on pandemic influenza and on influenza antiviral drugs
The World Health Organization provides information on influenza (in several languages) and has detailed guidelines on the use of vaccines and antivirals during influenza pandemics
The UK Health Protection Agency provides information on pandemic influenza
MedlinePlus provides a list of links to other information about influenza (in English and Spanish)
doi:10.1371/journal.pmed.1000085
PMCID: PMC2680070  PMID: 19440354
10.  Selective Chemical Inhibition of agr Quorum Sensing in Staphylococcus aureus Promotes Host Defense with Minimal Impact on Resistance 
PLoS Pathogens  2014;10(6):e1004174.
Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development.
Author Summary
New approaches are needed to lessen the burden of antibiotic resistant bacterial infections. One strategy is to develop therapies that target virulence which rely on host defense elements to clear the bacteria rather than direct antimicrobial killing. Quorum sensing is a bacterial signaling mechanism that often regulates virulence in medically relevant bacterial pathogens. Therefore, drugs that inhibit quorum sensing can promote host defense by rendering the pathogenic bacteria avirulent and/or less fit for survival within the host. Our work addressed this strategy in the pathogen Staphylococcus aureus which is the major cause of acute bacterial skin and soft tissue infections. We conducted a high throughput screen to identify compounds that could inhibit signaling by the quorum sensing operon, agr. We found a compound that we termed savirin (S. aureus virulence inhibitor) that could inhibit signaling by this operon. The drug helped the innate immune system in animals to clear bacteria that express this operon without affecting clearance of bacteria that do not have this operon. We addressed the mechanism of action of this compound and whether resistance or tolerance to this compound would likely develop. Our data indicate for the first time that host defense against S. aureus skin infections can be enhanced by chemical inhibition of agr-mediated quorum sensing.
doi:10.1371/journal.ppat.1004174
PMCID: PMC4055767  PMID: 24945495
11.  OPC-67683, a Nitro-Dihydro-Imidazooxazole Derivative with Promising Action against Tuberculosis In Vitro and In Mice 
PLoS Medicine  2006;3(11):e466.
Background
Tuberculosis (TB) is still a leading cause of death worldwide. Almost a third of the world's population is infected with TB bacilli, and each year approximately 8 million people develop active TB and 2 million die as a result. Today's TB treatment, which dates back to the 1970s, is long and burdensome, requiring at least 6 mo of multidrug chemotherapy. The situation is further compounded by the emergence of multidrug-resistant TB (MDR-TB) and by the infection's lethal synergy with HIV/AIDS. Global health and philanthropic organizations are now pleading for new drug interventions that can address these unmet needs in TB treatment.
Methods and Findings
Here we report OPC-67683, a nitro-dihydro-imidazooxazole derivative that was screened to help combat the unmet needs in TB treatment. The compound is a mycolic acid biosynthesis inhibitor found to be free of mutagenicity and to possess highly potent activity against TB, including MDR-TB, as shown by its exceptionally low minimum inhibitory concentration (MIC) range of 0.006–0.024 μg/ml in vitro and highly effective therapeutic activity at low doses in vivo. Additionally, the results of the post-antibiotic effect of OPC-67683 on intracellular Mycobacterium tuberculosis showed the agent to be highly and dose-dependently active also against intracellular M. tuberculosis H37Rv after a 4-h pulsed exposure, and this activity at a concentration of 0.1 μg/ml was similar to that of the first-line drug rifampicin (RFP) at a concentration of 3 μg/ml. The combination of OPC-67683 with RFP and pyrazinamide (PZA) exhibited a remarkably quicker eradication (by at least 2 mo) of viable TB bacilli in the lung in comparison with the standard regimen consisting of RFP, isoniazid (INH), ethambutol (EB), and PZA. Furthermore, OPC-67683 was not affected by nor did it affect the activity of liver microsome enzymes, suggesting the possibility for OPC-67683 to be used in combination with drugs, including anti-retrovirals, that induce or are metabolized by cytochrome P450 enzymes.
Conclusions
We concluded that based on these properties OPC-67683 has the potential to be used as a TB drug to help combat the unmet needs in TB treatment.
A nitro-dihydro-imidazooxazole derivative was shown to have the potential for use against tuberculosis.
Editors' Summary
Background.
One-third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB). Most infected people are healthy—the bacteria can remain latent for years, hidden within cells in the body. However, every year 8 million people develop active TB, a chronic disease that usually affects the lungs, and 2 million people die. For most of the second half of the 20th century, TB was in decline because of the powerful antibiotics that were developed from the 1940s onwards. The standard treatment for TB—four antibiotics that have to be taken several times a week for at least six months to flush out any latent M. tuberculosis bacteria—was introduced in the late 1970s and saved many lives. Recently, however, efforts to eradicate TB have been set back by the HIV/AIDS epidemic—people with damaged immune systems are very susceptible to TB—and the emergence of multi-drug resistant (MDR) bacteria.
Why Was This Study Done?
The treatment for TB is long and unpleasant, and patients who develop MDR-TB have to be treated with second-line drugs that are less effective, more expensive, and more toxic. In addition, for people infected with both HIV and TB, some antiretroviral and anti-TB drugs cannot be used at the same time. Many drugs are either activated or removed by enzymes in the liver, so combinations of these two classes of drugs sometimes alter liver function in a way that causes clinical problems. There is, therefore, an urgent need for new, effective anti-TB drugs that attack M. tuberculosis in a different way than do existing drugs. Such drugs should ideally be active against MDR M. tuberculosis, work quickly at low doses, be active against latent bacteria, and have minimal effects on the liver so that they can be used in patients co-infected with HIV. In this study, the researchers investigated a chemical called OPC-67683.
What Did the Researchers Do and Find?
The researchers identified a compound that inhibited the production of mycolic acid—an essential component of the cell wall of M. tuberculosis—and they tested its ability to kill the organism. They then tested in detail its ability to inhibit bacterial growth in dishes of antibiotic-sensitive and MDR M. tuberculosis and isolates from patients. OPC-67683 inhibited the growth of all these bugs at lower concentrations than the four antibiotics used in the standard TB treatment. It also killed bacteria hidden within human cells as well as or better than these drugs. Next, the researchers treated mice infected with M. tuberculosis with OPC-67683. They found that it reduced the number of bacteria in the lungs of both normal and immunocompromised mice at lower concentrations than the standard drugs. Furthermore, when combined with two of the standard drugs, it reduced the time taken to clear bacteria from the lungs by the standard drug regimen by two months. Finally, the researchers showed that OPC-67683 had no effects on the liver enzymes that metabolize antiretrovirals, and, conversely, that the activity of OPC-67683 was not affected by liver enzymes. Thus, this agent is unlikely to cause clinical problems or lose its efficacy in HIV patients who are receiving antiretroviral drugs.
What Do These Findings Mean?
These results from laboratory and animal experiments suggest that OPC-67683 could possibly fulfill the criteria for a new anti-TB drug. OPC-67683 is active against MDR-TB. It is also active against intracellular TB, which the authors postulate could be a positive link with the effective treatment of latent TB, and it works quickly in animals when combined with existing anti-TB drugs. Importantly, it also disables M. tuberculosis in a unique way and does not appear to have any major effects on the liver that might stop it from being used in combination with antiretrovirals. All these preclinical characteristics now need to be checked in people—many drugs do well in preclinical studies but fail in patients. These clinical studies need to be expedited given the upsurge in TB, and, write the researchers, OPC-67683 needs to be tested in combination with both conventional drugs and other new drugs so that the best regimen of new drugs for the treatment of TB can be found as soon as possible.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030466.
US National Institute of Allergy and Infectious Diseases patient fact sheet on tuberculosis
US Centers for Disease Control and Prevention information on tuberculosis
MedlinePlus encyclopedia entry on tuberculosis
NHS Direct Online patient information on tuberculosis from the UK National Health Service
World Health Organization information on the global elimination of tuberculosis
Global Alliance for TB Drug Development information on why new TB drugs are needed
doi:10.1371/journal.pmed.0030466
PMCID: PMC1664607  PMID: 17132069
12.  Evolution and Impact of Bacterial Drug Resistance in the Context of Cystic Fibrosis Disease and Nosocomial Settings 
Microbiology Insights  2013;6:29-36.
The use of antibiotics is unavoidable in trying to treat acute infections and in the prevention and control of chronic infections. Over the years, an ever increasing number of infections has escalated the use of antibiotics, which has necessitated action against an emerging bacterial resistance. There seems to be a continuous acquisition of new resistance mechanisms among bacteria that switch niches between human, animals, and the environment. An antibiotic resistant strain emerges when it acquires the DNA that confers the added capacity needed to survive in an unusual niche. Once acquired, a new resistance mechanism evolves according to the dynamics of the microenvironment; there is then a high probability that it is transferred to other species or to an avirulent strain of the same species. A well understood model for studying emerging antibiotic resistance and its impact is Pseudomonas aeruginosa, an opportunistic pathogen which is able to cause acute and chronic infections in nosocomial settings. This bacterium has a huge genetic repertoire consisting of genes that encode both innate and acquired antibiotic resistance traits. Besides acute infections, chronic colonization of P. aeruginosa in the lungs of cystic fibrosis (CF) patients plays a significant role in morbidity and mortality. Antibiotics used in the treatment of such infections has increased the longevity of patients over the last several decades. However, emerging multidrug resistant strains and the eventual increase in the dosage of antibiotic(s) is of major concern. Though there are various infections that are treated by single/combined antibiotics, the particular case of P. aeruginosa infection in CF patients serves as a reference for understanding the impact of overuse of antibiotics and emerging antibiotic resistant strains. This mini review presents the need for judicious use of antibiotics to treat various types of infections, protecting patients and the environment, as well as achieving a better treatment outcome.
doi:10.4137/MBI.S10792
PMCID: PMC3987750  PMID: 24826072
antibiotic resistance; bacterial adaptation; nosocomial infections; cystic fibrosis; chronic infections; biofilms
13.  The Impact of Different Antibiotic Regimens on the Emergence of Antimicrobial-Resistant Bacteria 
PLoS ONE  2008;3(12):e4036.
Backgroud
The emergence and ongoing spread of antimicrobial-resistant bacteria is a major public health threat. Infections caused by antimicrobial-resistant bacteria are associated with substantially higher rates of morbidity and mortality compared to infections caused by antimicrobial-susceptible bacteria. The emergence and spread of these bacteria is complex and requires incorporating numerous interrelated factors which clinical studies cannot adequately address.
Methods/Principal Findings
A model is created which incorporates several key factors contributing to the emergence and spread of resistant bacteria including the effects of the immune system, acquisition of resistance genes and antimicrobial exposure. The model identifies key strategies which would limit the emergence of antimicrobial-resistant bacterial strains. Specifically, the simulations show that early initiation of antimicrobial therapy and combination therapy with two antibiotics prevents the emergence of resistant bacteria, whereas shorter courses of therapy and sequential administration of antibiotics promote the emergence of resistant strains.
Conclusions/Significance
The principal findings suggest that (i) shorter lengths of antibiotic therapy and early interruption of antibiotic therapy provide an advantage for the resistant strains, (ii) combination therapy with two antibiotics prevents the emergence of resistance strains in contrast to sequential antibiotic therapy, and (iii) early initiation of antibiotics is among the most important factors preventing the emergence of resistant strains. These findings provide new insights into strategies aimed at optimizing the administration of antimicrobials for the treatment of infections and the prevention of the emergence of antimicrobial resistance.
doi:10.1371/journal.pone.0004036
PMCID: PMC2603320  PMID: 19112501
14.  Nongenetic Individuality in the Host–Phage Interaction 
PLoS Biology  2008;6(5):e120.
Isogenic bacteria can exhibit a range of phenotypes, even in homogeneous environmental conditions. Such nongenetic individuality has been observed in a wide range of biological processes, including differentiation and stress response. A striking example is the heterogeneous response of bacteria to antibiotics, whereby a small fraction of drug-sensitive bacteria can persist under extensive antibiotic treatments. We have previously shown that persistent bacteria enter a phenotypic state, identified by slow growth or dormancy, which protects them from the lethal action of antibiotics. Here, we studied the effect of persistence on the interaction between Escherichia coli and phage lambda. We used long-term time-lapse microscopy to follow the expression of green fluorescent protein (GFP) under the phage lytic promoter, as well as cellular fate, in single infected bacteria. Intriguingly, we found that, whereas persistent bacteria are protected from prophage induction, they are not protected from lytic infection. Quantitative analysis of gene expression reveals that the expression of lytic genes is suppressed in persistent bacteria. However, when persistent bacteria switch to normal growth, the infecting phage resumes the process of gene expression, ultimately causing cell lysis. Using mathematical models for these two host–phage interactions, we found that the bacteria's nongenetic individuality can significantly affect the population dynamics, and might be relevant for understanding the coevolution of bacterial hosts and phages.
Author Summary
Persistence of subpopulations of bacteria to antibiotic treatments is a major problem in recurrent infections. Unlike resistance, which is passed on to the next generations, persistence is a transient trait characterized by slow growth or dormancy. It has been suggested that the existence of both persister and non-persister bacteria within a given population might constitute a general strategy that bacterial populations use to cope with an ever-changing, stressful environment. Here, we studied the influence of persistence on the interaction between bacterial populations and viruses that infect bacteria, called phages. We found that persistence provides a clear advantage for lysogenic bacteria—in which the phage DNA has integrated into the host DNA but remains mostly inactive—as they enter the reversal of this state, typically in response to environmental stress. This suggests that persistence might have evolved in lysogenic bacteria under stressful conditions. In contrast, persister bacteria do not survive infections by lytic phages—which replicate until they cause the host cell to burst—any better than non-persister bacteria, but release the infectious phages on a significantly longer time scale. Mathematical analysis reveals that this host heterogeneity might substantially affect host–phage population dynamics and could be relevant for other predator–prey systems.
Mathematical analysis and single-cell observations of bacteria persistent to antibiotic treatments shed new light on the ecology of bacteria and phages.
doi:10.1371/journal.pbio.0060120
PMCID: PMC2386839  PMID: 18494559
15.  Cytological and Transcript Analyses Reveal Fat and Lazy Persister-Like Bacilli in Tuberculous Sputum 
PLoS Medicine  2008;5(4):e75.
Background
Tuberculous sputum provides a sample of bacilli that must be eliminated by chemotherapy and that may go on to transmit infection. A preliminary observation that Mycobacterium tuberculosis cells contain triacylglycerol lipid bodies in sputum, but not when growing in vitro, led us to investigate the extent of this phenomenon and its physiological basis.
Methods and Findings
Microscopy-positive sputum samples from the UK and The Gambia were investigated for their content of lipid body–positive mycobacteria by combined Nile red and auramine staining. All samples contained a lipid body–positive population varying from 3% to 86% of the acid-fast bacilli present. The recent finding that triacylglycerol synthase is expressed by mycobacteria when they enter in vitro nonreplicating persistence led us to investigate whether this state was also associated with lipid body formation. We found that, when placed in laboratory conditions inducing nonreplicating persistence, two M. tuberculosis strains had lipid body levels comparable to those found in sputum. We investigated these physiological findings further by comparing the M. tuberculosis transcriptome of growing and nonreplicating persistence cultures with that obtained directly from sputum samples. Although sputum has traditionally been thought to contain actively growing tubercle bacilli, our transcript analyses refute the hypothesis that these cells predominate. Rather, they reinforce the results of the lipid body analyses by revealing transcriptional signatures that can be clearly attributed to slowly replicating or nonreplicating mycobacteria. Finally, the lipid body count was highly correlated (R2 = 0.64, p < 0.03) with time to positivity in diagnostic liquid cultures, thereby establishing a direct link between this cytological feature and the size of a potential nonreplicating population.
Conclusion
As nonreplicating tubercle bacilli are tolerant to the cidal action of antibiotics and resistant to multiple stresses, identification of this persister-like population of tubercle bacilli in sputum presents exciting and tractable new opportunities to investigate both responses to chemotherapy and the transmission of tuberculosis.
Studying sputum from humans with pulmonary tuberculosis, Michael Barer and colleagues detect mycobacteria containing lipid bodies. Analyses linking this cytological feature to a slow-growing phenotype sheds light on persistence.
Editors' Summary
Background.
Every year, nearly nine million people develop tuberculosis—a contagious infection usually of the lungs—and about two million people die from the disease. Tuberculosis is caused by Mycobacterium tuberculosis, bacteria that are spread in airborne droplets when people with the disease cough or sneeze. The symptoms of tuberculosis include a persistent cough, weight loss, and night sweats. Diagnostic tests include chest X-rays, the tuberculin skin test, and sputum analysis. For the last of these tests, a sample of sputum (mucus and other matter brought up from the lungs by coughing) is collected and then taken to a laboratory where bacteriologists look for M. tuberculosis using special stains—tuberculosis-positive sputum contains “acid-fast bacilli”—and also try to grow bacteria from the sample. Tuberculosis can be cured by taking several powerful antibiotics for several months. It is very important that this treatment is completed to ensure that all the M. tuberculosis bacteria in the body are killed and to prevent the emergence of drug-resistant bacteria.
Why Was This Study Done?
Strenuous efforts are being made to reduce the global burden of tuberculosis but with limited success so far for many reasons. One barrier to success is the efficiency with which M. tuberculosis spreads from one person to another. Very little is known about this part of the bacteria's life cycle. If scientists could understand more about the transmission of M. tuberculosis between people, they might identify new therapeutic and preventative targets. In the study, therefore, the researchers examine the acid-fast bacilli in tuberculosis-positive sputum samples to get a snapshot of M. tuberculosis at the point of its transmission to a new person and ask how the characteristics of these bacilli compare with those of M. tuberculosis growing in the laboratory.
What Did the Researchers Do and Find?
The researchers collected sputum samples from patients with tuberculosis in the UK and The Gambia before they received any treatment, and looked for the presence of acid-fast bacilli containing “lipid bodies.” These small structures contain a fat called triacylglycerol. M. tuberculosis accumulates triacylglycerol when it is exposed to several stresses present during infection (for example, reduced oxygen or hypoxia) and the researchers suggest that the presence of this fat may help the bacteria survive during transmission and establish a new infection. They found that all the samples contained some lipid body–positive acid-fast bacilli. Next, the researchers showed that M. tuberculosis grown in the laboratory under hypoxic conditions, which induce the bacteria to enter an antibiotic-tolerant condition called a “nonreplicating persistent” (NRP) state, also accumulated lipid bodies. This result suggests that the lipid body–positive acid-fast bacilli in sputum might be in an NRP state. To test this idea, the researchers compared the pattern of mRNAs (the templates from which proteins are produced; the pattern of mRNAs is called the transcriptome and gives an idea of which proteins a cell is making under given conditions) made by growing cultures of M. tuberculosis, by M. tuberculosis maintained in the NRP state, and by the acid-fast bacilli in several sputum samples. The transcriptome of the sputum sample revealed production of many proteins made in the NRP state. Finally, the researchers showed that the time needed to grow M. tuberculosis from sputum samples increased as the proportion of lipid body–positive acid-fast bacilli in the sputum increased, just as one would suspect if the presence of lipid bodies signifies nongrowing cells.
What Do These Findings Mean?
It has been generally assumed that the acid-fast bacilli in sputum collected from patients with tuberculosis are rapidly replicating M. tuberculosis released from infected areas of the lungs. By identifying a population of bacteria that contain lipid bodies and that are in an NRP-like state in all the samples of sputum examined from two geographical sites, this study strongly challenges this assumption. The characteristics of this population of bacteria, the researchers suggest, might help them survive the adverse conditions that M. tuberculosis encounters during transmission between people and might partly explain why complete clearance of M. tuberculosis requires extended treatment with antibiotics. To establish the clinical significance of these findings, future studies will need to examine whether antibiotic treatment affects the frequency of lipid body–positive M. tuberculosis bacteria in patients' sputum and whether there is any relationship between this measurement and infectiousness, or clinical response to treatment.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050075.
The MedlinePlus encyclopedia contains pages on tuberculosis and on sputum culture (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases provides information on all aspects of tuberculosis
The US Centers for Disease Control and Prevention Division of Tuberculosis Elimination provides several fact sheets and other information resources about tuberculosis
The World Health Organization provides information on efforts to reduce the global burden of tuberculosis
doi:10.1371/journal.pmed.0050075
PMCID: PMC2276522  PMID: 18384229
16.  Structural and Molecular Basis for Resistance to Aminoglycoside Antibiotics by the Adenylyltransferase ANT(2″)-Ia 
mBio  2015;6(1):e02180-14.
ABSTRACT  
The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg2+ ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4′)-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules.
Importance   To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular problem among Gram-negative human pathogens, since there are very few therapeutic options for infections caused by these organisms. In order to successfully circumvent or inhibit the activity of aminoglycoside-modifying enzymes, and to thus rejuvenate the activity of the aminoglycoside antibiotics against Gram-negative pathogens, structural and mechanistic information is crucial. This study reveals the structure of a clinically prevalent aminoglycoside resistance enzyme [ANT(2″)-Ia] and depicts the molecular basis underlying modification of antibiotic substrates. Combined, these findings provide the groundwork for the development of broad-spectrum inhibitors against antibiotic nucleotidyltransferases.
Importance  
To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular problem among Gram-negative human pathogens, since there are very few therapeutic options for infections caused by these organisms. In order to successfully circumvent or inhibit the activity of aminoglycoside-modifying enzymes, and to thus rejuvenate the activity of the aminoglycoside antibiotics against Gram-negative pathogens, structural and mechanistic information is crucial. This study reveals the structure of a clinically prevalent aminoglycoside resistance enzyme [ANT(2″)-Ia] and depicts the molecular basis underlying modification of antibiotic substrates. Combined, these findings provide the groundwork for the development of broad-spectrum inhibitors against antibiotic nucleotidyltransferases.
doi:10.1128/mBio.02180-14
PMCID: PMC4313920  PMID: 25564464
17.  Evolutionary Trajectories of Beta-Lactamase CTX-M-1 Cluster Enzymes: Predicting Antibiotic Resistance 
PLoS Pathogens  2010;6(1):e1000735.
Extended-spectrum beta-lactamases (ESBL) constitute a key antibiotic-resistance mechanism affecting Gram-negative bacteria, and also an excellent model for studying evolution in real time. A shift in the epidemiology of ESBLs is being observed, which is characterized by the explosive diversification and increase in frequency of the CTX-M-type β-lactamases in different settings. This provides a unique opportunity for studying a protein evolutionary radiation by the sequential acquisition of specific mutations enhancing protein efficiency and fitness concomitantly. The existence of driver antibiotic molecules favoring protein divergence has been investigated by combining evolutionary analyses and experimental site-specific mutagenesis. Phylogenetic reconstruction with all the CTX-M variants described so far provided a hypothetical evolutionary scenario showing at least three diversification events. CTX-M-3 was likely the enzyme at the origin of the diversification in the CTX-M-1 cluster, which was coincident with positive selection acting on several amino acid positions. Sixty-three CTX-M-3 derivatives containing all combinations of mutations under positively selected positions were constructed, and their phenotypic efficiency was evaluated. The CTX-M-3 diversification process can only be explained in a complex selective landscape with at least two antibiotics (cefotaxime and ceftazidime), indicating the need to invoke mixtures of selective drivers in order to understand the final evolutionary outcome. Under this hypothesis, we found congruent results between the in silico and in vitro analyses of evolutionary trajectories. Three pathways driving the diversification of CTX-M-3 towards the most complex and efficient variants were identified. Whereas the P167S pathway has limited possibilities of further diversification, the D240G route shows a robust diversification network. In the third route, drift may have played a role in the early stages of CTX-M-3 evolution. Antimicrobial agents should not be considered only as selectors for efficient mechanisms of resistance but also as diversifying agents of the evolutionary trajectories. Different trajectories were identified using a combination of phylogenetic reconstructions and directed mutagenesis analyses, indicating that such an approach might be useful to fulfill the desirable goal of predicting evolutionary trajectories in antimicrobial resistance.
Author Summary
Antimicrobial resistance in bacterial organisms is a worldwide problem widely discussed from clinical, economical and social points of view. The number of new resistance mechanisms and microorganisms resistant to new drugs is increasing all over the world. The development and spread of antibiotic resistance in bacterial communities represents an excellent model for testing the predictive potential of evolutionary principles at short time-scales. A number of studies have tried to predict the selection of resistant variants when a new drug is commercialized. However, in many cases there is no correlation between in vitro predictions and in-field observations. For this reason, it can be suspected that the ability to predict the emergence of new resistant variants might be incomplete unless we know the evolutionary forces acting on the genetic diversification processes. Using the CTX-M β-lactamases as a model, a combination of molecular phylogenetic approaches and experimental site-specific mutagenesis has allowed us to establish evolutionary trajectories. We have demonstrated that two synthetic antibiotics, cefotaxime and ceftazidime, were the selective forces driving the diversification of CTX-M enzymes, but only when both antibiotics were simultaneously present in the environment. We also predict that, if the current selective landscape is not modified, variants carrying the mutation D240G will be more prevalent and diverse in the future.
doi:10.1371/journal.ppat.1000735
PMCID: PMC2809773  PMID: 20107608
18.  Pervasive Sign Epistasis between Conjugative Plasmids and Drug-Resistance Chromosomal Mutations 
PLoS Genetics  2011;7(7):e1002181.
Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)—including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.
Author Summary
Bacteria can become resistant to antibiotics by spontaneous mutation of chromosomal genes or through the acquisition of horizontally mobile genetic elements, mainly conjugative plasmids. Plasmid-borne resistance is widespread among bacterial pathogens. Plasmids generally entail a cost to the host, associated with the replication and maintenance of the genetic element and with the expression of its genes. Therefore, in the absence of antibiotic, both plasmids and resistance mutations are often deleterious and confer a fitness cost to the cell. Here we studied epistatic interactions between five natural conjugative plasmids and ten chromosomal mutations conferring resistance to three types of antibiotics, making a total of 50 different combinations of chromosomal mutations and conjugative plasmids. We show that sometimes plasmids confer an advantage to bacterial strains carrying resistance mutations in their chromosome. This occurs in 32% (16 out of 50) of tested combinations. Furthermore, in 5 out of 50 plasmid-mutations combinations studied (10%), we observed an increased fitness when a plasmid-bearing bacterial cell acquires a drug-resistant mutation. These examples of sign epistasis are highly unexpected. This work explains, at least in part, how multidrug resistance evolved so rapidly.
doi:10.1371/journal.pgen.1002181
PMCID: PMC3145620  PMID: 21829372
19.  Compensatory Evolution of pbp Mutations Restores the Fitness Cost Imposed by β-Lactam Resistance in Streptococcus pneumoniae 
PLoS Pathogens  2011;7(2):e1002000.
The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.
Author Summary
For many years, pneumococcal infections have been usually treated with β-lactams. However, the rapid emergence of β-lactam resistance has complicated the antimicrobial treatment of these infections in the last two decades. The emergence and stability of antibiotic resistance is a complex biological process driven by different factors, such as the volume of antibiotic used. Furthermore, many studies on the effect of a reduction in β-lactam consumption have reported a sustained resistance level to S. pneumoniae, suggesting that other factors contribute to the persistence of β-lactam resistance. By horizontal gene transfer, S. pneumoniae is able to acquire genes from resistant strains or the commensal streptococci, which confer β-lactam resistance. Here, we show that when certain resistance genes are acquired individually, an important cost results in the bacterial fitness. However, some clinical strains which have acquired genes that increase β-lactam resistance can also compensate the fitness cost imposed by this resistance, thereby producing a selective advantage and raising the potential spreading of β-lactam resistance. We suggest that pbp1a and pbp2x mutant alleles are acquired for their compensatory effect on fitness in addition to their contribution in developing higher β-lactam resistance levels, and that this process may occur even in the absence of antibiotics.
doi:10.1371/journal.ppat.1002000
PMCID: PMC3040684  PMID: 21379570
20.  A Multicentre Study of Shigella Diarrhoea in Six Asian Countries: Disease Burden, Clinical Manifestations, and Microbiology 
PLoS Medicine  2006;3(9):e353.
Background
The burden of shigellosis is greatest in resource-poor countries. Although this diarrheal disease has been thought to cause considerable morbidity and mortality in excess of 1,000,000 deaths globally per year, little recent data are available to guide intervention strategies in Asia. We conducted a prospective, population-based study in six Asian countries to gain a better understanding of the current disease burden, clinical manifestations, and microbiology of shigellosis in Asia.
Methods and Findings
Over 600,000 persons of all ages residing in Bangladesh, China, Pakistan, Indonesia, Vietnam, and Thailand were included in the surveillance. Shigella was isolated from 2,927 (5%) of 56,958 diarrhoea episodes detected between 2000 and 2004. The overall incidence of treated shigellosis was 2.1 episodes per 1,000 residents per year in all ages and 13.2/1,000/y in children under 60 months old. Shigellosis incidence increased after age 40 years. S. flexneri was the most frequently isolated Shigella species (1,976/2,927 [68%]) in all sites except in Thailand, where S. sonnei was most frequently detected (124/146 [85%]). S. flexneri serotypes were highly heterogeneous in their distribution from site to site, and even from year to year. PCR detected ipaH, the gene encoding invasion plasmid antigen H in 33% of a sample of culture-negative stool specimens. The majority of S. flexneri isolates in each site were resistant to amoxicillin and cotrimoxazole. Ciprofloxacin-resistant S. flexneri isolates were identified in China (18/305 [6%]), Pakistan (8/242 [3%]), and Vietnam (5/282 [2%]).
Conclusions
Shigella appears to be more ubiquitous in Asian impoverished populations than previously thought, and antibiotic-resistant strains of different species and serotypes have emerged. Focusing on prevention of shigellosis could exert an immediate benefit first by substantially reducing the overall diarrhoea burden in the region and second by preventing the spread of panresistant Shigella strains. The heterogeneous distribution of Shigella species and serotypes suggest that multivalent or cross-protective Shigella vaccines will be needed to prevent shigellosis in Asia.
A prospective, population-based study in six Asian countries showed thatShigella appears to be more ubiquitous in Asian impoverished populations than previously thought, and antibiotic-resistant strains have emerged.
Editors' Summary
Background.
Infections that cause diarrhea are a major public health problem in developing countries and other places where resources are scarce, particularly in young children. Although deaths from diarrhea have decreased considerably in recent decades, diarrheal illnesses continue to cause some 2.5 million deaths each year. Shigella, a group of rod-shaped bacteria closely related to those that normally live in the human intestine, is known to cause severe diarrhea in both developed and developing countries, but the global impact of Shigella infection (shigellosis) has not been well characterized. Shigella exists in more than 40 different varieties, an increasing number of cases have been found to be resistant to available antibiotics, and no vaccine is licensed except one oral vaccine in China.
Why Was This Study Done?
The best information available on the impact of shigellosis has been based on historical estimates, which are subject to inaccuracy. More recent studies suggest that the older reports may have underestimated the impact of shigellosis. The authors of this study wanted to obtain more accurate, current estimates of the impact of shigellosis in developing countries.
In addition, immunity to one type of Shigella does not necessarily provide protection against other types. Therefore, in order to develop an effective vaccine, researchers would need to know which types of Shigella are causing illness in affected parts of the world. Accordingly, the authors of this study also wanted to investigate the specific types of Shigella (called “serotypes” because they can be distinguished using serum from immune individuals) involved in cases of diarrhea.
What Did the Researchers Do and Find?
The researchers set up surveillance projects for diarrhea in six developing countries throughout Asia: at three rural or semirural sites (in China, Vietnam, and Thailand) and three urban slum sites (in Bangladesh, Pakistan, and Indonesia). They conducted information campaigns in each area to encourage residents to visit a participating clinic if they or their children developed diarrhea. Patients presenting with diarrhea were enrolled in the study and their medical findings were documented on standardized report forms. Stool or rectal swab specimens were obtained (with patient consent) and sent to laboratories to test for Shigella. When Shigella was identified, the bacteria was serotyped and tested for resistance to antibiotics. Because standard culture methods do not always detect Shigella when it is present, as a double-check, the researchers also tested some of the specimens for a type of DNA (called the ipaH gene) that serves as a molecular “footprint” of Shigella. Patients received treatment according to national guidelines.
The study involved approximately 600,000 participants over 1–3 years, and detected approximately 60,000 cases of diarrhea. Shigella was found in 5% of diarrhea episodes, meaning that two new cases of shigellosis occurred per 1,000 people (of all ages) per year. Rates were higher in children and in people over age 40. Among children less than 5 years old, there were 13 new cases per 1,000 children per year. Rates of shigellosis were higher in the Bangladesh site than in the China, Pakistan, and Indonesia sites, which in turn had higher rates than the Vietnam and Thailand sites.
In contrast to prior studies, no deaths were detected following episodes of shigellosis, and less than one-third of cases of shigellosis were associated with bloody diarrhea (dysentery).
The distribution of serotypes was found to differ from one site to another and within a given site over time. A high percentage of Shigella detected at all sites were resistant to two or more antibiotics. Testing for the ipaH gene was able to identify Shigella in half of patients with bloody diarrhea whose routine stool cultures did not reveal Shigella.
What Do These Findings Mean?
This study found that shigellosis occurs in these Asian sites at a rate approximately 100 times higher than in industrialized countries. The finding that shigellosis frequently occurs in the absence of bloody stool means that government data collections using dysentery as part of the case definition can be expected to miss the majority of shigellosis cases. Also, the increased rate of shigellosis above age 40 shows that older people share significantly in the burden (and most likely the transmission) of shigellosis.
The generally benign clinical course of Shigella-associated diarrhea calls into question the priority that this disease should receive in global vaccine development efforts, especially given the technological challenges posed by the complex and variable distribution of serotypes. Nonetheless, the emergence of multidrug-resistant strains clearly remains a threat, and raises the perennial issue of improved sanitation, rather than new antibiotics, as a long-term solution to the plethora of water-borne illnesses that disproportionately affect developing countries.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030353.
World Health Organization topic page on diarrhea
Centers for Disease Control and Prevention: Shigellosis
Wikipedia entry on Shigella (note: Wikipedia is a free Internet encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030353
PMCID: PMC1564174  PMID: 16968124
21.  Quinolone Resistance in Absence of Selective Pressure: The Experience of a Very Remote Community in the Amazon Forest 
Background
Quinolones are potent broad-spectrum bactericidal agents increasingly employed also in resource-limited countries. Resistance to quinolones is an increasing problem, known to be strongly associated with quinolone exposure. We report on the emergence of quinolone resistance in a very remote community in the Amazon forest, where quinolones have never been used and quinolone resistance was absent in 2002.
Methods
The community exhibited a considerable level of geographical isolation, limited contact with the exterior and minimal antibiotic use (not including quinolones). In December 2009, fecal carriage of antibiotic resistant Escherichia coli was investigated in 120 of the 140 inhabitants, and in 48 animals reared in the community. All fluoroquinolone-resistant isolates were genotyped and characterized for the mechanisms of plasmid- and chromosomal-mediated quinolone resistance.
Principal Findings
Despite the characteristics of the community remained substantially unchanged during the period 2002–2009, carriage of quinolone-resistant E. coli was found to be common in 2009 both in humans (45% nalidixic acid, 14% ciprofloxacin) and animals (54% nalidixic acid, 23% ciprofloxacin). Ciprofloxacin-resistant isolates of human and animal origin showed multidrug resistance phenotypes, a high level of genetic heterogeneity, and a combination of GyrA (Ser83Leu and Asp87Asn) and ParC (Ser80Ile) substitutions commonly observed in fluoroquinolone-resistant clinical isolates of E. coli.
Conclusions
Remoteness and absence of antibiotic selective pressure did not protect the community from the remarkable emergence of quinolone resistance in E. coli. Introduction of the resistant strains from antibiotic-exposed settings is the most likely source, while persistence and dissemination in the absence of quinolone exposure is likely mostly related with poor sanitation. Interventions aimed at reducing the spreading of resistant isolates (by improving sanitation and water/food safety) are urgently needed to preserve the efficacy of quinolones in resource-limited countries, as control strategies based only on antibiotic restriction policies are unlikely to succeed in those settings.
Author Summary
Quinolones are broad-spectrum antibiotics which bind to type II topoisomerases (DNA gyrase and topoisomerase IV) and inhibit DNA re-ligation after enzyme cut, exerting a rapid bactericidal activity. They are widely used for the treatment of several community- and hospital-acquired infections and have become increasingly important also in resource-limited countries, following the availability of generics (which have drastically reduced drug costs) and the remarkable increase of resistance to the oldest and cheapest antibiotic classes. Resistance to quinolones is an increasing worldwide problem that challenges the efficacy of these drugs against several bacterial pathogens and is known to be strongly associated with quinolone exposure. Restriction of quinolone consumption has been advocated as an important tool for the containment of quinolone resistance and has recently been proved to succeed in reducing resistance rates in clinical isolates of Escherichia coli in a community setting from an industrialized country. This study describes the dissemination of quinolone resistant E. coli in a very remote community in the Amazon forest, with a high level isolation and minimal antibiotic use, not including quinolones. These findings demonstrate that intervention strategies based only on quinolone restriction are unlikely to succeed in resource-limited countries, unless accompanied by measures for reducing dissemination of resistant isolates by improving sanitation.
doi:10.1371/journal.pntd.0001790
PMCID: PMC3429404  PMID: 22953012
22.  Pre-Disposition and Epigenetics Govern Variation in Bacterial Survival upon Stress 
PLoS Genetics  2012;8(12):e1003148.
Bacteria suffer various stresses in their unpredictable environment. In response, clonal populations may exhibit cell-to-cell variation, hypothetically to maximize their survival. The origins, propagation, and consequences of this variability remain poorly understood. Variability persists through cell division events, yet detailed lineage information for individual stress-response phenotypes is scarce. This work combines time-lapse microscopy and microfluidics to uniformly manipulate the environmental changes experienced by clonal bacteria. We quantify the growth rates and RpoH-driven heat-shock responses of individual Escherichia coli within their lineage context, stressed by low streptomycin concentrations. We observe an increased variation in phenotypes, as different as survival from death, that can be traced to asymmetric division events occurring prior to stress induction. Epigenetic inheritance contributes to the propagation of the observed phenotypic variation, resulting in three-fold increase of the RpoH-driven expression autocorrelation time following stress induction. We propose that the increased permeability of streptomycin-stressed cells serves as a positive feedback loop underlying this epigenetic effect. Our results suggest that stochasticity, pre-disposition, and epigenetic effects are at the source of stress-induced variability. Unlike in a bet-hedging strategy, we observe that cells with a higher investment in maintenance, measured as the basal RpoH transcriptional activity prior to antibiotic treatment, are more likely to give rise to stressed, frail progeny.
Author Summary
Individual organisms of identical genetic background, living in a homogeneous constant environment, may nonetheless exhibit observable differences dubbed phenotypic plasticity or variability. When such a population is challenged with an unforeseen stress, the disparity among individuals may increase, yielding different strategies in response. This work addresses the occurrence and propagation of phenotypic variation as it affects bacterial survival in response to mild antibiotic treatments. We recorded images of single bacterial cells as they divide prior to and during exposure to a sub-lethal level of streptomycin, a ribosome-targeted antibiotic. We found that individual differences increase upon stress to the extent that cells may either die or survive the treatment. Differentiation events were traced back prior to exposure. We suggest that a positive feedback loop, governed by increased membrane permeability, underlies the transient cell memory observed. Cells with relatively high basal stress-response levels prior to stress are not primed for better survival, but are rather more likely to succumb to antibiotic treatment. As pathogens commonly encounter sub-lethal doses of antibiotics, their survival may be better understood in light of this study.
doi:10.1371/journal.pgen.1003148
PMCID: PMC3527273  PMID: 23284305
23.  The Population and Evolutionary Dynamics of Homologous Gene Recombination in Bacteria 
PLoS Genetics  2009;5(8):e1000601.
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.
Author Summary
For many species of bacteria, recombination in the form of the acquisition and expression of genes and genetic elements acquired from other bacteria, eukaryotes, and archaea, HGT is an important source of variation for adaptive evolution. Not so clear is the contribution of recombination of homologous genes to adaptive evolution and as a selective pressure for the evolution and maintenance of HGT. Using computer simulations, we explore the role of HGR to adaptive evolution and selection for the evolution and maintenance of HGT. We demonstrate that under realistic conditions by shuffling genes within a bacterial population, HGR will increase its rate of adaptive evolution. Once established, this capacity to increase the rate of adaptive evolution can serve as a selective force for the maintenance of HGT. On the other hand, HGR cannot provide an advantage to a population when its density is low or when the recombining population is rare relative to non-recombining competitors. Thus, we postulate that it is unlikely that the only bacteria—rather than plasmid (or phage)—determined mechanism of HGR, transformation, evolved in response to selection for higher rates of evolution by gene shuffling.
doi:10.1371/journal.pgen.1000601
PMCID: PMC2717328  PMID: 19680442
24.  Sub-lethal antibiotic treatment leads to multidrug resistance via radical-induced mutagenesis 
Molecular cell  2010;37(3):311-320.
Summary
Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here we show that sub-lethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS, and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sub-lethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sub-lethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics.
doi:10.1016/j.molcel.2010.01.003
PMCID: PMC2840266  PMID: 20159551
25.  Strategies and molecular tools to fight antimicrobial resistance: resistome, transcriptome, and antimicrobial peptides 
The increasing number of antibiotic resistant bacteria motivates prospective research toward discovery of new antimicrobial active substances. There are, however, controversies concerning the cost-effectiveness of such research with regards to the description of new substances with novel cellular interactions, or description of new uses of existing substances to overcome resistance. Although examination of bacteria isolated from remote locations with limited exposure to humans has revealed an absence of antibiotic resistance genes, it is accepted that these genes were both abundant and diverse in ancient living organisms, as detected in DNA recovered from Pleistocene deposits (30,000 years ago). Indeed, even before the first clinical use of antibiotics more than 60 years ago, resistant organisms had been isolated. Bacteria can exhibit different strategies for resistance against antibiotics. New genetic information may lead to the modification of protein structure affecting the antibiotic carriage into the cell, enzymatic inactivation of drugs, or even modification of cellular structure interfering in the drug-bacteria interaction. There are still plenty of new genes out there in the environment that can be appropriated by putative pathogenic bacteria to resist antimicrobial agents. On the other hand, there are several natural compounds with antibiotic activity that may be used to oppose them. Antimicrobial peptides (AMPs) are molecules which are wide-spread in all forms of life, from multi-cellular organisms to bacterial cells used to interfere with microbial growth. Several AMPs have been shown to be effective against multi-drug resistant bacteria and have low propensity to resistance development, probably due to their unique mode of action, different from well-known antimicrobial drugs. These substances may interact in different ways with bacterial cell membrane, protein synthesis, protein modulation, and protein folding. The analysis of bacterial transcriptome may contribute to the understanding of microbial strategies under different environmental stresses and allows the understanding of their interaction with novel AMPs.
doi:10.3389/fmicb.2013.00412
PMCID: PMC3876575  PMID: 24427156
resistome; transcription; genetic; molecular modeling; antimicrobial peptides; NGS applications

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