Emergence and spread of antibiotic resistance following exposure to antibiotics

Within a susceptible wild-type population, a small fraction of cells, even <10(-9) , is not affected when challenged by an antimicrobial agent. This subpopulation has mutations that impede antimicrobial action, allowing their selection during clinical treatment. Emergence of resistance occurs in the frame of a selective compartment termed a mutant selection window (MSW). The lower margin corresponds to the minimum inhibitory concentration of the susceptible cells, whereas the upper boundary, named the mutant prevention concentration (MPC), restricts the growth of the entire population, including that of the resistant mutants. By combining pharmacokinetic/pharmacodynamic concepts and an MPC strategy, the selection of resistant mutants can be limited. Early treatment avoiding an increase of the inoculum size as well as a regimen restricting the time within the MSW can reduce the probability of emergence of the resistant mutants. Physiological and, possibly, genetic adaptation in biofilms and a high proportion of mutator clones that may arise during chronic infections influence the emergence of resistant mutants. Moreover, a resistant population can emerge in a specific selective compartment after acquiring a resistance trait by horizontal gene transfer, but this may also be avoided to some extent when the MPC is reached. Known linkage between antimicrobial use and resistance should encourage actions for the design of antimicrobial treatment regimens that minimize the emergence of resistance. Emergence of a resistant bacterial subpopulation within a susceptible wild-type population can be restricted with a regimen using an antibiotic dose that is sufficiently high to inhibit both susceptible and resistant bacteria.     

Exploring the role of the immune response in preventing antibiotic resistance

For many bacterial infections, drug resistant mutants are likely present by the time antibiotic treatment starts. Nevertheless, such infections are often successfully cleared. It is commonly assumed that this is due to the combined action of drug and immune response, the latter facilitating clearance of the resistant population. However, most studies of drug resistance emergence during antibiotic treatment focus almost exclusively on the dynamics of bacteria and the drug and neglect the contribution of immune defenses. Here, we develop and analyze several mathematical models that explicitly include an immune response. We consider different types of immune responses and investigate how each impacts the emergence of resistance. We show that an immune response that retains its strength despite a strong drug-induced decline of bacteria numbers considerably reduces the emergence of resistance, narrows the mutant selection window, and mitigates the effects of non-adherence to treatment. Additionally, we show that compared to an immune response that kills bacteria at a constant rate, one that trades reduced killing at high bacterial load for increased killing at low bacterial load is sometimes preferable. We discuss the predictions and hypotheses derived from this study and how they can be tested experimentally.     

Selection of resistant bacteria at very low antibiotic concentrations

The widespread use of antibiotics is selecting for a variety of resistance mechanisms that seriously challenge our ability to treat bacterial infections. Resistant bacteria can be selected at the high concentrations of antibiotics used therapeutically, but what role the much lower antibiotic concentrations present in many environments plays in selection remains largely unclear. Here we show using highly sensitive competition experiments that selection of resistant bacteria occurs at extremely low antibiotic concentrations. Thus, for three clinically important antibiotics, drug concentrations up to several hundred-fold below the minimal inhibitory concentration of susceptible bacteria could enrich for resistant bacteria, even when present at a very low initial fraction. We also show that de novo mutants can be selected at sub-MIC concentrations of antibiotics, and we provide a mathematical model predicting how rapidly such mutants would take over in a susceptible population. These results add another dimension to the evolution of resistance and suggest that the low antibiotic concentrations found in many natural environments are important for enrichment and maintenance of resistance in bacterial populations.     

Phage-resistant mutations impact bacteria susceptibility to future phage infections and antibiotic response

Bacteriophage (phage) therapy in combination with antibiotic treatment serves as a potential strategy to overcome the continued rise in antibiotic resistance across bacterial pathogens. Understanding the impacts of evolutionary and ecological processes to the phage-antibiotic-resistance dynamic could advance the development of such combinatorial therapy. We tested whether the acquisition of mutations conferring phage resistance may have antagonistically pleiotropic consequences for antibiotic resistance. First, to determine the robustness of phage resistance across different phage strains, we infected resistant Escherichia coli cultures with phage that were not previously encountered. We found that phage-resistant E. coli mutants that gained resistance to a single phage strain maintain resistance to other phages with overlapping adsorption methods. Mutations underlying the phage-resistant phenotype affects lipopolysaccharide (LPS) structure and/or synthesis. Because LPS is implicated in both phage infection and antibiotic response, we then determined whether phage-resistant trade-offs exist when challenged with different classes of antibiotics. We found that only 1 out of the 4 phage-resistant E. coli mutants yielded trade-offs between phage and antibiotic resistance. Surprisingly, when challenged with novobiocin, we uncovered evidence of synergistic pleiotropy for some mutants allowing for greater antibiotic resistance, even though antibiotic resistance was never selected for. Our results highlight the importance of understanding the role of selective pressures and pleiotropic interactions in the bacterial response to phage-antibiotic combinatorial therapy.     

The copper (II) ion as a carrier for the antibiotic capreomycin against Mycobacterium tuberculosis

In recent years, the bacterium responsible for tuberculosis has been increasing its resistance to antibiotics resulting in new multidrug resistant Mycobacterium tuberculosis (MR-TB) and extensively drug-resistant tuberculosis (XDR-TB). In this study we use several analytical techniques including NMR, FT-ICR, TOF-MS, LC–MS and UV/Vis to study the copper–capreomycin complex. The copper (II) cation is used as a carrier for the antibiotic capreomycin. Once this structure was studied using NMR, FT-ICR, and MALDI-TOF-MS, the NIH-NIAID tuberculosis cell line for several Tb strains (including antibiotic resistant strains) were tested against up to seven variations of the copper–capreomycin complex. Different variations of copper improved the efficacy of capreomycin against Tb up to 250 fold against drug resistant strains of Tb.     

Bacterial cheating drives the population dynamics of cooperative antibiotic resistance plasmids

Inactivation of β ‐lactam antibiotics by resistant bacteria is a ‘cooperative’ behavior that may allow sensitive bacteria to survive antibiotic treatment. However, the factors that determine the fraction of resistant cells in the bacterial population remain unclear, indicating a fundamental gap in our understanding of how antibiotic resistance evolves. Here, we experimentally track the spread of a plasmid that encodes a β ‐lactamase enzyme through the bacterial population. We find that independent of the initial fraction of resistant cells, the population settles to an equilibrium fraction proportional to the antibiotic concentration divided by the cell density. A simple model explains this behavior, successfully predicting a data collapse over two orders of magnitude in antibiotic concentration. This model also successfully predicts that adding a commonly used β ‐lactamase inhibitor will lead to the spread of resistance, highlighting the need to incorporate social dynamics into the study of antibiotic resistance.     

Isolated cell behavior drives the evolution of antibiotic resistance

Bacterial antibiotic resistance is typically quantified by the minimum inhibitory concentration (MIC), which is defined as the minimal concentration of antibiotic that inhibits bacterial growth starting from a standard cell density. However, when antibiotic resistance is mediated by degradation, the collective inactivation of antibiotic by the bacterial population can cause the measured MIC to depend strongly on the initial cell density. In cases where this inoculum effect is strong, the relationship between MIC and bacterial fitness in the antibiotic is not well defined. Here, we demonstrate that the resistance of a single, isolated cell—which we call the single‐cell MIC (scMIC)—provides a superior metric for quantifying antibiotic resistance. Unlike the MIC, we find that the scMIC predicts the direction of selection and also specifies the antibiotic concentration at which selection begins to favor new mutants. Understanding the cooperative nature of bacterial growth in antibiotics is therefore essential in predicting the evolution of antibiotic resistance.     

Heteroresistance to beta-lactam antibiotics may often be a stage in the progression to antibiotic resistance

Antibiotic resistance is a growing crisis that threatens many aspects of modern healthcare. Dogma is that resistance often develops due to acquisition of a resistance gene or mutation and that when this occurs, all the cells in the bacterial population are phenotypically resistant. In contrast, heteroresistance (HR) is a form of antibiotic resistance where only a subset of cells within a bacterial population are resistant to a given drug. These resistant cells can rapidly replicate in the presence of the antibiotic and cause treatment failures. If and how HR and resistance are related is unclear. Using carbapenem-resistant Enterobacterales (CRE), we provide evidence that HR to beta-lactams develops over years of antibiotic usage and that it is gradually supplanted by resistance. This suggests the possibility that HR may often develop before resistance and frequently be a stage in its progression, potentially representing a major shift in our understanding of the evolution of antibiotic resistance.

A study of heteroresistance to broad range of beta-lactam antibiotics in clinical isolates of E. coli suggests that it may be an intermediate stage in the development of full antibiotic resistance, representing a shift in our understanding of the evolution of antibiotic resistance.     

Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium

Most chromosomal mutations that cause antibiotic resistance impose fitness costs on the bacteria. This biological cost can often be reduced by compensatory mutations. In Salmonella typhimurium, the nucleotide substitution AAA42 --> AAC in the rpsL gene confers resistance to streptomycin. The resulting amino acid substitution (K42N) in ribosomal protein S12 causes an increased rate of ribosomal proofreading and, as a result, the rate of protein synthesis, bacterial growth and virulence are decreased. Eighty-one independent lineages of the low-fitness, K42N mutant were evolved in the absence of antibiotic to ameliorate the costs. From the rate of fixation of compensated mutants and their fitness, the rate of compensatory mutations was estimated to be > or = 10-7 per cell per generation. The size of the population bottleneck during evolution affected fitness of the adapted mutants: a larger bottleneck resulted in higher average fitness. Only four of the evolved lineages contained streptomycin-sensitive revertants. The remaining 77 lineages contained mutants that were still fully streptomycin resistant, had retained the original resistance mutation and also acquired compensatory mutations. Most of the compensatory mutations, resulting in at least 35 different amino acid substitutions, were novel single-nucleotide substitutions in the rpsD, rpsE, rpsL or rplS genes encoding the ribosomal proteins S4, S5, S12 and L19 respectively. Our results show that the deleterious effects of a resistance mutation can be compensated by an unexpected variety of mutations.     

The resumption of consumption -- a review on tuberculosis

Among all infectious diseases that afflict humans, tuberculosis (TB) remains the deadliest. At present, epidemiologists estimate that one-third of the world population is infected with tubercle bacilli, which is responsible for 8 to 10 million new cases of TB and 3 million deaths annually throughout the world. Approximately 95% of new cases and 98% of deaths occur in developing nations, generally due to the few resources available to ensure proper treatment and where human immunodeficiency virus (HIV) infections are common. In 1882, Dr Robert Koch identified an acid-fast bacterium, Mycobacterium tuberculosis, as the causative agent of TB. Thirty-nine years later, BCG vaccine was introduced for human use, and became the most widely used prophylactic strategy to fight TB in the world. The discovery of the properties of first-line antimycobacterial drugs in the past century yielded effective chemotherapies, which considerably decreased TB mortality rates worldwide. The later introduction of some additional drugs to the arsenal used to treat TB seemed to provide an adequate number of effective antimicrobial agents. The modern, standard short-course therapy for TB recommended by the World Health Organization is based on a four-drug regimen that must be strictly followed to prevent drug resistance acquisition, and relies on direct observation of patient compliance to ensure effective treatment. Mycobacteria show a high degree of intrinsic resistance to most antibiotics and chemotherapeutic agents due to the low permeability of its cell wall. Nevertheless, the cell wall barrier alone cannot produce significant levels of drug resistance. M. tuberculosis mutants resistant to any single drug are naturally present in any large bacterial population, irrespective of exposure to drugs. The frequency of mutants resistant to rifampicin and isoniazid, the two principal antimycobacterial drugs currently in use, is relatively high and, therefore, the large extra-cellular population of actively metabolizing and rapidly growing tubercle bacilli in cavitary lesions will contain organisms which are resistant to a single drug. Consequently, monotherapy or improperly administered two-drug therapies will select for drug-resistant mutants that may lead to drug resistance in the entire bacterial population. Thereby, despite the availability of effective chemotherapy and the moderately protective vaccine, new anti-TB agents are urgently needed to decrease the global incidence of TB. The resumption of TB, mainly caused by the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and HIV epidemics, led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. The latter should be effective to combat both drug-susceptible and MDR/XDR-TB.     

Dynamics of the interaction between Pseudomonas aeruginosa bacteriophage phimF81 and host bacteria]

The population interactions of Pseudomonas aeruginosa virulent bacteriophage phi mF81 with host bacterial cells were studied in dynamics under the conditions of continuous cultivation in the chemostat regime with glucose limitation. It was detected that a maintenance of the bacterium and its specific bacteriophage in the population was realized due to the successive appearance of bacterial mutants resistant to the phage and of phage mutants overcoming this resistance.     

Genetic antagonism and hypermutability in Mycobacterium smegmatis

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.     

Evolution of antibiotic resistance by human and bacterial niche construction

Antibiotic treatment by humans generates strong viability selection for antibiotic-resistant bacterial strains. The frequency of host antibiotic use often determines the strength of this selection, and changing patterns of antibiotic use can generate many types of behaviors in the population dynamics of resistant and sensitive bacterial populations. In this paper, we present a simple model of hosts dimorphic for their tendency to use/avoid antibiotics and bacterial pathogens dimorphic in their resistance/sensitivity to antibiotic treatment. When a constant fraction of hosts uses antibiotics, the two bacterial strain populations can coexist unless host use-frequency is above a critical value; this critical value is derived as the ratio of the fitness cost of resistance to the fitness cost of undergoing treatment. When strain frequencies can affect host behavior, the dynamics may be analyzed in the light of niche construction. We consider three models underlying changing host behavior: conformism, the avoidance of long infections, and adherence to the advice of public health officials. In the latter two, we find that the pathogen can have quite a strong effect on host behavior. In particular, if antibiotic use is discouraged when resistance levels are high, we observe a classic niche-construction phenomenon of maintaining strain polymorphism even in parameter regions where it would not be expected.     

Predicting antibiotic resistance

The treatment of bacterial infections is increasingly complicated because microorganisms can develop resistance to antimicrobial agents. This article discusses the information that is required to predict when antibiotic resistance is likely to emerge in a bacterial population. Indeed, the development of the conceptual and methodological tools required for this type of prediction represents an important goal for microbiological research. To this end, we propose the establishment of methodological guidelines that will allow researchers to predict the emergence of resistance to a new antibiotic before its clinical introduction.     

Microbial phenotypic heterogeneity and antibiotic tolerance

Phenotypic heterogeneity, defined as metastable variation in cellular parameters generated by epigenetic mechanisms, is crucial for the persistence of bacterial populations under fluctuating selective pressures. Diversity ensures that some individuals will survive a potentially lethal stress, such as an antibiotic, that would otherwise obliterate the entire population. The refractoriness of bacterial infections to antibiotic therapy has been ascribed to antibiotic-tolerant variants known as 'persisters'. The persisters are not drug-resistant mutants and it is unclear why they survive antibiotic pressure that kills their genetically identical siblings. Recent conceptual and technological advances are beginning to yield some surprising new insights into the mechanistic basis of this clinically important manifestation of phenotypic heterogeneity.     

Persistence of antibiotic resistance in bacterial populations

Unfortunately for mankind, it is very likely that the antibiotic resistance problem we have generated during the last 60 years due to the extensive use and misuse of antibiotics is here to stay for the foreseeable future. This view is based on theoretical arguments, mathematical modeling, experiments and clinical interventions, suggesting that even if we could reduce antibiotic use, resistant clones would remain persistent and only slowly (if at all) be outcompeted by their susceptible relatives. In this review, we discuss the multitude of mechanisms and processes that are involved in causing the persistence of chromosomal and plasmid-borne resistance determinants and how we might use them to our advantage to increase the likelihood of reversing the problem. Of particular interest is the recent demonstration that a very low antibiotic concentration can be enriching for resistant bacteria and the implication that antibiotic release into the environment could contribute to the selection for resistance. Several mechanisms are contributing to the stability of antibiotic resistance in bacterial populations and even if antibiotic use is reduced it is likely that most resistance mechanisms will persist for considerable times.     

Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440

The genome of the soil bacterium Pseudomonas putida strain KT2440 has been erased of various determinants of resistance to antibiotics encoded in its extant chromosome. To this end, we employed a coherent genetic platform that allowed the precise deletion of multiple genomic segments in a large variety of Gram-negative bacteria including (but not limited to) P. putida. The method is based on the obligatory recombination between free-ended homologous DNA sequences that are released as linear fragments generated upon the cleavage of the chromosome with unique I-SceI sites, added to the segment of interest by the vector system. Despite the potential for a SOS response brought about by the appearance of double stranded DNA breaks during the process, fluctuation experiments revealed that the procedure did not increase mutation rates - perhaps due to the protection exerted by I-SceI bound to the otherwise naked DNA termini. With this tool in hand we made sequential deletions of genes mexC, mexE, ttgA and ampC in the genome of the target bacterium, orthologues of which are known to determine various degrees of antibiotic resistance in diverse microorganisms. Inspection of the corresponding phenotypes demonstrated that the efflux pump encoded by ttgA sufficed to endow P. putida with a high-level of tolerance to β-lactams, chloramphenicol and quinolones, but had little effect on, e.g. aminoglycosides. Analysis of the mutants revealed also a considerable diversity in the manifestation of the resistance phenotype within the population and suggested a degree of synergism between different pumps. The directed edition of the P. putida chromosome shown here not only enhances the amenability of this bacterium to deep genomic engineering, but also validates the corresponding approach for similar handlings of a large variety of Gram-negative microorganisms.     

Influence of antibiotic selection on genetic composition of Escherichia coli populations from conventional and organic dairy farms

The widespread agricultural use of antimicrobials has long been considered a crucial influence on the prevalence of resistant genes and bacterial strains. It has been suggested that antibiotic applications in agricultural settings are a driving force for the development of antimicrobial resistance, and epidemiologic evidence supports the view that there is a direct link between resistant human pathogens, retail produce, farm animals, and farm environments. Despite such concerns, little is understood about the population processes underlying the emergence and spread of antibiotic resistance and the reversibility of resistance when antibiotic selective pressure is removed. In this study, hierarchical log-linear modeling was used to assess the association between farm type (conventional versus organic), age of cattle (calf versus cow), bacterial phenotype (resistant versus susceptible), and the genetic composition of Escherichia coli populations (E. coli Reference Collection [ECOR] phylogroup A, B1, B2, or D) among 678 susceptible and resistant strains from a previously published study of 60 matched dairy farms (30 conventional and 30 organic) in Wisconsin. The analysis provides evidence for clonal resistance (ampicillin resistance) and genetic hitchhiking (tetracycline resistance [Tet(r)]), estimated the rate of compositional change from conventional farming to organic farming (mean, 8 years; range, 3 to 15 years), and discovered a significant association between low multidrug resistance, organic farms, and strains of the numerically dominant phylogroup B1. These data suggest that organic farming practices not only change the frequency of resistant strains but also impact the overall population genetic composition of the resident E. coli flora. In addition, the results support the hypothesis that the current prevalence of Tet(r) loci on dairy farms has little to do with the use of this antibiotic.     

The food safety perspective of antibiotic resistance

Bacterial antimicrobial resistance in both the medical and agricultural fields has become a serious problem worldwide. Antibiotic resistant strains of bacteria are an increasing threat to animal and human health, with resistance mechanisms having been identified and described for all known antimicrobials currently available for clinical use. There is currently increased public and scientific interest regarding the administration of therapeutic and sub-therapeutic antimicrobials to animals, due primarily to the emergence and dissemination of multiple antibiotic resistant zoonotic bacterial pathogens. This issue has been the subject of heated debates for many years, however, there is still no complete consensus on the significance of antimicrobial use in animals, or resistance in bacterial isolates from animals, on the development and dissemination of antibiotic resistance among human bacterial pathogens. In fact, the debate regarding antimicrobial use in animals and subsequent human health implications has been going on for over 30 years, beginning with the release of the Swann report in the United Kingdom. The latest report released by the National Research Council (1998) confirmed that there were substantial information gaps that contribute to the difficulty of assessing potential detrimental effects of antimicrobials in food animals on human health. Regardless of the controversy, bacterial pathogens of animal and human origin are becoming increasingly resistant to most frontline antimicrobials, including expanded-spectrum cephalosporins, aminoglycosides, and even fluoroquinolones. The lion's share of these antimicrobial resistant phenotypes is gained from extra-chromosomal genes that may impart resistance to an entire antimicrobial class. In recent years, a number of these resistance genes have been associated with large, transferable, extra-chromosomal DNA elements, called plasmids, on which may be other DNA mobile elements, such as transposons and integrons. These DNA mobile elements have been shown to transmit genetic determinants for several different antimicrobial resistance mechanisms and may account for the rapid dissemination of resistance genes among different bacteria. The increasing incidence of antimicrobial resistant bacterial pathogens has severe implications for the future treatment and prevention of infectious diseases in both animals and humans. Although much scientific information is available on this subject, many aspects of the development of antimicrobial resistance still remain uncertain. The emergence and dissemination of bacterial antimicrobial resistance is the result of numerous complex interactions among antimicrobials, microorganisms, and the surrounding environments. Although research has linked the use of antibiotics in agriculture to the emergence of antibiotic-resistant foodborne pathogens, debate still continues whether this role is significant enough to merit further regulation or restriction.     

Status of pathogens,antibiotic resistance genes and antibiotic residues in wastewater treatment systems

Pathogens are becoming nearly untreatable due to the rise in gaining new resistance against standard antibiotics. Coexistence of microbial pathogens, antibiotics and antibiotic resistant genes (ARGs) in wastewater treatment plants (WWTP) provide favourable conditions for the development of new antibiotic resistant bacteria (ARB); facilitate horizontal gene transfer among pathogens and may also serve as a hotspot for the spread of ARB and genes into the environment. In this study, the current status of wastewater treatment systems in the removal of pathogens, ARGs, and antibiotic residues are discussed. WWTP are efficient in removing pathogens and antibiotic residues to a greater extend during secondary and tertiary treatment processes. Recent studies, however, have shown high variations in the presence of pathogens including ARB as well as antibiotic resistance genes (ARG) in the final effluent. Prolonged sludge retention time (SRT) and hydraulic retention time (HRT) during secondary treatment will facilitate antibiotic removal by adsorption and biodegradation. However, the above conditions can also lead to the enhancement of antibiotic resistance process in microbes. Therefore, optimum conditions for the operation of conventional WWTP for the efficient removal of antibiotics are yet to be established. The removal of antibiotic residues can be accelerated by combining conventional activated sludge (CAS) process with an additional treatment technology involving dosing with ozone. The advanced biological treatment method using membrane bioreactors (MBR) in combination with coagulation reportedly has the best ARG removal efficiency, and removes both ARB and extracellular ARGs. While studies have predicted the fate for ARGs in wastewater treatment plants, the mechanisms of ARGs acquisition remains to be conclusively established. Thus, strategies to investigate the underlying mechanism of acquisition of ARGs within the WWTP are also provided in this review.