High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

Theoretical considerations and empirical predictions of the pharmaco- and population dynamics of heteroresistance.

Antibiotics are considered one of the most important contributions to clinical medicine in the last century. Due to the use and overuse of these drugs, there have been increasing frequencies of infections with resistant pathogens. One form of resistance, heteroresistance, is particularly problematic; pathogens appear sensitive to a drug by common susceptibility tests. However, upon exposure to the antibiotic, resistance rapidly ascends, and treatment fails. To quantitatively explore the processes contributing to the emergence and ascent of resistance during treatment and the waning of resistance following cessation of treatment, we develop two distinct mathematical and computer-simulation models of heteroresistance. In our analysis of the properties of these models, we consider the factors that determine the response to antibiotic-mediated selection. In one model, heteroresistance is progressive, with each resistant state sequentially generating a higher resistance level. In the other model, heteroresistance is non-progressive, with a susceptible population directly generating populations with different resistance levels. The conditions where resistance will ascend in the progressive model are narrower than those of the non-progressive model. The rates of reversion from the resistant to the sensitive states are critically dependent on the transition rates and the fitness cost of resistance. Our results demonstrate that the standard test used to identify heteroresistance is insufficient. The predictions of our models are consistent with empirical results. Our results demand a reevaluation of the definition and criteria employed to identify heteroresistance. We recommend that the definition of heteroresistance should include a consideration of the rate of return to susceptibility.

Suppression of the E. coli rnpA49 conditionally lethal phenotype by different compensatory mutations.

RNase P is an essential enzyme found across all domains of life that is responsible for the 5'-end maturation of precursor tRNAs. For decades, numerous studies have sought to elucidate the mechanisms and biochemistry governing RNase P function. However, much remains unknown about the regulation of RNase P expression, the turnover and degradation of the enzyme, and the mechanisms underlying the phenotypes and complementation of specific RNase P mutations, especially in the model bacterium, Escherichia coli. In E. coli, the temperature-sensitive rnpA49 mutation in the protein subunit of RNase P has arguably been one of the most well-studied mutations for examining the enzyme's activity in vivo. Here, we report for the first time naturally-occurring temperature-resistant suppressor mutations of E. coli strains carrying the rnpA49 allele. We find that rnpA49 strains can partially compensate the temperature-sensitive defect via gene amplifications of either RNase P subunit (rnpA49 or rnpB) or by the acquisition of loss-of-function mutations in Lon protease or RNase R. Our results agree with previous plasmid overexpression and gene deletion complementation studies, and importantly suggest the involvement of Lon protease in the degradation and/or regulatory pathway(s) of the mutant protein subunit of RNase P. This work offers novel insights into the behavior and complementation of the rnpA49 allele in vivo and provides direction for follow-up studies regarding RNase P regulation and turnover in E. coli.

Low levels of tetracyclines select for a mutation that prevents the evolution of high-level resistance to tigecycline.

In a collection of Escherichia coli isolates, we discovered a new mechanism leading to frequent and high-level tigecycline resistance involving tandem gene amplifications of an efflux pump encoded by the tet(A) determinant. Some isolates, despite carrying a functional tet(A), could not evolve high-level tigecycline resistance by amplification due to the presence of a deletion in the TetR(A) repressor. This mutation impaired induction of tetA(A) (encoding the TetA(A) efflux pump) in presence of tetracyclines, with the strongest effect observed for tigecycline, subsequently preventing the development of tet(A) amplification-dependent high-level tigecycline resistance. We found that this mutated tet(A) determinant was common among tet(A)-carrying E. coli isolates and analysed possible explanations for this high frequency. First, while the mutated tet(A) was found in several ST-groups, we found evidence of clonal spread among ST131 isolates, which increases its frequency within E. coli databases. Second, evolution and competition experiments revealed that the mutation in tetR(A) could be positively selected over the wild-type allele at sub-inhibitory concentrations of tetracyclines. Our work demonstrates how low concentrations of tetracyclines, such as those found in contaminated environments, can enrich and select for a mutation that generates an evolutionary dead-end that precludes the evolution towards high-level, clinically relevant tigecycline resistance.

Pervasive Selection for Clinically Relevant Resistance and Media Adaptive Mutations at Very Low Antibiotic Concentrations.

Experimental evolution studies have shown that weak antibiotic selective pressures (i.e., when the antibiotic concentrations are far below the minimum inhibitory concentration, MIC) can select resistant mutants, raising several unanswered questions. First, what are the lowest antibiotic concentrations at which selection for de novo resistance mutations can occur? Second, with weak antibiotic selections, which other types of adaptive mutations unrelated to the antibiotic selective pressure are concurrently enriched? Third, are the mutations selected under laboratory settings at subMIC also observed in clinical isolates? We addressed these questions using Escherichia coli populations evolving at subMICs in the presence of either of four clinically used antibiotics: fosfomycin, nitrofurantoin, tetracycline, and ciprofloxacin. Antibiotic resistance evolution was investigated at concentrations ranging from 1/4th to 1/2000th of the MIC of the susceptible strain (MICsusceptible). Our results show that evolution was rapid across all the antibiotics tested, and selection for fosfomycin- and nitrofurantoin-resistant mutants was observed at a concentration as low as 1/2000th of MICsusceptible. Several of the evolved resistant mutants showed increased growth yield and exponential growth rates, and outcompeted the susceptible ancestral strain in the absence of antibiotics as well, suggesting that adaptation to the growth environment occurred in parallel with the selection for resistance. Genomic analysis of the resistant mutants showed that several of the mutations selected under these conditions are also found in clinical isolates, demonstrating that experimental evolution at very low antibiotic levels can help in identifying novel mutations that contribute to bacterial adaptation during subMIC exposure in real-life settings.

A novel type of colistin resistance genes selected from random sequence space.

Antibiotic resistance is a rapidly increasing medical problem that severely limits the success of antibiotic treatments, and the identification of resistance determinants is key for surveillance and control of resistance dissemination. Horizontal transfer is the dominant mechanism for spread of resistance genes between bacteria but little is known about the original emergence of resistance genes. Here, we examined experimentally if random sequences can generate novel antibiotic resistance determinants de novo. By utilizing highly diverse expression libraries encoding random sequences to select for open reading frames that confer resistance to the last-resort antibiotic colistin in Escherichia coli, six de novo colistin resistance conferring peptides (Dcr) were identified. The peptides act via direct interactions with the sensor kinase PmrB (also termed BasS in E. coli), causing an activation of the PmrAB two-component system (TCS), modification of the lipid A domain of lipopolysaccharide and subsequent colistin resistance. This kinase-activation was extended to other TCS by generation of chimeric sensor kinases. Our results demonstrate that peptides with novel activities mediated via specific peptide-protein interactions in the transmembrane domain of a sensory transducer can be selected de novo, suggesting that the origination of such peptides from non-coding regions is conceivable. In addition, we identified a novel class of resistance determinants for a key antibiotic that is used as a last resort treatment for several significant pathogens. The high-level resistance provided at low expression levels, absence of significant growth defects and the functionality of Dcr peptides across different genera suggest that this class of peptides could potentially evolve as bona fide resistance determinants in natura.

Molecular mechanisms of collateral sensitivity to the antibiotic nitrofurantoin.

Antibiotic resistance increasingly limits the success of antibiotic treatments, and physicians require new ways to achieve efficient treatment despite resistance. Resistance mechanisms against a specific antibiotic class frequently confer increased susceptibility to other antibiotic classes, a phenomenon designated collateral sensitivity (CS). An informed switch of antibiotic may thus enable the efficient treatment of resistant strains. CS occurs in many pathogens, but the mechanisms that generate hypersusceptibility are largely unknown. We identified several molecular mechanisms of CS against the antibiotic nitrofurantoin (NIT). Mutants that are resistant against tigecycline (tetracycline), mecillinam (ß-lactam), and protamine (antimicrobial peptide) all show CS against NIT. Their hypersusceptibility is explained by the overexpression of nitroreductase enzymes combined with increased drug uptake rates, or increased drug toxicity. Increased toxicity occurs through interference of the native drug-response system for NIT, the SOS response, with growth. A mechanistic understanding of CS will help to develop drug switches that combat resistance.

CombiANT: Antibiotic interaction testing made easy.

Antibiotic combination therapies are important for the efficient treatment of many types of infections, including those caused by antibiotic-resistant pathogens. Combination treatment strategies are typically used under the assumption that synergies are conserved across species and strains, even though recent results show that the combined treatment effect is determined by specific drug-strain interactions that can vary extensively and unpredictably, both between and within bacterial species. To address this problem, we present a new method in which antibiotic synergy is rapidly quantified on a case-by-case basis, allowing for improved combination therapy. The novel CombiANT methodology consists of a 3D-printed agar plate insert that produces defined diffusion landscapes of 3 antibiotics, permitting synergy quantification between all 3 antibiotic pairs with a single test. Automated image analysis yields fractional inhibitory concentration indices (FICis) with high accuracy and precision. A technical validation with 3 major pathogens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, showed equivalent performance to checkerboard methodology, with the advantage of strongly reduced assay complexity and costs for CombiANT. A synergy screening of 10 antibiotic combinations for 12 E. coli urinary tract infection (UTI) clinical isolates illustrates the need for refined combination treatment strategies. For example, combinations of trimethoprim (TMP) + nitrofurantoin (NIT) and TMP + mecillinam (MEC) showed synergy, but only for certain individual isolates, whereas MEC + NIT combinations showed antagonistic interactions across all tested strains. These data suggest that the CombiANT methodology could allow personalized clinical synergy testing and large-scale screening. We anticipate that CombiANT will greatly facilitate clinical and basic research of antibiotic synergy.

Dynamics of Extensive Drug Resistance Evolution of Mycobacterium tuberculosis in a Single Patient During 9 Years of Disease and Treatment.

Mycobacterium tuberculosis is one of the hardest to treat bacterial pathogens with a high capacity to develop antibiotic resistance by mutations. Here we have performed whole-genome sequencing of consecutive M. tuberculosis isolates obtained during 9 years from a patient with pulmonary tuberculosis. The infecting strain was isoniazid resistant and during treatment it stepwise accumulated resistance mutations to 8 additional antibiotics. Heteroresistance was common and subpopulations with up to 3 different resistance mutations to the same drug coexisted. Sweeps of different resistant clones dominated the population at different time points, always coupled to resistance mutations coinciding with changes in the treatment regimens. Resistance mutations were predominant and no hitch-hiking, compensatory, or virulence-increasing mutations were detected, showing that the dominant selection pressure was antibiotic treatment. The results highlight the dynamic nature of M. tuberculosis infection, population structure, and resistance evolution and the importance of rapid antibiotic susceptibility tests to battle this pathogen.

Prevalence of colistin heteroresistance in carbapenem-resistant Pseudomonas aeruginosa and association with clinical outcomes in patients: an observational study.

OBJECTIVES: To describe the prevalence of colistin heteroresistance in carbapenem-resistant Pseudomonas aeruginosa (CRPA) and evaluate the association with clinical outcomes. METHODS: Colistin heteroresistance was evaluated in CRPA isolates collected from patients without cystic fibrosis in Atlanta, Georgia, USA using two definitions: HR1, growth at 4 and 8 mg/L of colistin at a frequency ≥1 × 10-6 the main population; and HR2, growth at a colistin concentration ≥8× the MIC of the main population at a frequency ≥1 × 10-7. A modified population analysis profile (mPAP) technique was compared with reference PAP for detecting heteroresistance. For adults hospitalized at the time of or within 1 week of CRPA culture, multivariable logistic regression estimated the association between heteroresistance and 90 day mortality. RESULTS: Of 143 colistin-susceptible CRPA isolates, 8 (6%) met the HR1 definition and 37 (26%) met the HR2 definition. Compared with the reference PAP, mPAP had a sensitivity and specificity of 50% and 100% for HR1 and 32% and 99% for HR2. Of 82 hospitalized patients, 45 (56%) were male and the median age was 63 years (IQR 49-73). Heteroresistance was not associated with 90 day mortality using HR1 (0% in heteroresistant versus 22% in non-heteroresistant group; P = 0.6) or HR2 (12% in heteroresistant versus 24% in non-heteroresistant group; P = 0.4; adjusted OR 0.8; 95% CI 0.2-3.4). CONCLUSIONS: Colistin heteroresistance was identified in up to 26% of patients with CRPA in our sample, although the prevalence varied depending on the definition. We did not observe an apparent association between colistin heteroresistance and 90 day mortality.

Evolutionary Trajectories to Antibiotic Resistance.

The ability to predict the evolutionary trajectories of antibiotic resistance would be of great value in tailoring dosing regimens of antibiotics so as to maximize the duration of their usefulness. Useful prediction of resistance evolution requires information about (a) the mutation supply rate, (b) the level of resistance conferred by the resistance mechanism, (c) the fitness of the antibiotic-resistant mutant bacteria as a function of drug concentration, and (d) the strength of selective pressures. In addition, processes including epistatic interactions and compensatory evolution, coselection of drug resistances, and population bottlenecks and clonal interference can strongly influence resistance evolution and thereby complicate attempts at prediction. Currently, the very limited quantitative data on most of these parameters severely limit attempts to accurately predict trajectories of resistance evolution.

Evolution of a New Function by Fusion between Phage DNA and a Bacterial Gene.

Mobile genetic elements, such as plasmids, phages, and transposons, are important sources for evolution of novel functions. In this study, we performed a large-scale screening of metagenomic phage libraries for their ability to suppress temperature-sensitivity in Salmonella enterica serovar Typhimurium strain LT2 mutants to examine how phage DNA could confer evolutionary novelty to bacteria. We identified an insert encoding 23 amino acids from a phage that when fused with a bacterial DNA-binding repressor protein (LacI) resulted in the formation of a chimeric protein that localized to the outer membrane. This relocalization of the chimeric protein resulted in increased membrane vesicle formation and an associated suppression of the temperature sensitivity of the bacterium. Both the host LacI protein and the extracellular 23-amino acid stretch are necessary for the generation of the novel phenotype. Furthermore, mutational analysis of the chimeric protein showed that although the native repressor function of the LacI protein is maintained in this chimeric structure, it is not necessary for the new function. Thus, our study demonstrates how a gene fusion between foreign DNA and bacterial DNA can generate novelty without compromising the native function of a given gene.

Selection of Resistant Bacteria in Mallards Exposed to Subinhibitory Concentrations of Ciprofloxacin in Their Water Environment.

Emergence and selection of antibiotic resistance following exposure to high antibiotic concentrations have been repeatedly shown in clinical and agricultural settings, whereas the role of the weak selective pressures exerted by antibiotic levels below the MIC (sub-MIC) in aquatic environments due to anthropogenic contamination remains unclear. Here, we studied how exposure to sub-MIC levels of ciprofloxacin enriches for Escherichia coli with reduced susceptibility to ciprofloxacin using a mallard colonization model. Mallards were inoculated with two isogenic extended-spectrum-ß-lactamase (ESBL)-encoding E. coli strains, differing only by a gyrA mutation that results in increased MICs of ciprofloxacin, and exposed to different levels of ciprofloxacin in their swimming water. Changes in the ratios of mutant to parental strains excreted in feces over time and ESBL plasmid spread within the gut microbiota from individual birds were investigated. Results show that in vivo selection of gyrA mutants occurred in mallards during exposure to ciprofloxacin at concentrations previously found in aquatic environments. During colonization, resistance plasmids were readily transferred between strains in the intestines of the mallards, but conjugation frequencies were not affected by ciprofloxacin exposure. Our results highlight the potential for enrichment of resistant bacteria in wildlife and underline the importance of reducing antibiotic pollution in the environment.

Evolutionary consequences of drug resistance: shared principles across diverse targets and organisms.

Drug therapy has a crucial role in the treatment of viral, bacterial, fungal and protozoan infections, as well as the control of human cancer. The success of therapy is being threatened by the increasing prevalence of resistance. We examine and compare mechanisms of drug resistance in these diverse biological systems (using HIV and Plasmodium falciparum as examples of viral and protozoan pathogens, respectively) and discuss how factors ­ such as mutation rates, fitness effects of resistance, epistasis and clonal interference ­ influence the evolutionary trajectories of drug-resistant clones. We describe commonalities and differences related to resistance development that could guide strategies to improve therapeutic effectiveness and the development of a new generation of drugs.

Cellular hysteresis as a principle to maximize the efficacy of antibiotic therapy.

Antibiotic resistance has become one of the most dramatic threats to global health. While novel treatment options are urgently required, most attempts focus on finding new antibiotic substances. However, their development is costly, and their efficacy is often compromised within short time periods due to the enormous potential of microorganisms for rapid adaptation. Here, we developed a strategy that uses the currently available antibiotics. Our strategy exploits cellular hysteresis, which is the long-lasting, transgenerational change in cellular physiology that is induced by one antibiotic and sensitizes bacteria to another subsequently administered antibiotic. Using evolution experiments, mathematical modeling, genomics, and functional genetic analysis, we demonstrate that sequential treatment protocols with high levels of cellular hysteresis constrain the evolving bacteria by (i) increasing extinction frequencies, (ii) reducing adaptation rates, and (iii) limiting emergence of multidrug resistance. Cellular hysteresis is most effective in fast sequential protocols, in which antibiotics are changed within 12 h or 24 h, in contrast to the less frequent changes in cycling protocols commonly implemented in hospitals. We found that cellular hysteresis imposes specific selective pressure on the bacteria that disfavors resistance mutations. Instead, if bacterial populations survive, hysteresis is countered in two distinct ways, either through a process related to antibiotic tolerance or a mechanism controlled by the previously uncharacterized two-component regulator CpxS. We conclude that cellular hysteresis can be harnessed to optimize antibiotic therapy, to achieve both enhanced bacterial elimination and reduced resistance evolution.

Mutations that increase expression of the EmrAB-TolC efflux pump confer increased resistance to nitroxoline in Escherichia coli.

OBJECTIVES: To determine the mechanism of resistance to the antibiotic nitroxoline in Escherichia coli. METHODS: Spontaneous nitroxoline-resistant mutants were selected at different concentrations of nitroxoline. WGS and strain reconstruction were used to define the genetic basis for the resistance. The mechanistic basis of resistance was determined by quantitative PCR (qPCR) and by overexpression of target genes. Fitness costs of the resistance mutations and cross-resistance to other antibiotics were also determined. RESULTS: Mutations in the transcriptional repressor emrR conferred low-level resistance to nitroxoline [nitroxoline MIC (MICNOX)=16 mg/L] by increasing the expression of the emrA and emrB genes of the EmrAB-TolC efflux pump. These resistant mutants showed no fitness reduction and displayed cross-resistance to nalidixic acid. Second-step mutants with higher-level resistance (MICNOX=32-64 mg/L) had mutations in the emrR gene, together with either a 50 kb amplification, a mutation in the gene marA, or an IS upstream of the lon gene. The latter mutations resulted in higher-level nitroxoline resistance due to increased expression of the tolC gene, which was confirmed by overexpressing tolC from an inducible plasmid in a low-level resistance mutant. Furthermore, the emrR mutations conferred a small increase in resistance to nitrofurantoin only when combined with an nfsAB double-knockout mutation. However, nitrofurantoin-resistant nfsAB mutants showed no cross-resistance to nitroxoline. CONCLUSIONS: Mutations in different genes causing increased expression of the EmrAB-TolC pump lead to an increased resistance to nitroxoline. The structurally similar antibiotics nitroxoline and nitrofurantoin appear to have different modes of action and resistance mechanisms.

Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging.

The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (104 cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.

Structural and functional innovations in the real-time evolution of new (ßα)8 barrel enzymes.

New genes can arise by duplication and divergence, but there is a fundamental gap in our understanding of the relationship between these genes, the evolving proteins they encode, and the fitness of the organism. Here we used crystallography, NMR dynamics, kinetics, and mass spectrometry to explain the molecular innovations that arose during a previous real-time evolution experiment. In that experiment, the (ßα)8 barrel enzyme HisA was under selection for two functions (HisA and TrpF), resulting in duplication and divergence of the hisA gene to encode TrpF specialists, HisA specialists, and bifunctional generalists. We found that selection affects enzyme structure and dynamics, and thus substrate preference, simultaneously and sequentially. Bifunctionality is associated with two distinct sets of loop conformations, each essential for one function. We observed two mechanisms for functional specialization: structural stabilization of each loop conformation and substrate-specific adaptation of the active site. Intracellular enzyme performance, calculated as the product of catalytic efficiency and relative expression level, was not linearly related to fitness. Instead, we observed thresholds for each activity above which further improvements in catalytic efficiency had little if any effect on growth rate. Overall, we have shown how beneficial substitutions selected during real-time evolution can lead to manifold changes in enzyme function and bacterial fitness. This work emphasizes the speed at which adaptive evolution can yield enzymes with sufficiently high activities such that they no longer limit the growth of their host organism, and confirms the (ßα)8 barrel as an inherently evolvable protein scaffold.

Experimental Determination and Prediction of the Fitness Effects of Random Point Mutations in the Biosynthetic Enzyme HisA.

The distribution of fitness effects of mutations is a factor of fundamental importance in evolutionary biology. We determined the distribution of fitness effects of 510 mutants that each carried between 1 and 10 mutations (synonymous and nonsynonymous) in the hisA gene, encoding an essential enzyme in the l-histidine biosynthesis pathway of Salmonella enterica. For the full set of mutants, the distribution was bimodal with many apparently neutral mutations and many lethal mutations. For a subset of 81 single, nonsynonymous mutants most mutations appeared neutral at high expression levels, whereas at low expression levels only a few mutations were neutral. Furthermore, we examined how the magnitude of the observed fitness effects was correlated to several measures of biophysical properties and phylogenetic conservation.We conclude that for HisA: (i) The effect of mutations can be masked by high expression levels, such that mutations that are deleterious to the function of the protein can still be neutral with regard to organism fitness if the protein is expressed at a sufficiently high level; (ii) the shape of the fitness distribution is dependent on the extent to which the protein is rate-limiting for growth; (iii) negative epistatic interactions, on an average, amplified the combined effect of nonsynonymous mutations; and (iv) no single sequence-based predictor could confidently predict the fitness effects of mutations in HisA, but a combination of multiple predictors could predict the effect with a SD of 0.04 resulting in 80% of the mutations predicted within 12% of their observed selection coefficients.

Upregulation of PBP1B and LpoB in cysB Mutants Confers Mecillinam (Amdinocillin) Resistance in Escherichia coli.

Mecillinam (amdinocillin) is a ß-lactam antibiotic that inhibits the essential penicillin-binding protein 2 (PBP2). In clinical isolates of Escherichia coli from urinary tract infections, inactivation of the cysB gene (which encodes the main regulator of cysteine biosynthesis, CysB) is the major cause of resistance. How a nonfunctional CysB protein confers resistance is unknown, however, and in this study we wanted to examine the mechanism of resistance. Results show that cysB mutations cause a gene regulatory response that changes the expression of ∼450 genes. Among the proteins that show increased levels are the PBP1B, LpoB, and FtsZ proteins, which are known to be involved in peptidoglycan biosynthesis. Artificial overexpression of either PBP1B or LpoB in a wild-type E. coli strain conferred mecillinam resistance; conversely, inactivation of either the mrcB gene (which encodes PBP1B) or the lpoB gene (which encodes the PBP1B activator LpoB) made cysB mutants susceptible. These results show that expression of the proteins PBP1B and LpoB is both necessary and sufficient to confer mecillinam resistance. The addition of reducing agents to a cysB mutant converted it to full susceptibility, with associated downregulation of PBP1B, LpoB, and FtsZ. We propose a model in which cysB mutants confer mecillinam resistance by inducing a response that causes upregulation of the PBP1B and LpoB proteins. The higher levels of these two proteins can then rescue cells with mecillinam-inhibited PBP2. Our results also show how resistance can be modulated by external conditions such as reducing agents.