Clinically relevant fluoroquinolone resistance due to constitutive overexpression of the PatAB ABC transporter in Streptococcus pneumoniae is conferred by disruption of a transcriptional attenuator

OBJECTIVES: Constitutive overexpression of patAB has been observed in several unrelated fluoroquinolone-resistant laboratory mutants and clinical isolates; therefore, we sought to identify the cause of this overexpression. METHODS: Constitutive patAB overexpression in two clinical isolates and a laboratory-selected mutant was investigated using a whole-genome transformation approach. To determine the effect of the detected terminator mutations, the WT and mutated patA leader sequences were cloned upstream of a GFP reporter. Finally, mutation of the opposing base in the stem–loop structure was carried out. RESULTS: We identified three novel mutations causing up-regulation of patAB. All three of these were located in the upstream region of patA and affected the same Rho-independent transcriptional terminator structure. Each mutation was predicted to destabilize the terminator stem–loop to a different degree, and there was a strong correlation between predicted terminator stability and patAB expression level. Using a GFP reporter of patA transcription, these terminator mutations led to increased transcription of a downstream gene. For one mutant sequence, terminator stability could be restored by mutation of the opposing base in the stem–loop structure, demonstrating that transcriptional suppression of patAB is mediated by the terminator stem–loop structure. CONCLUSIONS: This study showed that a mutation in a Rho-independent transcriptional terminator structure confers overexpression of patAB and fluoroquinolone resistance. Understanding how levels of the PatAB efflux pump are regulated increases our knowledge of pneumococcal biology and how the pneumococcus can respond to various stresses, including antimicrobials

Functional genomics to identify the factors contributing to successful persistence and global spread of an antibiotic resistance plasmid

Background: The spread of bacterial plasmids is an increasing global problem contributing to the widespread dissemination of antibiotic resistance genes including β-lactamases. Our understanding of the details of the biological mechanisms by which these natural plasmids are able to persist in bacterial populations and are able to establish themselves in new hosts via conjugative transfer is very poor. We recently identified and sequenced a globally successful plasmid, pCT, conferring β-lactam resistance. Results: Here, we investigated six plasmid encoded factors (tra and pil loci; rci shufflon recombinase, a putative sigma factor, a putative parB partitioning gene and a pndACB toxin-antitoxin system) hypothesised to contribute to the 'evolutionary success' of plasmid pCT. Using a functional genomics approach, the role of these loci was investigated by systematically inactivating each region and examining the impact on plasmid persistence, conjugation and bacterial host biology. While the tra locus was found to be essential for all pCT conjugative transfer, the second conjugation (pil) locus was found to increase conjugation frequencies in liquid media to particular bacterial host recipients (determined in part by the rci shufflon recombinase). Inactivation of the pCT pndACB system and parB did not reduce the stability of this plasmid. Conclusions: Our findings suggest the success of pCT may be due to a combination of factors including plasmid stability within a range of bacterial hosts, a lack of a fitness burden and efficient transfer rates to new bacterial hosts rather than the presence of a particular gene or phenotype transferred to the host. The methodology used in our study could be applied to other 'successful' globally distributed plasmids to discover the role of currently unknown plasmid backbone genes or to investigate other factors which allow these elements to persist and spread

Efflux in Acinetobacter baumannii can be determined by measuring accumulation of H33342 (bis-benzamide)

10.1093/jac/dkt052Journal of Antimicrobial Chemotherapy6871594-160

Chlorpromazine and amitriptyline are substrates and inhibitors of the acrb multidrug efflux pump

Efflux is an important mechanism in Gram-negative bacteria conferring multidrug resistance. Inhibition of efflux is an encouraging strategy to restore the antibacterial activity of antibiotics. Chlorpromazine and amitriptyline have been shown to behave as efflux inhibitors. However, their mode of action is poorly under-stood. Exposure of Salmonella enterica serovar Typhimurium and Escherichia coli to chlorpromazine selected for mutations within genes encoding RamR and MarR, regu-lators of the multidrug tripartite efflux pump AcrAB-TolC. Further experiments with S. Typhimurium containing AcrB D408A (a nonfunctional efflux pump) and chlor-promazine or amitriptyline resulted in the reversion of the mutant acrB allele to the wild type. Together, this suggests these drugs are AcrB efflux substrates. Subsequent docking studies with AcrB from S. Typhimurium and E. coli, followed by molecular dynamics simulations and free energy calculations showed that chlorpromazine and amitriptyline bind at the hydrophobic trap, a preferred binding site for substrates and inhibitors within the distal binding pocket of AcrB. Based on these simulations, we suggest that chlorpromazine and amitriptyline inhibit AcrB-mediated efflux by in-terfering with substrate binding. Our findings provide evidence that these drugs are substrates and inhibitors of AcrB, yielding molecular details of their mechanism of action and informing drug discovery of new efflux inhibitors. IMPORTANCE Efflux pumps of the resistance nodulation-cell division (RND) super-family are major contributors to multidrug resistance for most of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acineto-bacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens. The development of inhibitors of these pumps would be highly desirable; how-ever, several issues have thus far hindered all efforts at designing new efflux in-hibitory compounds devoid of adverse effects. An alternative route to de novo design relies on the use of marketed drugs, for which side effects on human health have been already assessed. In this work, we provide experimental evidence that the antipsychotic drugs chlorpromazine and amitriptyline are inhibi-tors of the AcrB transporter, the engine of the major RND efflux pumps in Escherichia coli and Salmonella enterica serovar Typhimurium. Furthermore, in silico calculations have provided a molecular-level picture of the inhibition mechanism, allowing rationalization of experimental data and paving the way for similar studies with other classes of marketed compounds

HIV Drugs Inhibit Transfer of Plasmids Carrying Extended-Spectrum β-Lactamase and Carbapenemase Genes

More and more bacterial infections are becoming resistant to antibiotics. This has made treatment of many infections very difficult. One of the reasons this is such a large problem is that bacteria are able to share their genetic material with other bacteria, and these shared genes often include resistance to a variety of antibiotics, including some of our drugs of last resort. We are addressing this problem by using a fluorescence-based system to search for drugs that will stop bacteria from sharing resistance genes. We uncovered a new role for two drugs used to treat HIV and show that they are able to prevent the sharing of two different types of resistance genes in two unique bacterial strains. This work lays the foundation for future work to reduce the prevalence of resistant infections.Antimicrobial-resistant (AMR) infections pose a serious risk to human and animal health. A major factor contributing to this global crisis is the sharing of resistance genes between different bacteria via plasmids. The WHO lists Enterobacteriaceae, such as Escherichia coli and Klebsiella pneumoniae, producing extended-spectrum β-lactamases (ESBL) and carbapenemases as “critical” priorities for new drug development. These resistance genes are most often shared via plasmid transfer. However, finding methods to prevent resistance gene sharing has been hampered by the lack of screening systems for medium-/high-throughput approaches. Here, we have used an ESBL-producing plasmid, pCT, and a carbapenemase-producing plasmid, pKpQIL, in two different Gram-negative bacteria, E. coli and K. pneumoniae. Using these critical resistance-pathogen combinations, we developed an assay using fluorescent proteins, flow cytometry, and confocal microscopy to assess plasmid transmission inhibition within bacterial populations in a medium-throughput manner. Three compounds with some reports of antiplasmid properties were tested; chlorpromazine reduced transmission of both plasmids and linoleic acid reduced transmission of pCT. We screened the Prestwick library of over 1,200 FDA-approved drugs/compounds. From this, we found two nucleoside analogue drugs used to treat HIV, abacavir and azidothymidine (AZT), which reduced plasmid transmission (AZT, e.g., at 0.25 μg/ml reduced pCT transmission in E. coli by 83.3% and pKpQIL transmission in K. pneumoniae by 80.8% compared to untreated controls). Plasmid transmission was reduced by concentrations of the drugs which are below peak serum concentrations and are achievable in the gastrointestinal tract. These drugs could be used to decolonize humans, animals, or the environment from AMR plasmids

A Requirement of TolC and MDR Efflux Pumps for Acid Adaptation and GadAB Induction in Escherichia coli

BACKGROUND: The TolC outer membrane channel is a key component of several multidrug resistance (MDR) efflux pumps driven by H(+) transport in Escherichia coli. While tolC expression is under the regulation of the EvgA-Gad acid resistance regulon, the role of TolC in growth at low pH and extreme-acid survival is unknown. METHODS AND PRINCIPAL FINDINGS: TolC was required for extreme-acid survival (pH 2) of strain W3110 grown aerobically to stationary phase. A tolC deletion decreased extreme-acid survival (acid resistance) of aerated pH 7.0-grown cells by 10(5)-fold and of pH 5.5-grown cells by 10-fold. The requirement was specific for acid resistance since a tolC defect had no effect on aerobic survival in extreme base (pH 10). TolC was required for expression of glutamate decarboxylase (GadA, GadB), a key component of glutamate-dependent acid resistance (Gad). TolC was also required for maximal exponential growth of E. coli K-12 W3110, in LBK medium buffered at pH 4.5-6.0, but not at pH 6.5-8.5. The TolC growth requirement in moderate acid was independent of Gad. TolC-associated pump components EmrB and MdtB contributed to survival in extreme acid (pH 2), but were not required for growth at pH 5. A mutant lacking the known TolC-associated efflux pumps (acrB, acrD, emrB, emrY, macB, mdtC, mdtF, acrEF) showed no growth defect at acidic pH and a relatively small decrease in extreme-acid survival when pre-grown at pH 5.5. CONCLUSIONS: TolC and proton-driven MDR efflux pump components EmrB and MdtB contribute to E. coli survival in extreme acid and TolC is required for maximal growth rates below pH 6.5. The TolC enhancement of extreme-acid survival includes Gad induction, but TolC-dependent growth rates below pH 6.5 do not involve Gad. That MDR resistance can enhance growth and survival in acid is an important consideration for enteric organisms passing through the acidic stomach

Efflux Pump, the Masked Side of ß-Lactam Resistance in Klebsiella pneumoniae Clinical Isolates

International audienceBACKGROUND: Beta-lactamase production and porin decrease are the well-recognized mechanisms of acquired beta-lactam resistance in Klebsiella pneumoniae isolates. However, such mechanisms proved to be absent in K. pneumoniae isolates that are non susceptible to cefoxitin (FOX) and susceptible to amoxicillin+clavulanic acid in our hospital. Assessing the role of efflux pumps in this beta-lactam phenotype was the aim of this study. METHODOLOGY/FINDINGS: MICs of 9 beta-lactams, including cloxacillin (CLX), and other antibiotic families were tested alone and with an efflux pump inhibitor (EPI), then with both CLX (subinhibitory concentrations) and EPI against 11 unique bacteremia K. pneumoniae isolates displaying the unusual phenotype, and 2 ATCC strains. CLX and EPI-dose dependent effects were studied on 4 representatives strains. CLX MICs significantly decreased when tested with EPI. A similar phenomenon was observed with piperacillin+tazobactam whereas MICs of the other beta-lactams significantly decreased only in the presence of both EPI and CLX. Thus, FOX MICs decreased 128 fold in the K. pneumoniae isolates but also 16 fold in ATCC strain. Restoration of FOX activity was CLX dose-dependent suggesting a competitive relationship between CLX and the other beta-lactams with regard to their efflux. For chloramphenicol, erythromycin and nalidixic acid whose resistance was also due to efflux, adding CLX to EPI did not increase their activity suggesting differences between the efflux process of these molecules and that of beta-lactams. CONCLUSION: This is the first study demonstrating that efflux mechanism plays a key role in the beta-lactam susceptibility of clinical isolates of K. pneumoniae. Such data clearly evidence that the involvement of efflux pumps in beta-lactam resistance is specially underestimated in clinical isolates

Choice of Bacterial Growth Medium Alters the Transcriptome and Phenotype of Salmonella enterica Serovar Typhimurium

The type of bacterial culture medium is an important consideration during design of any experimental protocol. The aim of this study was to understand the impact of medium choice on bacterial gene expression and physiology by comparing the transcriptome of Salmonella enterica SL1344 after growth in the widely used LB broth or the rationally designed MOPS minimal medium. Transcriptomics showed that after growth in MOPS minimal media, compared to LB, there was increased expression of 42 genes involved in amino acid synthesis and 23 genes coding for ABC transporters. Seven flagellar genes had decreased expression after growth in MOPS minimal medium and this correlated with a decreased motility. In both MOPS minimal medium and MEM expression of genes from SPI-2 was increased and the adhesion of S. Typhimurium to intestinal epithelial cells was higher compared to the levels after growth in LB. However, SL1344 invasion was not significantly altered by growth in either MOPs minimal media or MEM. Expression of SPI-2 was also measured using chromosomal GFP reporter fusions followed by flow cytometry which showed, for the first time, that the reduction in SPI-2 transcript after growth in different media related to a reduction in the proportion of the bacterial population expressing SPI-2. These data highlight the profound differences in the global transcriptome after in vitro growth in different media and show that choice of medium should be considered carefully during experimental design, particularly when virulence related phenotypes are being measured

The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel