Subinhibitory Concentrations Of Antibiotics Research Articles

Diverse modulation of spa transcription by cell wall active antibiotics in Staphylococcus aureus

BackgroundThe aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus.FindingsLacZ promoter fusions of genes related to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding α-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently, expression of all three genes were repressed by aminoglycosides and induced by fluoroquinolones and penicillins. In contrast, the β-lactam sub-group cephalosporins enhanced expression of RNAIII and hla but diversely affected expression of spa. The compounds cefalotin, cefamandole, cefoxitin, ceftazidime and cefixine were found to up-regulate spa, while down-regulation was observed for cefuroxime, cefotaxime and cefepime. Interestingly, biofilm assays demonstrated that the spa-inducing cefalotin resulted in less biofilm formation compared to the spa-repressing cefotaxime.ConclusionsWe find that independently of the cephalosporin generation, cephalosporins oppositely regulate spa expression and biofilm formation. Repression of spa expression correlates with the presence of a distinct methyloxime group while induction correlates with an acidic substituted oxime group. As cephalosporines target the cell wall penicillin binding proteins we speculate that subtle differences in this interaction fine-tunes spa expression independently of agr.

Macrolides and β-Lactam Antibiotics Enhance C3b Deposition on the Surface of Multidrug-Resistant Streptococcus pneumoniae Strains by a LytA Autolysin-Dependent Mechanism

The emergence of Streptococcus pneumoniae strains displaying high levels of multidrug resistance is of great concern worldwide and a serious threat for the outcome of the infection. Modifications of the bacterial envelope by antibiotics may assist the recognition and clearance of the pathogen by the host immune system. Recognition of S. pneumoniae resistant strains by the complement component C3b was increased in the presence of specific anti-pneumococcal antibodies and subinhibitory concentrations of different macrolides and β-lactam antibiotics for all the strains investigated. However, C3b levels were unchanged in the presence of serum containing specific antibodies and sub-MICs of levofloxacin. To investigate whether LytA, the main cell wall hydrolase of S. pneumoniae, might be involved in this process, lytA-deficient mutants were constructed. In the presence of antibiotics, loss of LytA was not associated with enhanced C3b deposition on the pneumococcal surface, which confirms the importance of LytA in this interaction. The results of this study offer new insights into the development of novel therapeutic strategies using certain antibiotics by increasing the efficacy of the host immune response to efficiently recognize pneumococcal resistant strains.

Antibiotic susceptibility profile of Mycobacterium avium subspecies hominissuis is altered in low-iron conditions

Antibiotic susceptibility testing of the Mycobacterium avium complex is often characterized by a lack of correlation between in vitro results and clinical response. The reason for this discrepancy might lie in the difference between in vitro susceptibility testing conditions and the actual environment experienced by mycobacteria in the host. The availability of iron is one such difference, which is limited in host macrophages upon infection, but abundant in susceptibility testing media. Accordingly, the aim of our study was to determine whether iron limitation affects the antibiotic susceptibility profile of M. avium subspecies hominissuis. Susceptibilities to multiple antibiotics targeting various cellular processes were determined in media with normal- and low-iron concentrations using the resazurin microplate assay. Differences in susceptibilities were evaluated by monitoring changes in the MIC and growth inhibition at subinhibitory antibiotic concentrations (sub-MICs). Cultures grown in low-iron conditions were less susceptible to the DNA synthesis inhibitors 6-mercaptopurine and levofloxacin at sub-MICs. Decreased susceptibility to the protein synthesis inhibitors azithromycin (>2-fold) and streptomycin (at sub-MICs) was observed only during adaptation to low-iron conditions. On the contrary, increased susceptibility to antibiotics that interfere with cell wall synthesis [isoniazid (4-fold), d-cycloserine (2-fold) and ethambutol (at sub-MICs)], mycobactin synthesis [4-aminosalicylate (at sub-MICs)] and mRNA synthesis [rifampicin (4-fold)] was observed in low-iron conditions. The susceptibility profile in low-iron conditions significantly differs from that observed in normal-iron conditions. Mimicking the host environment in terms of iron availability should be considered for in vitro susceptibility testing of mycobacteria, especially for antibiotics interfering with iron metabolism, such as 4-aminosalicylate.

Effects of Antibiotics on Shiga Toxin 2 Production and Bacteriophage Induction by Epidemic Escherichia coli O104:H4 Strain

The role of antibiotics in treatment of enterohemorrhagic Escherichia coli (EHEC) infections is controversial because of concerns about triggering hemolytic-uremic syndrome (HUS) by increasing Shiga toxin (Stx) production. During the recent large EHEC O104:H4 outbreak, antibiotic therapy was indicated for some patients. We tested a diverse panel of antibiotics to which the outbreak strain is susceptible to interrogate the effects of subinhibitory antibiotic concentrations on induction of stx(2)-harboring bacteriophages, stx(2) transcription, and Stx2 production in this emerging pathogen. Ciprofloxacin significantly increased stx(2)-harboring phage induction and Stx2 production in outbreak isolates (P values of <0.001 to <0.05), while fosfomycin, gentamicin, and kanamycin insignificantly influenced them (P > 0.1) and chloramphenicol, meropenem, azithromycin, rifaximin, and tigecycline significantly decreased them (P ≤ 0.05). Ciprofloxacin and chloramphenicol significantly upregulated and downregulated stx(2) transcription, respectively (P < 0.01); the other antibiotics had insignificant effects (P > 0.1). Meropenem, azithromycin, and rifaximin, which were used for necessary therapeutic or prophylactic interventions during the EHEC O104:H4 outbreak, as well as tigecycline, neither induced stx(2)-harboring phages nor increased stx(2) transcription or Stx2 production in the outbreak strain. These antibiotics might represent therapeutic options for patients with EHEC O104:H4 infection if antibiotic treatment is inevitable. We await further analysis of the epidemic to determine if usage of these agents was associated with an altered risk of developing HUS.

Characterization of a novel gene related to antibiotic susceptibility in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a human pathogen with increased intrinsic resistance to a large number of antibiotics used in clinical therapy. Therefore, understanding the mechanisms of resistance and developing therapy alternatives for P. aeruginosa are of profound importance. Previous work from our laboratory demonstrated that several mutants have isolated with altered expression of the phzA1B1C1D1E1F1G1 (phzA1) operon in the presence of sub-inhibitory concentrations (SICs) of tetracycline (TET). The present study investigates the roles of the PA0011 gene in mediating phzA1 expression at SIC of TET. The PA0011 gene encodes 2-OH-lauroytransferase by controlling the synthesis of the cell envelope and the outer membrane. We found that the PA0011 mutant strain was susceptible to several different antibiotics and environmental stresses. Complementation in the PA0011 mutant restored these phenotypes to wild-type levels. In addition, expression of the PA0011 gene, as monitored through a luciferase reporter, is increased at SICs of antibiotics. Indeed, the expression of the PA0011 gene increased about threefold in pqsR and pqsH mutants compared with the wild-type PAO1. However, the PA0011 gene negatively regulates the quorum sensing (QS) system. Taken together, these data suggest that PA0011 is involved in susceptibility to antimicrobial agents in P. aeruginosa, and that its susceptibility effect maybe partly dependent on increased QS expression.

Are Subinhibitory Concentrations of Antibiotics the only Culprit of Antibiotic Resistance?

Evaluation of: Gullberg E, Cao S, Berg OG et al. Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathog. 7(7), e1002158 (2011). Subinhibitory concentrations of antibiotics and antifungals promote resistance. Antibiotic consumption including hospital use, and country use, including patients self-medications is one of the major drivers of antibiotic or antifungal resistance. However, consumption of antibiotics should be distinguished between the hospital and community. Hospital consumption, poor hospital hygiene and intrahospital transfer have been determined as major risk factors for development of resistance. The correlation between resistance and consumption in the community is not so clear. Therefore consumption of antibiotics and antifungals alone cannot explain the selection of resistant bacterial and fungal mutants and other factors have to be investigated.

Antibiotics Shaping Bacterial Genome: Deletion of an IS91 Flanked Virulence Determinant upon Exposure to Subinhibitory Antibiotic Concentrations

The nucleoid-associated proteins Hha and YdgT repress the expression of the toxin α-hemolysin. An Escherichia coli mutant lacking these proteins overexpresses the toxin α-hemolysin encoded in the multicopy recombinant plasmid pANN202-312R. Unexpectedly, we could observe that this mutant generated clones that no further produced hemolysin (Hly-). Generation of Hly- clones was dependent upon the presence in the culture medium of the antibiotic kanamycin (km), a marker of the hha allele (hha::Tn5). Detailed analysis of different Hly- clones evidenced that recombination between partial IS91 sequences that flank the hly operon had occurred. A fluctuation test evidenced that the presence of km in the culture medium was underlying the generation of these clones. A decrease of the km concentration from 25 mg/l to 12.5 mg/l abolished the appearance of Hly- derivatives. We considered as a working hypothesis that, when producing high levels of the toxin (combination of the hha ydgT mutations with the presence of the multicopy hemolytic plasmid pANN202-312R), the concentration of km of 25 mg/l resulted subinhibitory and stimulated the recombination between adjacent IS91 flanking sequences. To further test this hypothesis, we analyzed the effect of subinhibitory km concentrations in the wild type E. coli strain MG1655 harboring the parental low copy number plasmid pHly152. At a km concentration of 5 mg/l, subinhibitory for strain MG1655 (pHly152), generation of Hly- clones could be readily detected. Similar results were also obtained when, instead of km, ampicillin was used. IS91 is flanking several virulence determinants in different enteric bacterial pathogenic strains from E. coli and Shigella. The results presented here evidence that stress generated by exposure to subinhibitory antibiotic concentrations may result in rearrangements of the bacterial genome. Whereas some of these rearrangements may be deleterious, others may generate genotypes with increased virulence, which may resume infection.

Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis

BackgroundBurkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes.ResultsSpontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance.ConclusionsGlobal mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.

Modulation of virulence gene expression by cell wall active antibiotics in Staphylococcus aureus

To investigate the effect of subinhibitory concentrations of cell wall active antibiotics on virulence gene expression and biofilm formation in Staphylococcus aureus Newman and in laboratory strains. Promoter regions of spa, lukE and agr RNAIII were cloned upstream of a modified luxABCDE reporter. Using disc diffusion assays, the effects of antibiotics were observed on gene expression and quantitative real-time PCR was employed to confirm the results. Assays were performed to measure biofilm formation in wild-type S. aureus and respective spa-deficient and small colony variant mutants in the presence of subinhibitory concentrations of antibiotics. Expression of spa and lukE was stimulated by subinhibitory concentrations of penicillin and cefalotin, while agr RNAIII expression was not affected. Denser biofilms were formed by S. aureus Newman and its small colony variant in the presence of subinhibitory concentrations of cefalotin. Subinhibitory concentrations of certain antibiotics have been shown to stimulate virulence gene expression in S. aureus; this may alter the progression of infection and thus render antimicrobial therapy unreliable. The use of appropriate combinations of antibiotics might be an approach to avoiding this situation. Promoter-lux reporters are sensitive tools for studying the modulation of transcription by antibiotic inhibitors, and could be used to predict novel therapeutic combinations for the treatment of infection.

Vibrio cholerae Triggers SOS and Mutagenesis in Response to a Wide Range of Antibiotics: a Route towards Multiresistance

Antibiotic resistance development has been linked to the bacterial SOS stress response. In Escherichia coli, fluoroquinolones are known to induce SOS, whereas other antibiotics, such as aminoglycosides, tetracycline, and chloramphenicol, do not. Here we address whether various antibiotics induce SOS in Vibrio cholerae. Reporter green fluorescent protein (GFP) fusions were used to measure the response of SOS-regulated promoters to subinhibitory concentrations of antibiotics. We show that unlike the situation with E. coli, all these antibiotics induce SOS in V. cholerae.

Influence of ciprofloxacin and vancomycin on mutation rate and transposition of IS 256 in Staphylococcus aureus

In Staphylococcus aureus, the development of intermediate resistance to vancomycin is due to an accumulation of mutations. To elucidate the mechanisms involved here, a standard laboratory strain ( S. aureus HG001) and a clinical MRSA mutator strain ( S. aureus SA1450/94, which is characterized by a spontaneous insertion of IS 256 into the gene of the mismatch repair enzyme MutS) were incubated at subinhibitory concentrations of ciprofloxacin and vancomycin. Ciprofloxacin increased the mutation rates of both strains, but this effect was inhibited when the SOS response was blocked by the presence of a non-cleavable variant of the LexA repressor. In the presence of vancomycin, the mutation rate was slightly elevated in the mutator strain, and this increase also depended on the strain's ability to induce the SOS response. Furthermore, treatment with subinhibitory concentrations of both antibiotics resulted in an activation of transposition frequency of the insertion element IS 256 in S. aureus HG001. Transposition was dependent on the presence of a functional transposase, and the activation of transposition depended on the presence of the functional phosphatase RsbU, which activates SigB transcription activity. An in silico analysis indicated a putative antisense sigma B promoter sequence within the transposase gene. Scrambling of this promoter resulted in an about 20-fold activation of transposition activity of IS 256. These data indicate that sigma B is involved in the regulation of IS 256 activity by generation of an antisense RNA.

Survival patterns of some common pathogens in human plasma following exposure to a sub-inhibitory concentration of ceftriaxone sodium

The survival of Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa in human plasma was investigated following their exposure to a sub-inhibitory concentration of ceftriaxone sodium. The aim was to determine the possible effect of human immune agents present in the plasma on microorganisms that may have been previously exposed to sub-inhibitory concentrations of antibiotics. Microbial survival was assessed by viable cell counts conducted at hourly intervals over a period of 5 hours. Subsequently, surviving organisms were used to determine the minimal inhibitory concentration of the antibiotics. The results showed that the rate of replication of P. aeruginosa in plasma was significantly reduced after exposure to a subinhibitory concentration of ceftriaxone sodium. On the contrary, there was an increase in the minimum inhibitory concentration of the antibiotic after K. pneumoniae or E. coli were exposed to the sub-inhibitory antibiotic concentration. This implies that, while some organisms were weakened by sub-lethal doses of ceftriaxone sodium to the extent of susceptibility to human immune agents, others gained resistance to both the antibiotic and the immune system. Keywords: Minimum inhibitory concentration, ceftriaxone sodium, susceptibility, viable cell count, survival pattern.

YfiBNR Mediates Cyclic di-GMP Dependent Small Colony Variant Formation and Persistence in Pseudomonas aeruginosa

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

The Fast Track to Multidrug Resistance

In this issue of Molecular Cell, Kohanski et al. (2010) demonstrate that even subinhibitory concentrations of bactericidal antibiotics result in the generation of reactive oxygen species, leading to an increase in mutation rate and the emergence of multidrug-resistant bacterial strains.

PRODUCTION OF SHIGA‐LIKE TOXINS BY ESCHERICHIA COLI O157 : H7: EFFECTS OF OTHER BACTERIA AND ANALOGS OF QUORUM SENSING MOLECULES

ABSTRACT Accompanied with the growth of Escherichia coli O157 : H7, there was a production of Shiga‐like toxins by this pathogenic bacterium. Time‐course studies indicated that the accumulation of toxins in the medium occurred mainly at the stationary phase of cell growth. The growth of E. coli O157 : H7 in culture media was not significantly affected by the presence of other bacteria, e.g., E. coli K‐12, E. coli B6, Salmonella and Pseudomonas, even at high ratios. However, the production of Shiga‐like toxins by E. coli O157 : H7 could be reduced by certain other bacteria, e.g., E. coli K‐12, Pseudomonas aeruginosa but not Pseudomonas putida. These lowering effects by other background bacteria on the toxin production were also observed in experiments using regular and irradiated ground beef. The presence of analogs of quorum‐sensing molecules such as homoserine lactone (HSL) and indole, in general decreased the production of toxins by E. coli O157 : H7. This decrease could be partially reversed by the presence of subinhibitory concentrations of antibiotics. The complex nature of the control of Shiga toxin production is discussed.PRACTICAL APPLICATIONSThe toxic effects of pathogenic bacterium E. coli O157 : H7 are related to the production of Shiga‐like toxins by the pathogen. Current studies on the influence of environmental factors such as cell density, presence of other bacteria, chemicals and food components indicated a complex influence on the production of Shiga‐like toxins by E. coli O157 : H7. The observation that the presence of certain other bacteria could lower the ability of E. coli O157 : H7 to produce the toxins was of particular interest. The analyses of the results suggested several practical possibilities to lower the production of the toxins, and thus, the health risk of the pathogen to the general public.

Phenotypic changes in ciprofloxacin-resistant Staphylococcus aureus

The work described here continues our studies of the effects of subinhibitory concentrations of antibiotics on SOS and DNA repair gene expression in Staphylococcus aureus. Mitomycin C and the new-generation fluoroquinolone moxifloxacin induced expression of SOS response genes ( lexA, recA, sosA, and umuC) in a ciprofloxacin-resistant Cip rI strain of S. aureus. To examine phenotypic changes in Cip r strains mutated in CIP targets (GrlA and/or GyrA), we used Biolog Phenotype MicroArrays™. Two other Cip r strains mutated in the norA promoter region were used to study the effects of subinhibitory concentrations of DNA-damaging antibiotics on norA expression. We show that mitomycin C and moxifloxacin induced overexpression of the norA gene in Cip r strains. Finally, we confirm that subinhibitory concentrations of CIP increase mutation rates in S. aureus.

Second messenger signalling governs Escherichia coli biofilm induction upon ribosomal stress

Biofilms are communities of surface-attached, matrix-embedded microbial cells that can resist antimicrobial chemotherapy and contribute to persistent infections. Using an Escherichia coli biofilm model we found that exposure of bacteria to subinhibitory concentrations of ribosome-targeting antibiotics leads to strong biofilm induction. We present evidence that this effect is elicited by the ribosome in response to translational stress. Biofilm induction involves upregulation of the polysaccharide adhesin poly-beta-1,6-N-acetyl-glucosamine (poly-GlcNAc) and two components of the poly-GlcNAc biosynthesis machinery, PgaA and PgaD. Poly-GlcNAc control depends on the bacterial signalling molecules guanosine-bis 3', 5'(diphosphate) (ppGpp) and bis-(3'-5')-cyclic di-GMP (c-di-GMP). Treatment with translation inhibitors causes a ppGpp hydrolase (SpoT)-mediated reduction of ppGpp levels, resulting in specific derepression of PgaA. Maximal induction of PgaD and poly-GlcNAc synthesis requires the production of c-di-GMP by the dedicated diguanylate cyclase YdeH. Our results identify a novel regulatory mechanism that relies on ppGpp signalling to relay information about ribosomal performance to the Pga machinery, thereby inducing adhesin production and biofilm formation. Based on the important synergistic roles of ppGpp and c-di-GMP in this process, we suggest that interference with bacterial second messenger signalling might represent an effective means for biofilm control during chronic infections.

Side effects of antibiotics on genetic variability

In recent years, there has been accumulating evidence that antibiotics, besides their antimicrobial action, potentially have a number of undesired side effects that can, at least in some cases, promote genetic variability of bacteria. In addition to resistant variants, antibiotics have also been shown to select mutator clones, thus stimulating evolution towards further resistance. Furthermore, mutations, recombination and horizontal gene transfer have been reported to be somehow affected when bacteria are exposed to subinhibitory concentrations of certain antibiotics. These findings may have implications for the use of antibiotics, because they may have undesired side effects, such as enhancing antibiotic resistance evolution. Here we present data supporting (or not) this fearsome possibility and discuss whether this potential threat should be taken into consideration.

The YheI/YheH heterodimer from Bacillus subtilis is a multidrug ABC transporter

ABC (ATP-binding cassette) transporters form the largest family of membrane proteins in micro-organisms where they are able to transport a wide variety of substrates against a concentration gradient, in an ATP-dependent process. Two genes from the same putative Bacillus subtilis operon, yheI and yheH, encoding possibly two different ABC transporters, were overexpressed in Escherichia coli in high yield, either separately or jointly. Using membrane vesicles, it is shown here that both subunits were required to detect, (i) the transport of four structurally unrelated drugs, and (ii) a vanadate-sensitive ATPase activity. Mutation of the invariant Walker-A lysine to an alanine residue in both subunits led to an inactive transporter. Moreover, after membrane solubilization by detergent, both wild-type subunits co-purified on a Ni-Agarose affinity column while only the YheH subunit contained a hexa-histidine tag. This shows that YheI and YheH are indeed able to interact together to form a heterodimer. Importantly, expression of both yheI and yheH genes in B. subtilis could be strongly stimulated by addition of sub-inhibitory concentrations of various unrelated antibiotics. Therefore, B. subtilis YheI/YheH forms a new heterodimeric multidrug ABC transporter possibly involved in multiple antibiotic resistance in vivo.

Isothermal micro calorimetry – a new method for MIC determinations: results for 12 antibiotics and reference strains of E. coli and S. aureus

BackgroundAntimicrobial susceptibility testing of microorganisms is performed by either disc diffusion or broth dilution tests. In clinical use, the tests are often still performed manually although automated systems exist. Most systems, however, are based on turbidometric methods which have well-known drawbacks.ResultsIn this study we evaluated isothermal micro calorimetry (IMC) for the determination of minimal inhibitory concentrations (MICs) of 12 antibiotics for five micro-organisms. Here we present the data for the 12 antibiotics and two representative microorganisms E. coli (a Gram-) and S. aureus (a Gram+). IMC was able to determine the MICs correctly according to CLSI values. Since MICs require 24 hours, time was not reduced. However, IMC provided new additional data – a continuous record of heat-producing bacterial activity (e.g. growth) in calorimetry ampoules at subinhibitory antibiotic concentrations. Key features of the heatflow (P) and aggregate heat (Q) vs. time curves were identified (tdelay and ΔQ/Δt respectively). Antibiotics with similar modes of action proved to have similar effects on tdelay and/or ΔQ/Δt.ConclusionIMC can be a powerful tool for determining the effects of antibiotics on microorganisms in vitro. It easily provides accurate MICs – plus a potential means for analyzing and comparing the modes of action of antibiotics at subinhibitory concentrations. Also IMC is completely passive, so after evaluation, ampoule contents (media, bacteria, etc.) can be analyzed by any other method desired.