Sublethal Concentrations Of Antibiotics Research Articles

The importance of growth kinetic analysis in determining bacterial susceptibility against antibiotics and silver nanoparticles.

Routine antibiotics susceptibility testing still relies on standardized cultivation-based analyses, including measurement of inhibition zones in conventional agar diffusion tests and endpoint turbidity-based measurements. Here, we demonstrate that common off-line monitoring and endpoint determination after 18–24 h could be insufficient for reliable growth-dependent evaluation of antibiotic susceptibility. Different minimal inhibitory concentrations were obtained in 20- and 48 h microdilution plate tests using an Enterococcus faecium clinical isolate (strain UKI-MB07) as a model organism. Hence, we used an on-line kinetic assay for simultaneous cultivation and time-resolved growth analysis in a 96-well format instead of off-line susceptibility testing. Growth of the Enterococcus test organism was delayed up to 30 h in the presence of 0.25 μg mL-1 of vancomycin and 8 μg mL-1 of fosfomycin, after which pronounced growth was observed. Despite the delayed onset of growth, treatment with fosfomycin, daptomycin, fusidic acid, cefoxitin, or gentamicin resulted in higher maximum growth rates and/or higher final optical density values compared with antibiotic-free controls, indicating that growth stimulation and hormetic effects may occur with extended exposure to sublethal antibiotic concentrations. Whereas neither maximum growth rate nor final cell density correlated with antibiotic concentration, the lag phase duration for some antibiotics was a more meaningful indicator of dose-dependent growth inhibition. Our results also reveal that non-temporal growth profiles are only of limited value for cultivation-based antimicrobial silver nanoparticle susceptibility testing. The exposure to Ag(0) nanoparticles led to plasma membrane damage in a concentration-dependent manner and induced oxidative stress in Enterococcus faecium UKI-MB07, as shown by intracellular ROS accumulation.

Genomic analysis reveals distinct concentration-dependent evolutionary trajectories for antibiotic resistance in Escherichia coli.

Evolution of bacteria under sublethal concentrations of antibiotics represents a trade-off between growth and resistance to the antibiotic. To understand this trade-off, we performed in vitro evolution of laboratory Escherichia coli under sublethal concentrations of the aminoglycoside kanamycin over short time durations. We report that fixation of less costly kanamycin-resistant mutants occurred earlier in populations growing at lower sublethal concentration of the antibiotic, compared with those growing at higher sublethal concentrations; in the latter, resistant mutants with a significant growth defect persisted longer. Using deep sequencing, we identified kanamycin resistance-conferring mutations, which were costly or not in terms of growth in the absence of the antibiotic. Multiple mutations in the C-terminal end of domain IV of the translation elongation factor EF-G provided low-cost resistance to kanamycin. Despite targeting the same or adjacent residues of the protein, these mutants differed from each other in the levels of resistance they provided. Analysis of one of these mutations showed that it has little defect in growth or in synthesis of green fluorescent protein (GFP) from an inducible plasmid in the absence of the antibiotic. A second class of mutations, recovered only during evolution in higher sublethal concentrations of the antibiotic, deleted the C-terminal end of the ATP synthase shaft. This mutation confers basal-level resistance to kanamycin while showing a strong growth defect in the absence of the antibiotic. In conclusion, the early dynamics of the development of resistance to an aminoglycoside antibiotic is dependent on the levels of stress (concentration) imposed by the antibiotic, with the evolution of less costly variants only a matter of time.

Antibiotics and antibiotic resistance: A bitter fight against evolution

One of the most terrible consequences of Darwinian evolution is arguably the emergence and spread of antibiotic resistance, which is becoming a serious menace to modern societies. While spontaneous mutation, recombination and horizontal gene transfer are recognized as the main causes of this notorious phenomenon; recent research has raised awareness that sub-lethal concentrations of antibiotics can also foster resistance as an undesirable side-effect. They can produce genetic changes by different ways, including a raise of free radicals within the cell, induction of error-prone DNA-polymerases mediated by SOS response, imbalanced nucleotide metabolism or affect directly DNA. In addition to certain environmental conditions, subinhibitory concentrations of antimicrobials may increase, even more, the mutagenic effect of antibiotics. Here, we review the state of knowledge on antibiotics as promoters of antibiotic resistance.

Sub-lethal concentrations of antibiotics increase mutation frequency in the cystic fibrosis pathogen Pseudomonas aeruginosa

Approximately 80% of adult patients with cystic fibrosis (CF) become chronically infected with Pseudomonas aeruginosa and consequently require antibiotic therapy at intervals throughout their lives. Achieving lethal concentrations of antibiotics in the lung remains a challenge. Recent evidence from Escherichia coli and Staphylococcus aureus suggests that the generation of hydroxyl radicals by sublethal concentrations of antibiotics may induce mutagenesis and confer bacteria with resistance to a wide range of antimicrobials. As Ps. aeruginosa can persist for many years following colonization of the airways and during this time it is repeatedly exposed to bactericidal antibiotics, we tested whether its exposure to sublethal levels increases mutation frequency. We demonstrate that sublethal levels of three classes of bactericidal antibiotics commonly used against Ps. aeruginosa infections, β-lactams, aminoglycosides and quinolones lead to an increase in mutation frequency, varying between c. threefold increase with aminoglycosides and a c. 14-fold increase in mutation frequency with β-lactam antibiotics. These findings could be clinically significant because exposure to sublethal concentrations of antibiotics during chronic infection leading to increased mutation frequency may facilitate adaptive radiation of pathogenic bacteria in the heterogeneous environment of the CF lung.

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.

Francisella tularensis tmRNA system mutants are vulnerable to stress, avirulent in mice, and provide effective immune protection

Through targeted inactivation of the ssrA and smpB genes, we establish that the trans-translation process is necessary for normal growth, adaptation to cellular stress and virulence by the bacterial pathogen Francisella tularensis. The mutant bacteria grow slower, have reduced resistance to heat and cold shocks, and are more sensitive to oxidative stress and sublethal concentrations of antibiotics. Modifications of the tmRNA tag and use of higher-resolution mass spectrometry approaches enabled the identification of a large number of native tmRNA substrates. Of particular significance to understanding the mechanism of trans-translation, we report the discovery of an extended tmRNA tag and extensive ladder-like pattern of endogenous protein-tagging events in F. tularensis that are likely to be a universal feature of tmRNA activity in eubacteria. Furthermore, the structural integrity and the proteolytic function of the tmRNA tag are both crucial for normal growth and virulence of F. tularensis. Significantly, trans-translation mutants of F. tularensis are impaired in replication within macrophages and are avirulent in mouse models of tularemia. By exploiting these attenuated phenotypes, we find that the mutant strains provide effective immune protection in mice against lethal intradermal, intraperitoneal and intranasal challenges with the fully virulent parental strain.

Antibiotics affect the growth responses of Daphnia magna to poor food quality

We tested the hypothesis that exposure to antibiotics alters the growth and reproductive responses of Daphnia magna to changing stoichiometric food quality. To do so, we measured growth and reproduction of differentially P-nourished Daphnia in the presence and absence of sublethal concentrations of antibiotics. We found that exposure to an antibiotic cocktail significantly reduced an index of the microbial load of Daphnia and altered its growth responses to changing dietary P-content. Growth rates of Daphnia consuming the most P-rich and P-poor food increased with antibiotic exposure but were negatively or not affected in animals eating mildly to moderately P-limiting food. Similar effects were found in a subsequent experiment where daphnid neonates were exposed to natural bacterial communities prior to receiving antibiotics and being fed different food C:P ratios. In contrast, antibiotic effects on Daphnia reproduction were either not detected (number and size of broods) or were relatively minor (day of first reproduction). We also found no evidence that gut flora provides defense against pathogenic bacterial infection; instead, infection rates in Daphnia by a bacterial microparasite, Pasteuria ramosa, decreased in animals that had experienced prior antibiotic exposure. Our results demonstrate that antibiotic exposure reduced the microbial load and altered growth rates of an important zooplankton herbivore. Given the mediating role of animal’s food C:P ratio, our results show that interactions between Daphnia and its microbial symbionts vary in strength and nature partly with the host’s nutritional state.

Sublethal Antibiotic Treatment Leads to Multidrug Resistance via Radical-Induced Mutagenesis

Antibiotic resistance arises through mechanisms such as selection of naturally occurring resistant mutants and horizontal gene transfer. Recently, oxidative stress has been implicated as one of the mechanisms whereby bactericidal antibiotics kill bacteria. Here, we show that sublethal levels of bactericidal antibiotics induce mutagenesis, resulting in heterogeneous increases in the minimum inhibitory concentration for a range of antibiotics, irrespective of the drug target. This increase in mutagenesis correlates with an increase in ROS and is prevented by the ROS scavenger thiourea and by anaerobic conditions, indicating that sublethal concentrations of antibiotics induce mutagenesis by stimulating the production of ROS. We demonstrate that these effects can lead to mutant strains that are sensitive to the applied antibiotic but resistant to other antibiotics. This work establishes a radical-based molecular mechanism whereby sublethal levels of antibiotics can lead to multidrug resistance, which has important implications for the widespread use and misuse of antibiotics.

Adaptive functions of Escherichia coli polyamines in response to sublethal concentrations of antibiotics

Escherichia coli exposure to sublethal antibiotic concentrations induced an increase in cell polyamine contents. Maximum accumulation of putrescine and spermidine in response to antibiotics-induced oxidative stress preceded the increment of cadaverine, the content of which was dependent on the rpoS expression level and reached the maximum in response to fluoroquinolones. The polyamine positive modulating effects on rpoS expression increased in the following order: cadaverine-putrescine-spermidine. The reason for cadaverine accumulation was the increase in activities of lysine decarboxylases CadA and Ldc. High cadaverine accumulation in the cells exposed to fluoroquinolones and cephalosporins resulted in the reduction of porin permeability; so it was considered as a response aimed at cell protection against antibiotic penetration into the cell. Netilmycin, unlike other antibiotics, did not substantially affect the lysine decarboxylase activity and cellular polyamine pools.

Sublethal concentrations of antibiotics and bacterial adhesion.

Various antibiotics in sublethal concentrations markedly impair adhesion of Streptococcus pyogenes and Escherichia coli to human cells. In streptococcal cells penicillin G caused an enhances loss of lipoteichoic acid, the ligand (adhesion) that binds the organism to host cells, with consequent loss of their adhesive properties. In E coli sublethal concentrations of penicillin prevented the surface expression of the mannose-specific adhesion by distorting cell wall biosynthesis. In contrast to streptococci, E coli cells could not be made to lose their adhesions once their adhesions once they had been formed. Streptomycin in subinhibitory concentration similarly suppressed the acquisition of mannose-binding and adhesive activities in several strains of antibiotic-sensitive E. coli but not in isogenic derivatives with ribosomal mutation to high-level streptomycin resistance, rpsL, or in bacteria in the stationary phase of growth, suggesting that streptomycin exerted its sublethal suppressive effects by classic mechanisms of action on the bacterial ribosome. Strain VL2, derived from one streptomycin-resistant mutant, retained a high level (1000 microgram/ml) of resistance to streptomycin but reacquired sensitivity to the sublethal effect; growth in 30 microgram streptomycin/ml suppressed mannose-sensitive haemagglutination (less than 1% of control) as well as mannose-sensitive adhesion to epithelial cells (42%) or leucocytes (7%). Although these streptomycin-treated bacteria demonstrated an unaltered degree of fimbriation their fimbriae were significantly longer than those on the untreated bacteria. Furthermore, in contrast to the untreated bacteria, the fimbriae isolated from the drug-treated bacteria were found to lack mannose-binding activity as measured by haemagglutination. It therefore, appears that streptomycin can cause even resistant bacteria to produce an aberrant fimbrial protein, presumably by causing misreading in "competent" ribosomes. These studies indicate that the use of sublethal doses of certain antibiotics whose mode of action is well known may shed light on the genetic and chemical modulation of bacterial factors involved in mucosal colonization.

Biofilms strike back

Sublethal concentrations of antibiotics increase bacterial drug tolerance by promoting biofilm formation.

Effects of sub-MIC concentrations of antibiotics on growth of and toxin production by Clostridium difficile.

Effects on growth and toxin A production of sub-MIC concentrations of six different antibiotics were investigated in three strains of Clostridium difficile: reference strain NCTC 11223, a fully sequenced strain (630) and a locally endemic isolate (strain 338a). The antibiotics chosen for investigation were the agents used to treat C. difficile-associated disease (CDAD), i.e. vancomycin and metronidazole, and four antibiotics that are commonly involved in precipitating CDAD (amoxycillin, clindamycin, cefoxitin and ceftriaxone). Strains were cultured in sublethal concentrations of antibiotics (1/2, 1/4 and 1/8 MIC) over 104 h and growth and toxin A production were measured three times a day. Effects varied between strain and antibiotic. The most common effect on growth of the strains was to increase the initial lag period by approximately 4 h, compared with antibiotic-free controls; however, strain NCTC 11223, which has high-level clindamycin resistance (> or = 512 microg ml(-1)), showed no lag whatsoever in comparison with the controls when grown in this antibiotic. The most common effect on production of toxin A was in the onset of toxin elaboration. Normally, toxins began to appear at low levels in the early stationary phase, before accumulating to high levels by the start of decline. In the presence of sub-MIC antibiotics, this onset appeared before that of the antibiotic-free controls. This effect was seen with metronidazole, amoxycillin and clindamycin, rarely with vancomycin and never with cefoxitin. These results suggest a very complex, strain-dependent relationship between the effects of growth and toxin production.

Cell wall stress responses in Bacillus subtilis: the regulatory network of the bacitracin stimulon.

In response to sublethal concentrations of antibiotics, bacteria often induce an adaptive response that can contribute to antibiotic resistance. We report the response of Bacillus subtilis to bacitracin, an inhibitor of cell wall biosynthesis found in its natural environment. Analysis of the global transcriptional profile of bacitracin-treated cells reveals a response orchestrated by two alternative sigma factors (sigmaB and sigmaM) and three two-component systems (YvqEC, YvcPQ and BceRS). All three two-component systems are located next to target genes that are strongly induced by bacitracin, and the corresponding histidine kinases share an unusual topology: they lack about 100 amino acids in their extracellular sensing domain, which is almost entirely buried in the cytoplasmic membrane. Sequence analysis indicates that this novel N-terminal sensing domain is a characteristic feature of a subfamily of histidine kinases, found almost entirely in Gram-positive bacteria and frequently linked to ABC transporters. A systematic mutational analysis of bacitracin-induced genes led to the identification of a new bacitracin-resistance determinant, bceAB, encoding a putative ABC transporter. The bcrC bacitracin resistance gene, which is under the dual control of sigmaX and sigmaM, was also induced by bacitracin. By comparing the bacitracin and the vancomycin stimulons, we can differentiate between loci induced specifically by bacitracin and those that are induced by multiple cell wall-active antibiotics.

Anti-adhesion therapy of bacterial diseases: prospects and problems

The alarming increase in drug-resistant bacteria makes a search for novel means of fighting bacterial infections imperative. An attractive approach is the use of agents that interfere with the ability of the bacteria to adhere to tissues of the host, since such adhesion is one of the initial stages of the infectious process. The validity of this approach has been unequivocally demonstrated in experiments performed in a wide variety of animals, from mice to monkeys, and recently also in humans. Here we review various approaches to anti-adhesion therapy, including the use of receptor and adhesin analogs, dietary constituents, sublethal concentrations of antibiotics and adhesin-based vaccines. Because anti-adhesive agents are not bactericidal, the propagation and spread of resistant strains is much less likely to occur than as a result of exposure to bactericidal agents, such as antibiotics. Anti-adhesive drugs, once developed, may, therefore, serve as a new means to fight infectious diseases.

Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo

The ability of magainin 2 to augment antibiotic therapy was examined. Susceptibility to magainin 2 was determined on Escherichia coli incubated in the presence and absence of sublethal concentrations of antibiotics both in vitro and in vivo. Experiments in buffer and normal human serum revealed that E. coli exposed to sublethal amounts of cefepime, a beta-lactam antibiotic, was significantly more susceptible to the antimicrobial activity of magainin 2. Bacteria incubated with subinhibitory concentrations of other beta-lactam type antibiotics, but not amikacin (an aminoglycoside) or ciprofloxacin (a quinolone), were also more susceptible to magainin 2 in normal human serum. Bacteria were less susceptible to magainin 2 when they were examined in heat-inactivated serum. Complement was shown to be required for magainin 2 activity in serum by using C8-deficient sera. The combination of magainin 2 and cefepime was shown to be more antimicrobial in normal human serum for a variety of bacterial strains. Magainin 2 was completely inactive as a therapeutic agent when it was administered alone (2 mg per mouse) but significantly increased the survival of mice when it was administered with a low level of cefepime.

Influence of Subinhibitory Concentrations of Cephalosporins on the Serum Sensitivity of Pseudomonas aeruginosa

The effect of sublethal concentrations of antibiotics on the serum sensitivity of Pseudomonas aeruginosa was examined. Cefepime, ceftazidime, and imipenem but not amikacin nor ciprofloxacin increased the serum bactericidal activity of pooled normal human serum. Killing was both serum- and antibiotic dose-dependent. Increased sensitivity to the bactericidal action of serum in the presence of cefepime was observed for several different clinical isolates. With the use of C8-deficient sera, the late components of the complement pathway were shown to be essential for bacterial killing. A significant increase in the amount of [125I]C9 based on bacterial mass was observed with bacteria incubated with cefepime compared with non-antibiotic- or amikacin-treated controls. No major change in the amount of type of lipopolysaccharide was observed when cefepime-treated and control bacteria were compared. The data show that cefepime and other cephalosporins at sublethal concentrations increase the complement-mediated bactericidal activity of serum against P. aeruginosa.

Effects of subminimal inhibitory concentrations of antibiotics on the adherence of Pseudomonas aeruginosa to tracheobronchial mucin.

Bacterial adherence to mucins may be important in tracheobronchial infections in cystic fibrosis. Sublethal concentrations of antibiotics reduce bacterial adherence to epithelial cells and mucins. This reduction in adherence may be a component of antimicrobial effects in infections at anatomical sites where bactericidal concentrations of antibiotics are difficult to achieve. We therefore tested the effects of sublethal concentrations of an aminoglycoside, tobramycin, and a beta-lactam antibiotic, ceftazidime, on the adherence of Pseudomonas aeruginosa to tracheobronchial mucin, since mucus secretions are often colonized by P. aeruginosa in cystic fibrosis. Adherence of the mucoid strains tested was inhibited by ceftazidime, but not by tobramycin. This effect of ceftazidime may partially explain its efficacy in patients with cystic fibrosis despite variables achieved in sputum.

The Antibacterial Effects of Low Concentrations of Antibiotics

The effects of sublethal concentrations of antibiotics on bacteria are varied and for the most part uncharacterized at the molecular level. Low concentrations of antibiotics have antimicrobial activity and may also exhibit immunological side effects in the host by either directly inhibiting or stimulating immune-response mechanisms. Exposure of bacteria to low concentrations of β-lactam antibiotics usually leads to growth inhibition accompanied by the production of abnormal morphological forms. However, this is not always so as reports are now emerging to show that certain β-lactam antibiotics are capable either of inducing the synthesis of novel penicillin-binding proteins (PBPs), or altering the balance of existing PBPs to permit normal growth in the presence of β-lactam antibiotics. Despite gaps in the understanding of the molecular basis of the changes, data are accumulating showing that low-level, or single-dose, antibiotic administration can be used for the successful therapy of certain bacterial infections.

Adherent interactions which may affect microbial ecology in the mouth.

Bacteria attach to teeth and oral mucosal surfaces in a surprisingly selective manner, and attachment appears to be the first step in the colonization process. Attachment is thought to involve lectin-like and/or hydrophobic ligands, called adhesins, often present in bacterial surface appendages which interact with receptors on oral tissues. A variety of factors can influence bacterial attachment, and therefore have the potential to affect host-parasite interactions in the mouth. Factors discussed include salivary components, dietary lectins, and sublethal concentrations of antibiotics.

Influence of sublethal antibiotic concentrations on bacterial adherence to saliva-treated hydroxyapatite.

The influence of growth in the presence of sublethal concentrations of nine antibiotics on the ability of certain potentially odontopathic bacteria to attach to saliva-treated hydroxyapatite surfaces which mimic teeth was studied. Cells of Actinomyces viscosus LY7 and S2, Bacteroides gingivalis 381, Capnocytophaga ochraceus 6, and Actinobacillus actinomycetemcomitans N27 attached in lower numbers to saliva-treated hydroxyapatite when grown in the presence of 50% of the minimum inhibitory concentration of tetracycline. Electron microscopic observations of negatively stained preparations indicated that tetracycline-grown A. viscosus LY7 cells had fewer fimbriae than did untreated cells, which may account for the impaired ability of the treated cells to attach. However, cells of Actinomyces naeslundii L13 and S4 attached in higher numbers when grown in the presence of tetracycline, clindamycin, erythromycin, chloramphenicol, or neomycin. Streptococcus mutans strains H12 and JBP also exhibited increased adherence to saliva-treated hydroxyapatite when grown in the presence of 50 or 25% of the minimum inhibitory concentration of penicillin. Thus, growth in the presence of sublethal antibiotic concentrations could increase as well as decrease the adherence of bacteria to saliva-treated hydroxyapatite. Antibiotic-grown cells of the Actinomyces strains showed enhanced hemagglutination activity, but this did not correlate with their ability to attach to saliva-treated hydroxyapatite. Sublethal concentrations of antibiotics in the growth media also affected the coaggregation reactions of several organisms; the effects were specific for one member of the coaggregation pair.