Subinhibitory Concentrations Of Ampicillin Research Articles

Influence of Selected Factors on Biofilm Formation by Salmonella enterica Strains.

Biofilm formed by S. enterica on the surface of gallstones or biomaterials promotes the development and spread of chronic infection. The aim of the study was to assess biofilm formation on the surface of polystyrene depending on nutritional conditions and the effect of 0.5, 1.0, and 2.0% glucose and 3.0% bile and sub-inhibitory concentrations of ampicillin on biofilm formation of S. enterica. Sixty-nine clinical strains of S. enterica isolated from feces (92.8%) and blood (7.2%) collected from patients (66.7%) and carriers (33.3%) were used in the study. Assessment of forming 24-h biofilm by these strains was performed on the surface of polystyrene 96-well plates at 37 °C. In this study, it was indicated that 1.0% glucose and 3.0% bovine bile inhibit biofilm formation. Biofilm formation was inhibited in all examined sub-MIC of ampicillin. Biofilm formation is varied in different conditions, depending on the serovar.

Upregulation of the clpB gene in response to heat shock and beta-lactam antibiotics in Acinetobacter baumannii.

The role of the clpB gene encoding HSP/chaperone ClpB was evaluated in the multiresistant antibiotic cells of Acinetobacter baumannii (RS4 strain) under stress-induced heat shock and different beta-lactams. The expression of the clpB gene was assessed by qPCR during heat shock at 45°C and subinhibitory concentrations of ampicillin (30μgmL-1), amoxicillin + sulbactam (8/12μgmL-1), cefepime (30μgmL-1), sulfamethoxazole + trimethoprim (120/8μgmL-1) and meropenem (18μgmL-1). The results indicated a transient increase in clpB transcription in all treatments except cefepime. Both in the presence of ampicillin and amoxicillin/sulbactam for 20min, the mRNA-clpB synthesis was 1.4 times higher than that of the control at time zero. Surprisingly, the mRNA-clpB levels were more than 30-fold higher after 10min of incubation with meropenem and more than eightfold higher in the presence of trimethoprim/sulfamethoxazole. In addition, western blot assays showed that the RS4 strain treated with meropenem showed a marked increase in ClpB protein expression. Our data indicate that during exposure to beta-lactams, A. baumannii adjusts the transcription levels of the clpB mRNA and protein to respond to stress, suggesting that the chaperone may act as a key cellular component in the presence of antibiotics in this bacterium.

The effect of antimicrobials on verocytotoxin bacteriophage transduction under bovine rumen fluid and broth conditions

AbstractThe verocytotoxin genes in verocytotoxigenicEscherichia coli(VTEC) are carried by bacteriophages, incorporated into the bacterial genome (prophage). Antibiotics may promote phage replication and release to infect other cells (transduction), thus leading to the emergence of new VTEC strains. This study investigated transduction of a verocytotoxin2-encoding bacteriophage (3538(vtx2::cat)) under laboratory conditions, including the effect of antibiotic treatments. Luria-Bertani Miller broth and rumen fluid (raw and sterilised by irradiation) were inoculated with the donor (C600φ3538(Δvtx2::cat)) and recipient (E. coli C600::kanamycinR) strains (4 log10cfu/mL) and incubated at 38°C. Antibiotic treatments (minimal inhibitory and sub-inhibitory concentrations of ampicillin, cefquinome, oxytetracycline and sodium sulfamethazine) were applied after 3 h. Samples were tested for donor, recipient, cell-free phage and transductants at times t = 0, 3, 4, 6, 27 (24 h post-antibiotic treatment) and 51 h. Free phage was detected in the untreated broth and rumen samples, as were the transductants confirmed by polymerase chain reaction. The antibiotic treatments did not significantly (P > 0.01) increase the concentrations of free phage or transductants detected. It was therefore concluded that, under laboratory conditions, the antibiotics tested did not induce bacteriophage lysis, release and infection of new bacterial cells beyond that constitutively found in the phage population.

Proteomic analysis reveals that a global response is induced by subinhibitory concentrations of ampicillin

ABSTRACTIn this study, a recipient-donor co-culture system was used to research the effect of subinhibitory concentrations of antibiotics on horizontal transmission in bacteria and the influence of antibiotics on protein expression. We employed two-dimensional gel electrophoresis combined with mass spectrometry to compare the protein expression profiles in systems with or without 0.5 × the minimum inhibitory concentration of ampicillin. RT-PCR was used to assess the transcriptional levels of the differentially expressed genes. Fifty-seven different proteins were induced or suppressed. The upregulated proteins were involved in transcription and translation, cell wall synthesis, bacterial SOS response, and detoxifying functions, and the downregulated proteins were involved in metabolism. These results indicated that a global response was induced in the recipient-donor co-culture system by the subinhibitory concentration of ampicillin. Further analysis revealed that a global regulatory network based on key pathways was induced in the system in response to the antibiotic pressure. These findings provide a new, more comprehensive view for research on antibiotic-resistance mechanisms in recipient-donor co-culture.

Bacteroides fragilis response to subinhibitory concentrations of antimicrobials includes different morphological, physiological and virulence patterns after in vitro selection

As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host–bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.

Raman spectroscopic identification of single bacterial cells under antibiotic influence

The identification of pathogenic bacteria is a frequently required task. Current identification procedures are usually either time-consuming due to necessary cultivation steps or expensive and demanding in their application. Furthermore, previous treatment of a patient with antibiotics often renders routine analysis by culturing difficult. Since Raman microspectroscopy allows for the identification of single bacterial cells, it can be used to identify such difficult to culture bacteria. Yet until now, there have been no investigations whether antibiotic treatment of the bacteria influences the Raman spectroscopic identification. This study aims to rapidly identify bacteria that have been subjected to antibiotic treatment on single cell level with Raman microspectroscopy. Two strains of Escherichia coli and two species of Pseudomonas have been treated with four antibiotics, all targeting different sites of the bacteria. With Raman spectra from untreated bacteria, a linear discriminant analysis (LDA) model is built, which successfully identifies the species of independent untreated bacteria. Upon treatment of the bacteria with subinhibitory concentrations of ampicillin, ciprofloxacin, gentamicin, and sulfamethoxazole, the LDA model achieves species identification accuracies of 85.4, 95.3, 89.9, and 97.3 %, respectively. Increasing the antibiotic concentrations has no effect on the identification performance. An ampicillin-resistant strain of E. coli and a sample of P. aeruginosa are successfully identified as well. General representation of antibiotic stress in the training data improves species identification performance, while representation of a specific antibiotic improves strain distinction capability. In conclusion, the identification of antibiotically treated bacteria is possible with Raman microspectroscopy for diverse antibiotics on single cell level.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of β-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host–bacteria relationship.

Subinhibitory concentrations of antibiotics affect stress and virulence gene expression inListeria monocytogenesand cause enhanced stress sensitivity but do not affect Caco-2 cell invasion

Antibiotics can act as signal molecules and affect bacterial gene expression, physiology and virulence. The purpose of this study was to determine whether subinhibitory antibiotic concentrations alter gene expression and physiology of Listeria monocytogenes. Using an agar-based screening assay with promoter fusions, 14 of 16 antibiotics induced or repressed expression of one or more stress and/or virulence genes. Despite ampicillin-induced up-regulation of PinlA-lacZ expression, Caco-2 cell invasion was not affected. Subinhibitory concentrations of ampicillin and tetracycline caused up- and down-regulation of stress response genes, respectively, but both antibiotics caused increased sensitivity to acid stress. Six combinations of gene-antibiotic were quantified in broth cultures and five of the six resulted in the same expression pattern as the agar-based assay. Antibiotics affect virulence and/or stress gene expression; however, altered expression could not predict changes in phenotypic behaviour. Subinhibitory concentrations of antibiotics led to increased acid sensitivity, and we speculate that this is attributed to changes in cell envelope or reduced σ(B) -dependent gene expression. Although subinhibitory concentrations of antibiotics affect gene expression in L. monocytogenes, the changes did not increase virulence but did enhance the acid sensitivity.

Violacein and biofilm production in Janthinobacterium lividum

To analyse the environmental stimuli modulating violacein and biofilm production in Janthinobacterium lividum. Violacein and biofilm production by J. lividum DSM1522(T) was assayed in different growth conditions. Our data suggest that violacein and biofilm production is controlled by the carbon source, being inhibited by glucose and enhanced by glycerol. J. lividum produced violacein also in the presence of different sub-inhibitory concentrations of ampicillin. As opposite, the production of N-acylhomoserine lactone(s), quorum sensing regulators was shown to be positively regulated by glucose. Moreover, violacein-producing cultures of J. lividum showed higher CFU counts than violacein-nonproducing ones. Taken together, our results suggest that violacein and biofilm production could be regulated by a common metabolic pathway and that violacein as well as biofilm could represent a response to environmental stresses and a key factor in the survival mechanisms of J. lividum. Although several recent studies disclosed a number of interesting biological properties of violacein, few data are reported on the physiologic function of violacein in J. lividum. This paper adds new information on the complex mechanisms allowing and regulating bacterial life in hostile environments.

Adhesion of Enterococcus faecalis 1131 grown under subinhibitory concentrations of ampicillin and vancomycin to a hydrophilic and a hydrophobic substratum

The effect of two subinhibitory antibiotic concentrations of ampicillin and vancomycin during growth on the adhesion of Enterococcus faecalis 1131 to glass and silicone rubber was studied in a parallel plate flow chamber. Initial deposition rates and numbers of adhering bacteria after 4 h were higher on hydrophilic glass than on hydrophobic silicone rubber, regardless of growth conditions. The presence of 1/4 minimal inhibitory concentration (MIC) of ampicillin during growth reduced enterococcal adhesion to both substrata, but growth in the presence of 1/4 MIC vancomycin did not affect the adhesion of E. faecalis. Moreover, enterococcal adhesion increased after growth in the presence of 1/8 MIC vancomycin. The increased adhesion after growth in the presence of subinhibitory concentrations of vancomycin may have strong implications for patients living with implanted biomaterials, as they may suffer adverse effects from use of this antibiotic, especially since bacteria once adhered are less sensitive to antibiotics.

Adhesion of Gardnerella vaginalis to vaginal epithelial cells: variables affecting adhesion and inhibition by metronidazole.

Variables affecting the adherence of Gardnerella vaginalis to human vaginal epithelial cells were examined in vitro. Adherence depended on pH, with maximum attachment occurring between pH 5 and pH 6. Preincubation of the bacteria at 56 degrees C for 30 minutes and ultraviolet irradiation resulted in a noticeable decrease in adherence. In contrast, adherence was not altered by preincubating the epithelial cells under these conditions. Periodate oxidation of the vaginal cells caused an appreciable reduction in subsequent adherence of G vaginalis. None of the 19 single carbohydrates tested inhibited adherence completely. Metronidazole at subinhibitory concentrations for G vaginalis, appreciably reduced the adhesive capacity of G vaginalis, whereas subinhibitory concentrations of ampicillin did not.

Kinetic studies of ampicillin action on Escherichia coli and their spheroplasts.

Kinetic studies of ampicillin action were made on exponentially growing Escherichia coli and on E. coli-spheroplasts using a range of inhibitory and subinhibitory concentrations of ampicillin. For each concentration, the value (ko--ka) representing the difference in generation rates of ampicillin-free culture (ko) and the generation rate of the culture with ampicillin (ka) was calculated and plotted against ampicillin concentration. A straight line relation was obtained with E. coli cells, its intersection with the abscissa, where (ko--ka) = o, give the concentration of ampicillin which exerts no inhibitory action on the cells (0.25 microgram/ml). When ka was plotted against ampicillin concentration, the relation was also linear. Its intersection with the abscissa gives the minimum lethal concentration of ampicillin on the bacterial cells (1.05 microgram/ml). With E. coli-spheroplasts such plots were non-linear which means that a different order of reaction was involved. This difference is probably due to a different mechanism of ampicillin action as revealed by the electroscanning microscopy.

Decrease in adhesion of Escherichia coli to human urinary tract epithelial cells in vitro by subinhibitory concentrations of ampicillin

Low levels of ampicillin, 1/4 the minimal inhibitory concentration, decrease the ability of Escherichia coli to attach to human urinary tract epithelial cells in vitro. Such adhesion may be a virulence factor inEscherichia coli producing symptomatic urinary tract infections, since strains from the urine of patients with acute pyelonephritis and acute cystitis attach more than strains from patients with asymptomatic bacteriuria or normal faecalE. coli.