Дисертації з теми "Sub-Inhibitory concentrations of antibiotics"

Infections caused by Staphylococcus aureus are a major concern to public health due to multifactorial virulence of the bacteria and increasing resistance to antimicrobial therapy. The bacterial cell wall continues to be the primary target for antibiotics used to treat staphylococcal infections. I have used promoter-lux reporter constructs to study the effects of sub-inhibitory concentrations of cell wall active antibiotics on virulence gene expression in S. aureus. Constructs made for virulence genes encoding Spa, an adhesin, RNAIII, a regulatory RNA molecule, and LukE, a leukotoxin E, were introduced into several strains of S. aureus. It was found that the effects of sub-inhibitory concentrations of antibiotics differed depending on the strain, and antibiotics affected the expression of virulence genes differently in the same strain. Based on the results with S. aureus strains lacking virulence regulators, SarA and SarS, it was concluded that sub-inhibitory concentrations of antibiotics modulate the expression of virulence regulators, which affects transcription of the downstream genes, spa and lukE. A speculative model for the mechanism of transcription modulation by antibiotics was proposed. The effect of sub-inhibitory concentrations of antibiotics on S. aureus biofilm formation was also studied. Finally, promoter-lux reporter constructs were used to investigate effects of various antibiotic combinations on gene expression in S. aureus.

Pseudomonas aeruginosa is a notorious nosocomial opportunist. Planktonic forms of this pathogen have been traditionally studied for its pathogenicity. Such studies have shown that sub-minimal inhibitory concentrations (sub-MICs) of antibiotics are able to negatively modulate pathogenicity. However, more recent findings suggest a biofilm basis of infection. In this study, monospecies and binary biofilms of Pseudomonas aeruginosa ATCC 15692 (PAOl) and Escherichia coli ATCC 10000 were investigated for their pathogenic potential using resistance and virulence as key pathogenic determinants, in the presence of sub-MICs of selected antibiotics (Ampicillin, Nalidixic acid and Streptomycin). MICs of biofilms were observed to be at least 7-fold greater than those of the corresponding planktonic form of the same species (as judged from results obtained from MIC experiments). SDS-PAGE and 2D-PAGE analysis indicate alteration of outer membrane proteins (OMPs) within the envelope of the pathogen in sub-MIC antibiotic treated samples. The observed rearrangement of lipopolysaccharide (LPS; as observed in LPS gel experiments) may also contribute to the pathogens increased tolerance to antibiotics within the biofilm state. While LPS changes may possibly help the biofilm bacteria escape host immune system in vivo, more direct evidence of increases in virulence of the pathogen comes from investigation of its secreted proteases and cytotoxins (leucocidin). Virulence-specific azocasein and micro-culture tetrazolium (MTT) assays against both monospecies and binary biofilms of Pseudomonas aeruginosa indicate significant increases in virulence potential of proteases and cytotoxins, respectively. These results were further substantiated in phase contrast microscopy images showing advanced stages of oncosis in tissue cultured mouse spleen myeloma (Sp2) cells treated with leucocidin isolated from Ps. aeruginosa treated with sub-MIC of ampicillin (8 pg mL'1). The results reported in this thesis provide evidence of observed increases in virulence and pathogenicity in biofilm cells of Pseudomonas aeruginosa in the presence of sub-MICs of selected antibiotics, in vitro. Although these findings are those of in vitro experiments, they may have significant implications regarding the usage and therapeutic control of antibiotics in clinical situations.

This study examined the effect of iron deprivation and sub-inhibitory concentrations of antifungal agents on yeast cell surface antigen recognition by antibodies from patients with Candida infections. Separation of cell wall surface proteins by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and immunological detection by immunoblotting, revealed that antigenic profiles of yeasts were profoundly influenced by the growth environment. Cells grown under iron-depleted conditions expressed several iron-regulated proteins that were recognized by antibodies from patient sera. An attempt to characterize these proteins by lectin blotting with concanavalin A revealed that some could be glycoprotein in nature. Furthermore, these proteins which were located within cell walls and on yeast surfaces, were barely or not expressed in yeasts cultivated under iron-sufficient conditions. The magnitude and heterogeneity of human antibody responses to these iron-regulated proteins were dependent on the type of Candida infection, serum antibody class and yeast strain. Hydroxamate-type siderophores were also detected in supernatants of iron depleted yeast cultures. This evidence suggests that Candida albicans expresses iron-regulated proteins/glycoproteins in vitro which may play a role in siderophore-mediated iron uptake in Candida albicans. Sequential monitoring of IgG antibodies directed against yeast surface antigens during immunization of rabbits revealed that different antigens were recognized particularly during early and later stages of immunization in iron-depleted cells compared to iron-sufficient cells. In vitro and in vivo adherence studies demonstrated that growth phase, yeast strain and growth conditions affect adhesion mechanisms. In particular, growth under iron-depletion in the presence of sub-inhibitory concentrations of polyene and azole antifungals enhanced the hydrophobicity of C.albicans. Growth conditions also influenced MICs of antifungals, notably that of ketoconazole. Sub-inhibitory concentrations of amphotericin B and fluconazole had little effect on surface antigens, whereas nystatin induced profound changes in surface antigens of yeast cells. The effects of such drug concentrations on yeast cells coupled with host defence mechanisms may have a significant affect on the course of Candida infections.

Les intégrons de résistance (IR) de classe 1 sont des éléments génétiques bactériens qui permettent l’acquisition et l’expression de gènes de résistance aux antibiotiques. Cette étude examine comment la présence d'un IR chromosomique et le mode de vie bactérien, planctonique ou biofilm, affectent l'évolution des populations bactériennes exposées à des concentrations sub-inhibitrices d'antibiotiques. Pour ce faire, deux IRs synthétiques contenant trois cassettes de gènes, exprimées à partir des variants faible (PcW) ou fort (PcS), ont été intégrés dans la souche de E. coli, R3A. Les souches R3A (sans IR), R3Aw et R3As (avec IR) ont été cultivées en conditions planctonique et biofilm, avec ou sans antibiotiques, sur 15 transferts. Les résultats montrent qu'après 15 passages, l’évolution des 3 souches en planctonique en présence de Tobramycine (Tob) supplémentée ou non de Ciprofloxacine (Cip), a induit une augmentation de la résistance à la Tob. De même, la formation du biofilm a augmenté, pour toutes les populations évoluées (PE) en planctonique, et pour les populations R3As en biofilm. La caractérisation génomique des PE en biofilm, n’a montré aucune mutation fixée chez R3Aw, et une seule (gène srkA) chez R3A exposée à la Tob. Pour les PE en planctonique, une mutation a été fixée chez R3A propagée en Tob +/- Cip (fusA) et une chez R3A évoluée sans Tob (rpoC). Pour R3Aw, toutes les PE en Tob +/- Cip présentaient des mutations identiques dans 5 gènes dont intI1. Dans celui-ci, la présence de deux mutations a permis l'évolution du promoteur PcW vers PcS. La caractérisation génomique des populations R3As est encore en cours. Cette étude met ainsi en lumière des trajectoires évolutives distinctes des populations bactériennes selon la présence d’IR, l'exposition aux antibiotiques et leur mode de vie
Class 1 resistance integrons (RI) are bacterial genetic elements that enable the acquisition andexpression of antibiotic resistance genes. This study examines how the presence of a chromosomal RI and the bacterial planktonic or biofilm lifestyle affect the evolution of bacterial populations exposedto sub-inhibitory concentrations of antibiotics. To this end, two synthetic RI containing three genecassettes, expressed from weak (PcW) or strong (PcS) variants, were integrated into the E. coli R3Astrain. Strains R3A (without RE), R3Aw and R3As (with RI) were grown under planktonic and biofilmconditions, with or without antibiotics, over 15 transfers. The results show that after 15 passages, theevolution of the 3 strains in planktonic conditions in the presence of Tobramycin (Tob) supplementedor not with Ciprofloxacin (Cip) induced an increase in resistance to Tob. Similarly, biofilm formationincreased for all evolved population (EP) in planktonic, and for R3As biofilm populations. Genomiccharacterization of EP in biofilm showed no fixed mutations in R3Aw, and only one (srkA gene) in Tobexposed R3A. For planktonic EP, one mutation was fixed in R3A propagated in Tob +/- Cip (fusA) and one in R3A evolved without Tob (rpoC). For R3Aw, all Tob +/- Cip EP had identical mutations in 5 genes, including intI1. In the latter, the presence of two mutations led to the evolution of the PcW promoter towards PcS. Genomic characterization of the R3As populations is still in progress. This study highlights distinct evolutionary trajectories of bacterial populations according to the presence of RI, antibiotic exposure and lifestyle

Antibiotics are today one of the most important cornerstones in modern healthcare when it comes to treating bacterial infections. It is an asset human kind have been leaning on for the last century, but excessive and widespread misuse of antibiotics have left deep scars in the form of multi resistant pathogenic strains of bacteria that we soon will not be able to treat. A lot of research have been invested in understanding the mechanisms and spread of resistance within bacteria living in planktonic form, overlooking the fact that there are more lifestyles that causes problems. In this study, focus has been put on antibiotic resistance within bacteria living as biofilms, a lifestyle that causes problems in chronic infections and prosthetics/medical implants. By constructing resistant mutants derived from a biofilm forming strain of Escherichia coli, the minimal selection concentration has been investigated in both planktonic and biofilm assays for Streptomycin and Ciprofloxacin. By comparing the results, it is possible to evaluate if and how the antibiotic resistance properties differ between the two lifestyles. Focus has been put on concentrations of antibiotics below the minimal inhibitory concentration with the objective to see how selection of antibiotic resistant mutants take place with the susceptible strain still growing, although with reduced growth rate. The hope is that the results gained in this study will provide a foundation for future research regarding antibiotic resistance in biofilms, and be part of the solution to the excessive resistance problem before it is too late.

Staphylococcus epidermidis biofilm formation is a primary cause of medical device infections, which are persistent and difficult to eradicate because biofilms intrinsically exhibit a naturally high level of antibiotic resistance. Although biofilm antibiotic resistance or tolerance is a multifactorial process, some mechanisms such as limited diffusion, low metabolic activity and persister cells, contribute to the failure of antibiotics in the treatment of biofilm infections. Current, antibiotic treatment strategies may provide biofilm infections with intermittent exposure to sub-minimum inhibitory concentrations (sub-MIC) of antibiotics. Biofilms have been shown to display an increase in antibiotic tolerance when exposed to antibiotics at sub-MIC. Such mechanisms of adaptive antibiotic resistance are not well characterized but are of extreme clinical importance. This project showed that exposure to sub-MIC vancomycin increases the virulence of S. epidermidis biofilms because it induces vancomycin tolerance. BODIPY FL-vancomycin (fluorescent vancomycin conjugate) and confocal microscopy were used to show that the penetration of vancomycin through sub-MIC vancomycin pre-treated S. epidermidis biofilms was impeded, when compared to control, untreated biofilms. In addition, the results showed that a wide range of sub-MIC vancomycin concentrations induced an increased amount of extracellular DNA (eDNA) within the matrix of sub-MIC vancomycin treated biofilms. Finally, a set of ex vivo experiments using extracted exogenous S. epidermidis DNA revealed that exogenous S. epidermidis DNA binds vancomycin. Collectively these findings suggest that sub-MIC vancomycin exposure increase the abundance of eDNA in the matrix of S. epidermidis biofilms, which protects the biofilm community from subsequent vancomycin exposure by binding vancomycin as it travels through the matrix. Therefore the work in this project provides details of an eDNA-based mechanism of adaptive antibiotic tolerance in sub-MIC vancomycin treated S. epidermidis biofilms, which might be an important factor in the persistence of biofilms infections.

The extensive medical and agricultural use and misuse of antibiotics during the last 70 years has caused an enrichment of resistant pathogenic bacteria that now severely threatens our capacity to efficiently treat bacterial infections. While is has been known for a long time that high concentrations of antibiotics can select for resistant mutants, less is known about the lower limit at which antibiotics can be selective and enrich for resistant bacteria. In this thesis we investigated the role of low concentrations of antibiotics and heavy metals in the enrichment and evolution of antibiotic resistance. Selection was studied using Escherichia coli and Salmonella enterica serovar Typhimurium LT2 with different resistance mutations, different chromosomal resistance genes as well as large conjugative multidrug resistance plasmids. Using very sensitive competition experiments, we showed that antibiotic and heavy metal levels more than several hundred-fold below the minimal inhibitory concentration of susceptible bacteria can enrich for resistant bacteria. Additionally, we demonstrated that subinhibitory levels of antibiotics can select for de novo resistant mutants, and that these conditions can select for a new spectrum of low-cost resistance mutations. The combinatorial effects of antibiotics and heavy metals can cause an enrichment of a multidrug resistance plasmid, even if the concentration of each compound individually is not high enough to cause selection. These results indicate that environments contaminated with low levels of antibiotics and heavy metals such as, for example, sewage water or soil fertilized with sludge or manure, could provide a setting for selection, enrichment and transfer of antibiotic resistance genes. This selection could be a critical step in the transfer of resistance genes from environmental bacteria to human pathogens.

The adherence of three Candida species to human buccal epithelial cells (BECs) following treatment of the yeast with sub-inhibitory concentrations of amphotericin B, nystatin, miconazole nitrate, 5-fluorocytosine, octenidine and pirtenidine was investigated in vitro. Pre-incubation of C. albicans (two strains), C. tropicalis or C.kefyr with these antifungal drugs inhibited their adherence to varying degrees (reduction between 17% and 78% of the control value). Pre-treatment of yeast for a short period (l hr) had less effect on adhesion than pre-treatment for a long period (24 hr). Furthermore, treating C. albicans with a combination of amphotericin B plus 5-fluorocytosine, both at 1/8 MIC level, led to stronger adherence inhibition than that obtained for yeast pre-treated with either one alone at 1/4 MIC levels. In addition, the pre-treatment of either Candida or BECs or both types of cells with the drugs reduced adherence, the reduction being greatest when both types of cells were pre-treated. No difference in adherence between stationary or exponential phase yeast to BEC was observed and the drugs were effective in reducing the adherence of cells from either growth phase.

Background Broth microdilution (BMD) is a gold-standard reference method to determine minimum inhibitory concentration (MIC) of antibiotics. For this, a standardized concentration of bacterial inoculum (2e5–8e5 colony-forming units, CFU/ml) is added to progressively higher concentrations of antibiotics. Bacteria stop growing at a particular antibiotic concentration termed MIC. Like other assays, various biological and/or technical factors can affect BMD results.   Aims To investigate the effects of inoculum concentration (5e4–5e6 CFU/ml), growth-medium concentration (cation-adjusted Mueller-Hinton Broth (CAMHB)), ranging 0.5x to 2x (1x as standard)) and age (<6-months or >1-year old) of fastidious medium on MIC results. And to compare BMD results using 5 different brands of CAMHBs and 1 cation-non-adjusted MH-broth (non-CAMHB).   Methods 12 isolates of bacteria (gram-positive (n=3), gram-negative(n=5), fastidious isolates (n=7)) and custom-made antibiotics-containing plates for gram-positive (11 antibiotics) or gram-negative bacteria (10 antibiotics) were used. Overnight-grown colonies were used to prepare BMD solutions (MH-broth + inoculum +/- fastidious) which were plated on antibiotic-plates as well as diluted prior to plating on agar-plates. Antibiotic- and agar-plates were incubated (18–20hr, 35°C) and used to determine MICs (following European Committee on Antimicrobial Susceptibility Testing instructions) and actual number of viable bacteria in BMD solutions, respectively.   Results Increasing inoculum concentration increased MICs of all antibiotics except cefoxitin. Piperacillin–tazobactam, levofloxacin, benzylpenicillin and ampicillin were especially sensitive to increase in inoculum and showed a 4-fold increase in >50% isolates. MICs for tobramycin, tigecycline and gentamicin increased by 2-fold in >50% isolates every time MH-broth concentration increased. Age of fastidious medium had no decipherable pattern of effects on MIC. All MH-broths gave similar results except when testing daptomycin which gave higher MICs with non-CAMHB compared to CAMHB.    Conclusion This research reveals some technical factors affecting MIC results. These results could help define parameters for automated BMD-performing-systems. However, this research shows only trends as more replicates are needed to determine statistically significant results.

Oral Biology
M.S.
Objectives: Systemic antibiotics are generally recognized as providing a beneficial impact in treatment of both aggressive and chronic periodontitis. Since strains of periodontal pathogens among periodontitis patients may vary in their antibiotic drug resistance, the American Academy of Periodontology recommends antimicrobial susceptibility testing of suspected periodontal pathogens prior to administration of systemic periodontal antibiotic therapy, to reduce the risk of a treatment failure due to pathogen antibiotic resistance. E-test and MIC Test Strip assays are two in vitro antimicrobial susceptibility testing systems employing plastic- and paper-based, respectively, carriers loaded with predefined antibiotic gradients covering 15 two-fold dilutions. To date, no performance evaluations have been carried out comparing the Etest and MIC Test Strip assays in their ability to assess the in vitro antimicrobial susceptibility of periodontal bacterial pathogens. As a result, the purpose of this study was to compare the in vitro performance of E-test and MIC Test Strip assays in assessing minimal inhibitory concentration (MIC) values of four antibiotics frequently utilized in systemic periodontal antibiotic therapy against 11 fresh clinical subgingival isolates of the putative periodontal pathogen, Prevotella intermedia/ nigrescens, and to compare the distribution of P. intermedia/ nigrescens strains identified with interpretative criteria as "susceptible" and "resistant" to each of the four antibiotics using MIC values determined by the two antimicrobial susceptibility testing methods. Methods: Standardized cell suspensions, equivalent to a 2.0 McFarland turbidity standard, were prepared with 11 fresh clinical isolates of P. intermedia/nigrescens, each recovered from the subgingival microbiota of United States chronic periodontitis subjects, and plated onto to the surfaces of culture plates containing enriched Brucella blood agar. After drying, pairs of antibiotic-impregnated, quantitative, gradient diffusion strips from two manufacturers (E-test, bioMérieux, Durham, NC, USA, and MIC Test Strip, Liofilchem s.r.l., Roseto degli Abruzzi, Italy) for amoxicillin, clindamycin, metronidazole, and doxycycline were each placed apart from each other onto the inoculated enriched Brucella blood agar surfaces, so that an antibiotic test strip from each manufacturer was employed per plate against each P. intermedia/ nigrescens clinical isolate for antibiotic susceptibility testing. After 48-72 hours anaerobic jar incubation, individual MIC values for each antibiotic test strip against P. intermedia/nigrescens were read in μg/ml at the point where the edge of the bacterial inhibition ellipse intersected with the antibiotic test strip. MIC50, MIC90, and MIC range were calculated and compared for each of the test antibiotics, with essential agreement (EA) values determined per test antibiotic for the level of outcome agreement between two antimicrobial susceptibility testing methods. In addition, the identification of antibiotic "susceptible" and "resistant" strains among the P. intermedia/nigrescens clinical isolates was determined for each test antibiotic using MIC interpretative criteria from the MIC interpretative standards developed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for gram-negative anaerobic bacteria for amoxicillin, clindamycin, and metronidazole findings, and from the French Society of Microbiology breakpoint values for anaerobic disk diffusion testing for doxycycline data. Results: For amoxicillin, higher MIC50 and MIC90 values against the P. intermedia/ nigrescens strains were found with the MIC Test Strip assay than with E-test strips, resulting in a relatively low EA value of 45.5% between the two susceptibility testing methods. A higher percentage of amoxicillin "resistant" P. intermedia/nigrescens strains (72.7%) were identified by MIC Test Strips as compared to E-test strips (54.5%), although both methods found the same proportion of amoxicillin "susceptible" strains (27.3%). For clindamycin, both susceptibility testing methods provided identical MIC values (EA value = 100%), and exactly the same distributions of "susceptible" and "resistant" strains of P. intermedia/nigrescens. For metronidazole, only very poor agreement (EA value = 9.1%) was found between the two susceptibility testing methods, with MIC Test Strips exhibiting markedly higher MIC50 and MIC90 values against P. intermedia/nigrescens as compared to E-test strips. However, the distribution of "susceptible" and "resistant" P. intermedia/ nigrescens were identical between the two susceptibility testing methods. For doxycycline, relatively good agreement (EA value = 72.7%) was found in MIC concentrations between the two susceptibility testing methods, although generally lower MIC values were associated with MIC Test Strips. In addition, identical distributions of "susceptible" and "resistant" P. intermedia/nigrescens were provided by both susceptibility testing methods. Conclusions: Relative to MIC values measured against periodontal strains of P. intermedia/nigrescens, MIC Test Strips gave higher MIC values with amoxicillin and metronidazole, equal MIC values with clindamycin, and lower MIC values with doxycycline, as compared to MIC values measured with the E-test assay. Relative to the identification of antibiotic "susceptible" periodontal P. intermedia/ nigrescens strains, both susceptibility testing methods provided identical findings, suggesting that both methods appear to be interchangeable for clinical decision making in regard to identification of antibiotic-sensitive strains of periodontal P. intermedia/nigrescens. However, for epidemiologic surveillance of drug susceptibility trends, where exact MIC values are important to track over time, the relatively higher proportion of non-exact MIC differences between the two susceptibility testing methods argues against using them interchangeably. Instead, one or the other method should be used consistently for such studies. Further comparative studies of the E-test and MIC Test Strip assays are indicated using other periodontopathic bacterial species besides P. intermedia/ nigrescens, and to assess the reproducibility of MIC values provided by both in vitro susceptibility testing methods over time.
Temple University--Theses

Levofloxacin is in a class of antibiotics known as fluoroquinolones, which treat infections by killing the bacteria that cause them. A physiologically-based pharmacokinetic (PBPK) model was developed to investigate the uptake, distribution, and elimination of Levofloxacin after a single dose. PBPK modeling uses parameters such as body weight, blood flow rates, partition coefficients, organ volumes, and several other parameters in order to model the distribution of a particular drug throughout the body. Levofloxacin is only moderately bound in human blood plasma, and, thus, for the purposes of this paper, linear bonding is incorporated into the model because the free or unbound portion of the drug is the only portion that is considered to be medicinally effective. Parameter estimation is then used to estimate the two unknown parameters given clinical data from literature on the total concentration of Levofloxacin in the blood over time. Once an adequate model is generated, the effects of varying Body Mass Index are tested for the absorption and distribution of Levofloxacin throughout the body.

Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.

Aquaculture is the fastest growing food industry in the world and it accounts for roughly half of the world's fish supply. The majority of global aquaculture production occurs in freshwater systems that are increasingly subject to multiple uses by different stakeholders. Given the overall scarcity of freshwater on a global scale, freshwater aquaculture will face increasing environmental constraints that will demand an ever better understanding of its potential impacts on the aquatic environment and human health. This thesis consists of a series of studies that, collectively, contribute to further our understanding on the effects of freshwater aquaculture effluents on aquatic ecosystems, on the effects and environmental safety of antibiotics used in freshwater aquaculture on aquatic bacterial communities and on the link between antibiotic pollution and antibiotic resistance. Chapter 2 reviews the effects of freshwater aquaculture effluents on stream ecosystems using land-based salmonid farms as a case study. In this chapter I discuss relevant considerations related to the temporal and spatial scales of effluent discharge and ecological effects that highlight the need to characterize the patterns of stressor discharge when assessing environmental impacts and designing ecological effects studies. I also discuss the potential role of multiple stressors - with an emphasis on veterinary medicines - in disrupting ecosystem structure and function. Overall, the critical analysis presented in this chapter indicates that further research on the effects of veterinary medicines using relevant exposure scenarios would significantly contribute to our understanding of their impact in relation to other effluent stressors. Chapter 3 is a general methods chapter that describes the stream microcosm system used to assess the effects of erythromycin thiocyanate (ERT) and florfenicol (FFC) on bacterial communities of stream biofilms. This chapter presents the results of preliminary experiments whose results provided relevant information on the overall operation of the microcosms and on the variability of major physical and biological variables. This information guided the experimental designs used to assess the effects of FFC and ERT on the bacterial community structure of stream biofilms. Chapter 4 presents the results of the experiment conducted to assess the effects of FFC on the bacterial community structure of developing biofilms. The objective was to assess changes in bacterial community structure along a gradient of FFC concentrations that could provide insight into the type and magnitude of effects that could be expected from episodic exposure of stream biofilms to FFC in headwater streams. At 10 and 20 days of biofilm development, bacterial community structure differentiated in a pattern consistent with the FFC concentration gradient and there was a positive relationship between bacterial richness and bacterial diversity with FFC concentration. At 15 days of biofilm development there was also a positive relationship between FFC concentration and the surface coverage of bacteria and extracellular polymeric substances. These trends declined as the biofilm developed a more complex architecture, in terms of thickness and in the surface coverage of algae. The results are consistent with an initial stimulatory effect of FFC on biofilm formation that triggered changes in bacterial community structure that were gradually compressed as the development of a complex biofilm architecture increased the relative importance of autogenic ecological processes. The results suggest that the co-occurrence of FFC with bacterial pathogens in effluents and wastewaters may favour their persistence in the environment by enhancing biofilm formation. Chapter 5 presents the results of the experiment conducted to assess the effects of ERT on the bacterial community structure of developing biofilms. Currently, Aquamycin® 100 - a Type A medicated article (i.e., Premix) containing 100 g ERT lb-1 and used to produce a Type C medicated feed - is a candidate drug for approval by the US FDA to control mortality associated with bacterial kidney disease in freshwater salmonids. The objective of this experiment was to assess the effects of ERT on the bacterial community structure of stream biofilms using an exposure period consistent with the 28-day treatment regime suggested for Aquamycin® 100. The results provide no evidence to suggest that a 30-day exposure to ERT concentrations in the range of 10 μg L-1 (i.e., 7.3 ± 3.9 μg L-1) would lead to changes in the bacterial community structure or overall bacterial abundance of stream biofilms, while they suggest that these effects may occur at concentrations in the range of 100 μg L-1 (i.e., 87.2 ± 31.1 μg L-1). Chapter 6 attempts to determine whether environmental concentrations of antibiotics and concentrations representing action limits used in environmental risk assessment may exert a selective pressure on clinically relevant bacteria in the environment. In this chapter I use bacterial inhibition as an assessment endpoint to link antibiotic selective pressures to the prevalence of resistance in bacterial populations. Species sensitivity distributions were derived for three antibiotics by fitting log-logistic models to endpoints calculated from minimum inhibitory concentration (MIC) distributions based on worldwide data collated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Bacteria represented in these distributions were placed in a broader context by performing a brief phylogenetic analysis. The potentially affected fraction of bacterial genera at measured environmental concentrations of antibiotics and environmental risk assessment action limits was used as a proxy for antibiotic selective pressure. Measured environmental concentrations and environmental risk assessment action limits were also directly compared to wild-type cut-off values. Results suggest that measured environmental concentrations of antibiotics and concentrations representing environmental risk assessment action limits are high enough to exert a selective pressure on clinically relevant bacteria that may lead to an increase in the prevalence of resistance. Chapter 7 presents the results of an exploratory analysis conducted to assess the abundance of class 1 integrons in stream biofilms exposed to FFC and ERT. There was no pattern in the abundance of intI1 genes consistent with the treatment of FFC and ERT, suggesting either the absence of gene cassettes involved in dealing with selective pressures caused by these antibiotics or that the concentrations tested were below those required to give them a selective advantage. Chapter 8 is a brief general discussion that brings together the findings of the thesis and makes suggestions for future research. Key areas identified for future research include assessing in further detail the stimulatory effect of FFC on biofilm formation in complex bacterial communities, the interactive effects of multiple aquaculture effluent stressors on aquatic bacterial communities and their potential effects on the development of antibiotic resistance, the fate of FFC and ERT in stream ecosystems, and further developing the analysis based on MIC distributions presented in chapter 6 to assess the potential effects of antibiotic pollution on the selection of multi-drug resistance in the environment.

Os Mollicutes causam doenças em várias espécies animais de importância econômica, inclusive em bovinos. Neste estudo, foi avaliada por concentração inibitória mínima (CIM) e citometria de fluxo, a atividade de oito agentes antibacterianos (enrofloxacina, ciprofloxacina, gentamicina, claritromicina, cloranfenicol, oxitetraclina, tiamulina e tilosina) contra Ureaplasma diversum. Foram analisadas 24 amostras de isolados de campo oriundas da mucosa genital de fêmeas bovinas. As amostras foram confirmadas por crescimento em caldo, placa e por PCR. Os inóculos foram submetidos à analise de suscetibilidade aos antibióticos pelo método da microdiluição em microplaca e posteriormente analisados pelo citômetro de fluxo a fim de avaliar a atividade antimicrobiana nas células. A claritromicina apresentou os maiores índices de inibição in vitro, sendo a gentamicina considerada o antibiótico de menor espectro de ação nesse estudo. De acordo com as análises do citômetro, a gentamicina apresentou o menor número de células viáveis enquanto a tiamulina apresentou o maior número. Embora haja resultados destoantes entre as técnicas utilizadas, o citômetro de fluxo pode ser utilizado como uma boa ferramenta para auxiliar a avaliação da suscetibilidade desses microrganismos a antibióticos.
The Mollicutes cause disease in several economically important species, including cattle. In this study, was evaluated by minimum inhibitory concentration (MIC) and flow cytometry, the activity of eight antibacterial agents (enrofloxacin, ciprofloxacin, gentamicin, clarithromycin, chloramphenicol, oxitetraclina, tiamulin and tylosin) against Ureaplasma diversum. We analyzed 24 samples of field isolates originating from the genital mucosa of cows. The samples were confirmed by growth in broth, plate, and PCR. The inoculations were subjected to analysis of susceptibility to antibiotics by the method of micro-dilution plate and then analyzed by flow cytometry to assess the antimicrobial activity in cells. Clarithromycin showed the highest levels of inhibition in vitro, the antibiotic gentamicin considered lower spectrum of action in this study. According to the analysis of the flow cytometer, gentamicin showed the lowest number of viable cells as tiamulin showed the greatest number. Although there are divergent results between the techniques used, flow cytometry can be used as a good tool even help assess the susceptibility of microorganisms to antibiotics.

This work was aimed at developing novel tools that utilize HINT, an empirical forcefield capable of quantitating both hydrophobic and hydrophilic (hydropathic) interactions, for implementation in theoretical biology and drug discovery/design. The role of hydrophobicity in determination of macromolecular structure and formation of complexes in biological molecules is undeniable and has been the subject of research across several decades. Hydrophobicity is introduced, with a review of its history and contemporary theories. This is followed by a description of various methods that quantify this all-pervading phenomenon and their use in protein folding and contemporary drug design projects – including a detailed overview of the HINT forcefield. The specific aim of this dissertation is to introduce our attempts at developing new methods for use in the study of antibacterial drug resistance and antiviral drug discovery. Multidrug efflux is commonly regarded as a fast growing problem in the field of medicine. Several species of microbes are known to have developed resistance against almost all classes of antibiotics by various modes-of-action, which include multidrug transporters (a.k.a. efflux pumps). These proteins are present in both gram-positive and gram-negative bacteria and extrude molecules of various classes. They protect the efflux pump-expressing bacterium from harmful effects of exogenous agents by simply evacuating the latter. Perhaps the best characterized mechanism amongst these is that of the AcrA-AcrB-TolC efflux pump. Data is available in literature and perhaps also in proprietary databases available with pharmaceutical companies, characterizing this pump in terms of the minimum inhibitory concentration ratios (MIC ratios) for various antibiotics. We procured a curated dataset of 32 β-lactam and 12 antibiotics of other classes from this literature. Initial attempts at studying the MIC ratios of β-lactam antibiotics as a function of their three dimensional topology via 3D-quantitative structure activity relationship (3D-QSAR) technology yielded seemingly good models. However, this methodology is essentially designed to address single receptor-ligand interactions. Molecules being transported by the efflux pump must undoubtedly be involved in multiple interactions with the same. Notably, such methods require a pharmacophoric overlap of ligands prior to the generation of models, thereby limiting their applicability to a set of structurally-related compounds. Thus, we designed a novel method that takes various interactions between antibiotic agents and the AcrA-AcrB-TolC pump into account in conjunction with certain properties of the drugs. This method yielded mathematical models that are capable of predicting high/low efflux with significant efficiency (>93% correct). The development of this method, along with the results from its validation, is presented herein. A parallel aim being pursued by us is to discover inhibitors for hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 3 (HPIV3) by in silico screening. The basis for targeting HN is explored, along with commentary on the methodology adopted during this effort. This project yielded a moderate success rate of 34%, perhaps due to problems in the computational methodology utilized. We highlight one particular problem – that of emulating target flexibility – and explore new avenues for overcoming this obstacle in the long run. As a starting point towards enhancing the tools available to us for virtual screening in general (and for discovering antiviral compounds in specific), we explored the compatibility between sidechain rotamer libraries and the HINT scoring function. A new algorithm was designed to optimize amino acid residue sidechains, if provided with the backbone coordinates, by generating sidechain positions using the Dunbrack and Cohen backbone-dependent rotamer library and scoring them with the HINT scoring function. This rotamer library was previously used by its developers previously to design a very successful sidechain optimization algorithm called SCWRL. Output structures from our algorithm were compared with those from SCWRL and showed extraordinary similarities as well as significant differences, which are discussed herein. This successful implementation of HINT in our sidechain optimization algorithm establishes the compatibility between this forcefield and sidechain rotamer libraries. Future aims in this project include enhancement of our current algorithm and the design of a new algorithm to explore partial induced-fit in targets aimed at improving current docking methodology. This work shows significant progress towards the implementation of our hydropathic force field in theoretical modeling of biological systems in order to enhance our ability to understand atomistic details of inter- and intramolecular interactions which must form the basis for a wide variety of biological phenomena. Such efforts are key to not only to understanding the said phenomena, but also towards a solid basis for efficient drug design in the future.

A mastite bovina apresenta alta prevalência nos rebanhos leiteiros e a antibioticoterapia é o procedimento mais utilizado no tratamento da mesma. O Staphylococcus aureus, agente etiológico mais frequentemente isolado em casos de mastite, tem demonstrado em diversos estudos aumento crescente no padrão de resistência aos antimicrobianos. O desenvolvimento de bactérias resistentes e a presença de resíduos de antimicrobianos no leite estimulam o desenvolvimento de novos tratamentos que solucionem estes problemas. O muco dos moluscos Achatina fulica é composto por uma glicoproteína, denominada achacin, com atividade antimicrobiana sobre bactérias Gram positivas. Sendo assim, os objetivos deste estudo foram avaliar a atividade antimicrobiana in vitro do muco de A. fulica sobre S. aureus, bem como determinar e avaliar a concentração inibitória mínima (CIM) do muco de A. fulica purificado sobre S. aureus ATCC 25923 e isolados de S. aureus provenientes de infecções intramamárias bovina. O muco foi purificado mediante cromatografia líquida de alta eficiência e a fração com atividade antimicrobiana foi submetida à quantificação protéica e microdiluição em caldo. A CIM do muco purificado determinada para a cepa ATCC 25923 correspondeu a 50 µg/mL e para os isolados de S. aureus, variou entre 12,5 e 100 µg/mL. Sendo que destes, dois isolados (6,7 %) apresentaram CIM de 12,5 µg/mL, três (10 %) CIM de 25µg/mL, vinte e três (76,6 %) CIM de 50 µg/mL e dois (6,7 %) apresentaram CIM de 100 µg/mL. A CIM50 para os isolados correspondeu a 50 µg/mL e a CIM90 a 100 µg/mL. No presente estudo observou-se que o muco purificado apresentou atividade antimicrobiana com ação bactericida. Estes resultados proporcionam perspectivas para a compreensão e otimização de protocolos microbiológicos deste potencial biofármaco com a finalidade de empregá-lo futuramente na terapêutica veterinária para o controle de infecções intramamárias causadas por S. aureus.
The bovine mastitis is highly prevalent in dairy herds and antibiotic therapy is the procedure most commonly used to treat it. Staphylococcus aureus is the most frequently isolated etiologic agent in cases of mastitis and it has been shown in several studies the pattern of increasing antimicrobial resistance. Due to the development of resistant bacterias and the presence of antimicrobial residues in the milk the development of new treatments to solve those problems are required. The mucus of the molluscs Achatina fulica contains a glycoprotein called achacin that presents antimicrobial activity against Gram positive bacteria. The aim of this study was the evaluation of the in vitro antimicrobial activity of the A. fulica mucus of on S. aureus, as well the determination of the minimum inhibitory concentration (MIC) of A. fulica mucus purified on S. aureus ATCC 25923 and isolates of S. aureus from bovine intramammary infections. The mucus was purified by high performance liquid chromatography. The protein content in portion presenting the was quantified and microdiluted. The MIC determined for the purified mucus strain ATCC 25923 was 50 µg/mL and for the isolates of S. aureus ranged between 12,5 and 100 µg/mL. Among these samples, two isolates (6,7 %) showed MIC of 12,5 µg/mL, three (10 %) of MIC 25 µg/mL, twenty-three (76,6 %) MIC of 50 µg/mL and two (6,7 %) had an MIC of 100 µg/mL. The MIC50 for isolates was 50 µg/mL and MIC90 to100 µg/mL. It was found that the purified mucus showed bactericidal activity. The results showed in the present work improved the understanding and optimization of the biopharmaceutical microbiological potential that could be applied in new protocols in future veterinary therapy for the control of intramammary infections caused by S. aureus.

博士
國立陽明大學
微生物及免疫學研究所
104
Mycobacterium abscessus has emerged as the most pathogenic and chemotherapy-resistant rapid-growing mycobacterium. The mechanism of antibiotic resistance in M. abscessus was well studied, but effect of antibiotics on virulence of M. abscessus was still unknown. In this study, we observed that colony morphology of M. abscessus was transiently altered in the presence of aminoglycoside antibiotics; meanwhile their virulence and biofilm formation was also enhanced. Amikacin pretreatment contributed to ability of anti-phagocytosis, TNF-α cytokine stimulation and persistence to human macrophage-mediated killing. In addition, aminoglycoside antibiotics promoted the drug tolerance in M. abscessus. Sub-MIC amikacin activated whiB7Mabs gene expression. Overexpression of WhiB7Mabs triggered the colony morphotype switch, and enhanced the persistence against macrophage-mediated killing. Globally, aminoglycoside antibiotics switched the avirulent smooth morphotype into invasive rough morphotype of M. abscessus through WhiB7-mediated pathway. Thus, these findings suggested that sub-MIC aminoglycoside treatment could predispose the M. abscessus infection patients to more severe illness and may concern the guideline of antibiotic therapy.

Biofilms are complex communities of microbial cells encased in a self-produced extracellular matrix which are associated with recurrent infections in clinical settings and are difficult treat. Many microorganisms including Staphylococcus species form biofilms. The difficulty of treating biofilms is due to tolerance phenotypes which include reduced growth rate, the extracellular matrix reducing antibiotic penetration and adaptive responses to environmental stresses. Sub-inhibitory concentrations of antibiotics have been reported to influence eDNA-dependent biofilm formation in species of S. aureus with poor biofilm forming ability in an eDNA-dependent manner. However, gene expression studies into the regulation of eDNA on strains which respond have not been conducted. The aim of this study was to evaluate whether there is a link between a positive biofilm response to sub-inhibitory concentrations of antibiotics and up-regulation of genes involved in cell lysis and DNA degradation. A number of strains of Staphylococcus aureus were screened for their ability to form a biofilm. Those which showed poor biofilm forming ability were exposed to further analysis at sub-inhibitory concentrations of oxacillin and vancomycin. Two strains, S. aureus WSU2 and WSU3 showed a response to 1/2 MIC of vancomycin and 1/4 MIC of oxacillin respectively. S. aureus WSU2 showed a 1.8 times increase in eDNA with a similar increase in biofilm production whilst S. aureus WSU3 showed a doubling of eDNA and biofilm production. Reverse Transcription Real Time PCR was used to analyse the expression of genes which have been linked to cell death and lysis in S. aureus. These genes included cidA and lrgA, which act on murein hydrolases, the autolysin atlA, and the staphylococcal nuclease genes nuc 1 and nuc 2. The polysaccharide gene, icaA was also measured to see if it was linked to increased biofilm production in the test strains. There were small changes in gene regulation for both S. aureus WSU2 and WSU3 for all genes tested. S. aureus WSU2 treated with vancomycin showed different expression of genes compared to S. aureus WSU3 treated with oxacillin. In most cases the nucleases genes were unaffected, however in S. aureus WSU3 the nuclease genes were found to be significantly downregulated. The autolysin gene was significantly upregulated 1.6 fold in S. aureus WSU2 treated with 1/2 MIC vancomycin. This upregulation of the autolysin was consistent with the increase in eDNA observed for the strain. This effect was also observed when 24 hour biofilms were exposed to a single 90 minute antibiotic treatment. The gene expression changes observed in this study, although are less than 2-fold, do appear to show a consistent trend and suggest that atlA could play an important role in eDNA-dependent biofilm formation. With further investigation, the link between gene expression and eDNA-dependent biofilm formation under sub-inhibitory doses of antibiotics can be further understood. The increase in biofilm formation and alteration of eDNA within the biofilm matrix can potentially impact S. aureus biofilm-associated infections. Therefore, further studies may give a better understanding of the effect of sub-inhibitory antibiotic exposure on biofilm formation which could lead to improved treatment strategies.

Emergence of resistance is never far behind the introduction of new antimicrobials. In response to antimicrobial challenge, bacteria have the capacity to alter their genomes in order to survive. Antibiotic environments play an important but poorly defined role in mediating such changes. The primary focus of this research was to further define the role of sub-inhibitory antimicrobials in the bacterial response to antimicrobial challenge, with particular attention to adaptive resistance responses. A greater understanding of this biological signature will help in evaluating dosing regimes, understanding mechanism of action and in designing new antimicrobials. The effect of sub-inhibitory ciprofloxacin was studied here, beginning with a global analysis using custom DNA microarray technology. Before conducting this analysis, a custom DNA microarray to 5,378 of the 5,570 open reading frames in the Pseudomonas aeruginosa genome was designed, constructed and validated, along with the accompanying set of RNA isolation, cDNA preparation and labelling, and microarray hybridization protocols. Analysis of the P. aeruginosa transcriptome following exposure to sub-inhibitory ciprofloxacin found expression changes in numerous genes. Prominent among these changes was up-regulation of the SOS DNA-repair response and the R2/F2 pyocin region. These changes were confirmed at both the transcription and protein level. Mutants in the R2/F2 pyocin region were found to be resistant to quinolones as well as other DNA damaging agents like mitomycin C, highlighting a role for this region in mediating susceptibility to DNA damage. I also found that sub-inhibitory ciprofloxacin, particularly 0.3μg/ml ciprofloxacin, induced adaptive resistance responses in P. aeruginosa. Similar responses were not observed in a R2/F2 pyocin mutant strain, although the extent of the role of the R2/F2 pyocin region in mediating adaptive resistance to ciprofloxacin awaits further characterization. Overall, this study indicated that sub-inhibitory concentrations of ciprofloxacin antimicrobial do play an important role in the development of resistance in P. aeruginosa, and highlight the clinical importance of better understanding bacterial-antimicrobial interactions.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of biological and medical sciences Study program: Pharmacy Autor: Klára Kopečná Supervisor: Mgr. Klára Konečná, Ph.D. Title of diploma thesis: Evaluation of activity of potencional antibiotic substances through the use of microdilution broth method II Background: To evaluate the antibacterial activity of the compounds synthesized by the Department of Pharmaceutical Chemistry and Drug control, Faculty of Pharmacy in Hradec Králové, Charles University in Prague under the leadership of prof. PharmDr. Martin Doležal, Ph.D. Methods: Using broth microdilution method was tested antibacterial activity at eight bacterial strains, namely Staphylococcus aureus, Staphylococcus aureus methicilin resistant, Staphylococcus epidermidis, Enterococcus sp., Escherichia coli, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL positive and Pseudomonas aeruginosa. Results: Test substances were divided into 5 groups according to similarities in the chemical structure. The best antibacterial activity was observed in the group of derivatives of N-benzyl- 6-(alkylamino)pyrazine-2-carboxamide. Conclusion: Antibacterial activity was detected in 10 of 24 tested compounds. From active substances, all but one danced only Gram-positive bacteria. Among...

This study is a part of a project at Q-linea that aims to present a rapid diagnostic instrument to speed up the process of identification of pathogens and determination of MIC-values (Minimal Inhibitory Concentration) of antibiotic needed to treat patients with sepsis. Specifically, this report is aimed to describe the development and implementation of algorithms that examine susceptibility profiles ofsepsis related pathogens where the bacteria have been exposed to different antibiotics and by different lapse of concentrations. The developed algorithms are based on a clustering technique that identify inhibited growth and present the lowest concentration needed to slow down the growth of the pathogen. The implemented solution was tested on sepsis related pathogens and the determined MIC values were compared to MIC values generated with a method commonly used in healthcare today. Approximately 90% of instances were correctly classified based on data from six hours long tests which is significantly faster than the reference method which takes 16-24 hours to complete. Furthermore, each result comes with a set of quality measures for validation of the algorithm results. Although, further studies are necessary to increase the performance at the four-hour target time, and more data is needed to validate the developed quality measures.

Saskatchewan is the only province in Canada to have routinely tested the antimicrobial susceptibility of all provincially reported human cases of campylobacteriosis. From 1999 to 2006, 1378 human Campylobacter species infections were tested for susceptibility at the Saskatchewan Disease Control Laboratory using the Canadian Integrated Program for Antimicrobial Resistance Surveillance panel and minimum inhibitory concentration (MIC) breakpoints. Of these, 1200 were C. jejuni, 129 were C. coli, with the remaining made up of C. lari, C. laridis, C. upsaliensis and undifferentiated Campylobacter species. Campylobacter coli had significantly higher prevalences of ciprofloxacin resistance (CIPr), erythromycin resistance (ERYr), combined CIPr-ERYr resistance and multidrug resistance (to three or greater drug classes) than C. jejuni. Logistic regression models indicated that CIPr in C. jejuni decreased from 1999 to 2004 and subsequently increased in 2005 and 2006. The risk of CIPr was significantly increased in the winter months (January to March) compared to other seasons. A comparison of logistic regression and Cox proportional hazard survival models found that the latter were better able to detect significant temporal trends in CIPr and tetracycline resistance by directly modeling MICs, but that these trends were more difficult to interpret. Scan statistics detected significant spatial clusters of CIPr C. jejuni infections in urban centers (Saskatoon and Regina) and temporal clusters in the winter months; the space-time permutation model did not detect any space-time clusters. Bernoulli scan tests were computationally the fastest for cluster detection, compared to ordinal MIC and multinomial antibiogram models. eBURST analysis of antibiogram patterns showed a marked distinction between case and non-case isolates from the scan statistic clusters. Multilevel logistic regression models detected significant individual and regional contextual risk factors for infection with CIPr C. jejuni. Patients infected in the winter, that were between the ages of 40-45 years of age, that lived in urban regions and that lived in regions of moderately high poultry density had higher risks of a resistant infection. These results advance the epidemiologic knowledge of CIPr C. jejuni in Saskatchewan and provide novel analytical methods for antimicrobial resistance surveillance data in Canada.
Saskatchewan Disease Control Laboratory (Saskatchewan Ministry of Health); Laboratory for Foodborne Zoonoses (Public Health Agency of Canada); Centre for Foodborne, Environmental and Zoonotic Infectious Diseases (Public Health Agency of Canada); Ontario Veterinary College Blake Graham Fellowship