The objective of this longitudinal study was to study the relationship between changes in prevalence of resistant enteric bacteria associated with mean monthly doses (MMD) of various antibiotics used in each of two host species. From January 2004 – January 2007, monthly composite swine fecal samples and human wastewater samples representing various production and occupational cohorts, respectively, were collected from 19 geographically unique locations in east- and south-central Texas. Bacterial isolates cultivated on CHROMagar-E.coliTM and DifcoTM mEnterococcus (ME) were tested for susceptibility to multiple antibiotics by microbroth dilution using the SensititreTM system. The relationship between the prevalence of resistant bacteria, sampling period, and antibiotic use within each host species was assessed in a generalized linear model adjusted for the dependence of responses within location using a binomial distribution and logit link function in STATA® ver. 9.2. For the swine E. coli isolates, the relationship between tetracycline resistance and level of chlortetracycline (CTC) use in swine illustrated a dose-response relationship, with odds ratios (OR) of 1.20 and 1.81 (P < 0.05) for second- and third-level categories of MMD relative to baseline (zero-use) respectively. When considered by swine production groups, intake boar isolates had an elevated relative odds of resistance to tetracycline (OR = 1.51, P < 0.05), and the nursery units had an elevated odds (OR = 2.61, P < 0.05) of exhibiting resistance to ceftiofur, relative to pigs housed in the farrowing barns. Regarding swine Enterococci isolates, those swine from locations that utilized tylosin had an elevated OR of 3.54 (P < 0.05) of exhibiting resistance to tylosin, relative to those locations that used no tylosin. At this juncture, an apparent occupational risk of harboring tetracycline-resistant E. coli, and the apparent sparing effect (Enterococcus spp.) associated with exposure to swine production, remain unexplained. This study demonstrated that the prevalences of tetracycline- and tylosin-resistant enteric bacteria swine were dependent on CTC and tylosin use in feed, respectively. Swine production group-effects on the prevalence of tetracycline, ceftiofur, and erythromycin resistance were also important. This study provides a better understanding of the relationships between antibiotic prescribing practices at the ecologic level and the relative odds of carriage of resistant bacteria within two host species in a vertically integrated agri-food system.

The essay investigates the economic burden of skin, soft tissue, bone and lung infections in Sweden 2003. The cost-of-illness method, based on the human capital theory, is used in the estimation. A prevalence approach and a top-down method were chosen for direct as well as indirect costs. Also there is a discussion concerning health economic aspects of antibiotic resistance and evidence of the increasing costs because of it. The lack of data leads to a result within a large interval of uncertainty; the direct costs are estimated to 1 072 million SEK and indirect costs are estimated to 4 655 million SEK.

The study of protein evolution and adaptation resides at the junction between the disciplines of biological chemistry and evolutionary biology. We chose the B. thetaiotaomicron tetracycline resistant enzyme TetX2, as our model system to study the biophysical basis for adaptation to antibiotics; a phenomenon that continuously poses global health challenges. In the work presented here, experimental evolution and biophysical characterization were used to identify and link the physicochemical properties of TetX2 and its adaptive mutants to increased resistance to minocycline. Bacteroides thetaiotaomicron TetX2 was previously identified as a novel oxidoreductase with broad activity against tetracyclines. Experimental evolution of E. coli expressing a chromosomal copy of tet(X2) was used to identify an adaptive mutation (TetX2 T280A ) that confers higher resistance to minocycline and tigecycline. In addition to TetX2 T280A , a family of variants of TetX2 with single amino acid changes in TetX2 sequence that conferred equal or higher resistance towards MCN was identified by error-prone mutagenesis. Changes in fitness of E. coli carrying a single chromosomal copy of either wild-type or one of the mutant alleles were assessed by growth rate assays over a range of minocycline concentrations. Despite similar in vivo performances of TetX2 T280A and two other variants (TetX2 N371I and TetX2 N371T ), TetX2 T280A was the only successful mutant in the adaption experiment suggesting that mutational supply may play an important role in evolutionary dynamics of populations undergoing adaptation. The most surprising result is that the differences in growth rates among TetX2 variants arise from small changes in in vitro catalytic activity and in vivo protein expression. The steady-state kinetic studies with minocycline and NADPH suggest a binary mechanism for antibiotic inactivation by TetX2 which is supported by the structural characteristics of the enzyme. The atomic structures of the best adaptive mutant TetX2 T280A in complex with minocycline and tigecycline reveal the details of substrate recognition and show that the site of the mutation is ∼18 Å away from the active site suggesting an indirect mechanism for improved catalysis. Taken together, our data show that very small changes in the in vitro biochemical properties and expression levels can have surprisingly large fitness effects and are important during adaption. In addition, a promising preliminary mathematical model suggests that based on kinetic activity and in vivo expression levels the success of bacteria undergoing adaptation to antibiotics can be predicted.

Acinetobacter baumannii is an opportunistic pathogen which has been implicated in a variety of nosocomial infections among immunocompromised patients worldwide. Recently, Multi-drug resistant (MDR) isolates of A. baumannii have been isolated from military personnel returning from service in Iraq and Afghanistan. Antibiotic resistance of A. baumannii has limited the number of active antibacterial, making very difficult to treat these types of infections. This work investigated the role of Resistance-Nodulation-cell Division (RND) efflux pumps in the antibiotic resistance mechanism of A. baumannii. Expression of six different RND pumps was analyzed in clinical isolates of A. baumannii. A novel RND family pump, AdeMNO, was found to be present in a majority of isolates. The adeMNO operon was cloned, sequenced, and characterized using the single copy gene expression system in an efflux sensitized surrogate Pseudomonas aeruginosa strain. Antibiotics, trimethoprim, chloramphenicol, and clindamicin were identified as the substrates of this pump. In order to understand the mechanisms of regulation of adeMNO operon, a putative regulator belonging to the lysR-family was identified, cloned, and sequenced from the upstream region of the operon. Promoter regions of the adeMNO operon were also sequenced from various clinical isolates and sequence polymorphisms identified that could be implicated in the regulation of adeMNO expression. / UOIT

The continued propagation of antibiotic resistance requires the development of new therapeutics. The lipopeptide antibiotic enduracidin has demonstrated high activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus. In addition to a lack of cross-resistance with existing antibiotic classes, enduracidin has no known transferrable resistance mechanism. The development of enduracidin as a human therapeutic is hampered by its poor solubility in plasma. Utilizing chemical and genetic techniques, analogs of enduracidin have been produced and evaluated for biological activity. Making use of the hydroxyphenylglycine (Hpg) biosynthetic pathway, fluorine was incorporated into enduracidin with minimal to no loss of bioactivity. The semisynthetic chemical modification of enduracidin proved to be challenging. The chemical nitration of the Hpg residues was unsuccessful. Modifications to the lipid tail by cleavage at the C2-olefin with ozone and the use of Diels-Alder reagents to react with the lipid tail diene also proved unsuccessful. However, the reduction and dihydroxylation modifications of the lipid tail diene were successful. Introduction of polar hydroxyl groups onto the alkyl tail reduced bioactivity while reduction of the diene had no significant effect. Analysis of the biosynthetic pathways involved in producing the lipid tail and the unusual amino acid enduracididine yielded some insights into the formation of the antibiotic. Through complementation of mutants having disruptions in the biosynthetic gene cluster and crystallographic data, the function of EndR as a cyclase was established. Additionally, the use of 4-hydroxyarginine as an intermediate in enduracididine biosynthesis was demonstrated. The ability of EndQ to function as a transaminase on both 4-hydroxyarginine and 2-ketoenduracididine was also established. The specific functions of EndP and EndQ have not been determined. The introduction of the lipid tail diene by the three enzymes Orf39, Orf44 and Orf45 was confirmed. Orf45 functions as a CoA ligase and a dehydrogenase to introduce the C2 double bond. The functions of Orf39 and Orf44 appear to be the introduction of the C4 double bond and isomerization of the C2 olefin. / Graduation date: 2013

In India, there is no regulation of antibiotics and allegedly the use has doubled since 2006. Indiscriminate use of antibiotics gives rise to development of resistant bacteria. The media has, according to the theories used in this study, a responsibility to educate and empower the people to make personal judgments about health risks. This study focuses on the extent to which two of the largest English-language newspapers in India, the Hindu and Times of India, report on antibiotic resistance; and also, how the journalists and editors on these newspapers look upon their profession and responsibilities when it comes to reporting on health issues. In addition to the quantitative content analysis, which comprises 162 articles about antibiotic resistance published between 2006 and 2012, six in-depth interviews were conducted. The results show that the amount of coverage on antibiotic resistance increased 2010 when the Lancet published a report on new findings of multi-resistant bacteria in India. This indicates that an event was needed to qualify antibiotic resistance for the news pages. Our study also shows that preventive measures which can be taken to reduce the emerge of resistant bacteria are often included in the articles and that they are addressed to doctors as well as to the general public. On the other hand, information on the magnitude of the problem is rarely presented. Scientists are often quoted or referred to, and the journalists of the investigated newspapers state that they have a great confidence in them. Furthermore, the respondents express that they have a responsibility to report on health issues. They believe that their newspapers have a major influence on its readership, and that their reporting can make a difference in the health situation in India. Some of them mention, however, that their overall impact is limited since their newspapers only reach the literate middle-class.

3,3'-Neotrehalosadiamine (NTD) is a diaminosugar that possesses a rare alpha,beta-1,1'-linked glycosidic bond and has been reported to possess antimicrobial activity against Staphylococcus aureus. The ntdABC operon contains three structural genes that are necessary for the production of NTD in certain mutants of Bacillus subtilis. The gene predicted to be the first in the NTD biosynthetic pathway, ntdC, was subcloned into pET-28b as the hexa-histidine tagged fusion. The gene product was expressed, purified to homogeneity, and found to be an NAD+-dependent glucose 6-phosphate 3-dehydrogenase, likely operating according to a ternary complex mechanism and possessing a catalytic dyad composed by D176 and H180. The advent of this knowledge suggests that additional genes are required for the biosynthesis of NTD aside from the three encoded by the ntdABC operon.

<p>Bistable switches are commonly observed in the regulation of critical processes such as cell cycles and differentiation. The switches possess two fundamental properties: memory and bimodality. Once switched ON, the switches can remember their ON state despite a drastic drop in stimulus levels. Furthermore, at intermediate stimulus levels with cellular noise, the switches can cause a population to exhibit bimodal distribution of cell states. Till date, experimental studies have focused primarily on cellular mechanisms that generate bistable switches and their impact on cellular dynamics. </p><p>Here, I study emergent bistability due to bacterial interactions with either synthetic gene circuits or antibiotics. A synthetic gene circuit is often engineered by considering the host cell as an invariable "chassis". Circuit activation, however, may modulate host physiology, which in turn can drastically impact circuit behavior. I illustrate this point by a simple circuit consisting of mutant T7 RNA polymerase (T7 RNAP*) that activates its own expression in bacterium Escherichia coli. Although activation by the T7 RNAP* is noncooperative, the circuit caused bistable gene expression. This counterintuitive observation can be explained by growth retardation caused by circuit activation, which resulted in nonlinear dilution of T7 RNAP* in individual bacteria. Predictions made by models accounting for such effects were verified by further experimental measurements. The results reveal a novel mechanism of generating bistability and underscore the need to account for host physiology modulation when engineering gene circuits.</p><p>In the context of antibiotic treatment, I investigate bistability as the underlying mechanism of inoculum effect. The inoculum effect refers to the decreasing efficacy of an antibiotic with increasing bacterial density. Despite its implication for the design of antibiotic treatment strategies, its mechanism remains poorly understood. Here I show that, for antibiotics that target the core replication machinery, the inoculum effect can be explained by bistable bacterial growth. My results suggest that a critical requirement for this bistability is sufficiently fast turnover of the core machinery induced by the antibiotic via the heat shock response. I further show that antibiotics that exhibit the inoculum effect can cause a "band-pass" response of bacterial growth on the frequency of antibiotic treatment, whereby the treatment efficacy drastically diminishes at intermediate frequencies. The results have implications on optimal design of antibiotic treatment.</p> / Dissertation

The mammalian intestine contains a dense and diverse community of microorganisms. The resident microbiota makes contributions to host to promote proper immune system development and limit pathogen colonization. In this study, the effects of microbiota disruption with or without TLRs stimulation on intestinal permeability and immunity were examined in C57BL/6 mice receiving antibiotic treatment for 6 days and in antibiotics-treated mice received dead E. coli or S. aureus at day 4. The results showed that antibiotic treatment significantly decreased the total number of bacteria including specific aerobic group Enterobacteriaceae and Enterococcus, and specific anaerobic group Lactococcus/Bifidobacterium in intestinal mucosa and lumen. Although only a slight increase in the intestinal permeability and no change in caspase-3 activity of intestinal mucosa were observed after antibiotic treatment, the bacterial translocation (BT) to mesenteric lymph nodes (MLN) increased significantly. Subsequent experiments showed that antibiotic treatment decreased the mucosal killing activity and the expression of non-defensin family including RegIII£], RegIII£^, CRP-ductin and RELM£] but not the defensin family, and increased the translocation of pathogen K. pneumoniae significantly, suggesting that the increase of BT to MLN after antibiotic treatment is likely due to a reduction in gut immunity rather than an increase of intestinal permeability. Moreover, stimulation of TLR4 reversed the effect of antibiotic treatment, suggesting that the functioning of TLR4 in intestinal epithelium is required to prevent pathogenic invasion and maintain intestinal homeostasis.

The objective of this longitudinal study was to study the relationship between changes in prevalence of resistant enteric bacteria associated with mean monthly doses (MMD) of various antibiotics used in each of two host species. From January 2004 – January 2007, monthly composite swine fecal samples and human wastewater samples representing various production and occupational cohorts, respectively, were collected from 19 geographically unique locations in east- and south-central Texas. Bacterial isolates cultivated on CHROMagar-E.coliTM and DifcoTM mEnterococcus (ME) were tested for susceptibility to multiple antibiotics by microbroth dilution using the SensititreTM system. The relationship between the prevalence of resistant bacteria, sampling period, and antibiotic use within each host species was assessed in a generalized linear model adjusted for the dependence of responses within location using a binomial distribution and logit link function in STATA® ver. 9.2. For the swine E. coli isolates, the relationship between tetracycline resistance and level of chlortetracycline (CTC) use in swine illustrated a dose-response relationship, with odds ratios (OR) of 1.20 and 1.81 (P < 0.05) for second- and third-level categories of MMD relative to baseline (zero-use) respectively. When considered by swine production groups, intake boar isolates had an elevated relative odds of resistance to tetracycline (OR = 1.51, P < 0.05), and the nursery units had an elevated odds (OR = 2.61, P < 0.05) of exhibiting resistance to ceftiofur, relative to pigs housed in the farrowing barns. Regarding swine Enterococci isolates, those swine from locations that utilized tylosin had an elevated OR of 3.54 (P < 0.05) of exhibiting resistance to tylosin, relative to those locations that used no tylosin. At this juncture, an apparent occupational risk of harboring tetracycline-resistant E. coli, and the apparent sparing effect (Enterococcus spp.) associated with exposure to swine production, remain unexplained. This study demonstrated that the prevalences of tetracycline- and tylosin-resistant enteric bacteria swine were dependent on CTC and tylosin use in feed, respectively. Swine production group-effects on the prevalence of tetracycline, ceftiofur, and erythromycin resistance were also important. This study provides a better understanding of the relationships between antibiotic prescribing practices at the ecologic level and the relative odds of carriage of resistant bacteria within two host species in a vertically integrated agri-food system.