Dissertations / Theses on the topic 'Carbapenemase gram negative bacteria'

Atividades antropogênicas relacionadas ao uso massivo de antibacterianos tem favorecido para que ambientes aquáticos sejam importantes locais para seleção e/ou disseminação de bactérias multirresistentes (MR). O objetivo do estudo foi monitorar a ocorrência de bactérias MRs em ambientes aquáticos públicos no estado de SP, caracterizando genótipos de resistência adquirida. Foram analisados 50 isolados de bactérias gram-negativas, recuperadas de amostras de água, sendo que 70% dos isolados apresentaram perfil de MR, identificando-se os genes blaCTX-M-2 (n= 5), blaCTX-M-9 (n= 1), blaCTX-M-15 (n= 2), blaKPC-2 (n= 3), qnrB (n= 1), oqxA (n= 3), oqxB (n= 3) e, aac(6)1b-cr (n= 7). Destaca-se a primeira detecção de A. calcoaceticus produtora de KPC-2. Ambientes aquáticos de acesso público podem ser importantes fontes para a disseminação e/ou transmissão de uma ampla variedade de espécies bacterianas MRs tanto para seres humanos como para ecossistemas associados, sendo que a presença de genótipos endêmicos sugere contaminação por esgoto doméstico e/ou hospitalar.<br>Anthropogenic activities related to the massive use of antibacterial has contributed to the selection and spread of multidrug-resistant (MDR) into the aquatic environment. This study aimed to monitor the occurrence of MDR bacteria in public aquatic environments, in the state of São Paulo, characterizing genotypes of acquired resistance. Of the 50 gram-negative isolates, recovered of water samples, 70% exhibited a MDR profile. Indeed, blaCTX-M-2 (n= 5), blaCTX-M-9 (n= 1), blaCTX-M-15 (n= 2)-, blaKPC-2 (n= 3)-, qnrB (n= 1)-, oqxA (n= 3)-, oqxB (n= 3)- and aac(6)-1b-cr (n = 7) genes were identified. Noteworthy is the first the detection of KPC-2-producing Acinetobacter calcoaceticus. Aquatic environments, with public access, may be important sources for the dissemination and/or transmission of a wide variety of MDR bacterial species to both humans and associated ecosystems. Moreover, the presence of endemic genotypes suggests contamination by domestic and/or hospital sewage.

The increasing emergence and spread of antibiotic-resistant bacteria is a serious threat to public health. Of particular concern are Gram-negative bacteria such as Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Some of these strains are resistant to a large number of antibiotics and thus our treatment options are rapidly declining. In addition to the increasing number of antibiotic-resistant bacteria, a major problem is that many of the antibiotics at our disposal are ineffective against Gram-negative bacteria. This is partly due to the properties of the outer membrane (OM) which prevents efficient uptake. The overarching goal of this thesis was to investigate how the OM of the Gram-negative bacterium E. coli could be weakened to improve the activity of antibiotics. In the first two papers of my thesis (paper I + II), I investigated the periplasmic chaperone network which consists of the two parallel pathways SurA and Skp/DegP. This network is essential for the integrity of the OM and strains lacking either SurA or Skp are defective in the assembly of the OM, which results in an increased sensitivity towards vancomycin and other antimicrobials. We identified a novel component of the periplasmic chaperone network, namely YfgM, and showed that it operates in the same network as Skp and SurA/DegP. In particular, we demonstrated that deletion of YfgM in strains with either a ΔsurA or Δskp background further compromised the integrity of the OM, as evidenced by an increased sensitivity towards vancomycin. In the remaining two papers of my thesis (paper III + IV), the goal was to characterize small molecules that permeabilize the OM and thus could be used to improve the activity of antibiotics. Towards this goal, we performed a high-throughput screen and identified an inhibitor of the periplasmic chaperone LolA, namely MAC-13243, and showed that it can be used to permeabilize the OM of E. coli (paper III). We further demonstrated that MAC-13243 can be used to potentiate the activity of antibiotics which are normally ineffective against E. coli. In the last paper of my thesis (paper IV), we undertook a more specific approach and wanted to identify an inhibitor against the glycosyltransferase WaaG. This enzyme is involved in the synthesis of LPS and genetic inactivation of WaaG results in a defect in the OM, which leads to an increased sensitivity to various antibiotics. In this paper, we identified a small molecular fragment (compound L1) and showed that it can be used to inhibit the activity of WaaG in vitro. To summarize, this thesis provides novel insights into how the OM of the Gram-negative bacterium E. coli can be weakened by using small molecules. We believe that the two identified small molecules represent important first steps towards the design of more potent inhibitors that could be used in clinics to enhance the activity of antibiotics.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Two β-lactamase gene sequences encoded by <i>Ps. aeruginosa</i> RMS 149 plasmid and <i>Rps. capsulata</i> sp 108 were investigated. The genes were located using DNA recombinant techniques and their nucleic acid sequences were determined using the Sanger dideoxy-sequencing technique. The amino acid sequences were identified and compared with other characterised β-lactamases. They are both class A enzymes (Ambler classification). The pseudomonad plasmid encoded enzyme is expressed constitutively, but its gene sequence has an attenuator sequence - reminiscent of inducible bacterial synthetic operons. It also has three putative loop-forming sequences in the middle of the gene. RNA mapping studies indicate that the attenuator is read through from an upstream promoter. There is low level initiation from its own promoter and the transcripts sometimes terminate around the second internal stem-loop. The full message is also made. Thus, it is likely that the pseudomonad gene is normally highly regulated. Its constitutive expression may be as a result of some control mutation. The rhodopseudomonad enzyme is unlike other characterised Gram-negative class β-lactamases because it is inducible. Gene hybridization experiments suggest that it may be chromosomally encoded in strain sp 108 as well as in the Pen^S strain sp 109. β-lactamase active bands were also observed in Pen<SUP>S</SUP> <i>Rps. capsulata</i> St. Louis and <i>Rps. sphaeroides</i>. If this is the usual state of affairs in photosynthetic bacteria which are not normally subject to the selective pressures of the presence of β-lactam antibiotics by virtue of their aquatic habitat, the sp 108 strain may also be producing the enzyme in large quantities due to some control mutation. It is postulated then, that β-lactamase genes in Gram-negative bacteria may be of two kinds - one that is chromosomal and is highly regulated, and another which has lost the regulation and over-expresses the enzyme. The latter may be representative of the common plasmid-borne β-lactamase genes.

Post-transcriptional regulation plays a pivotal role in gene expression control as it ensures a fast response to environmental changes. This is particularly important in unicellular organisms that are exposed to environmental changes. During my PhD, I studied different aspects of this type of regulation in two Gram-negative bacteria: Escherichia coli and the opportunistic pathogen Pseudomonas aeruginosa. In E. coli I investigated the role of the ribosomal protein S1 in the interplay between translation and mRNA decay. In particular we showed that S1 not associated to 30S can sequester and protect mRNA from RNase E, the main endonuclease in E. coli. Another aspect that I considered in my research has been regulation by RNA molecules in P. aeruginosa. In the last decade, it has become clear how RNA-based regulatory mechanisms are important in controlling bacterial gene expression. However, little is known about it in this relevant human pathogen. By RNA deep sequencing we identified more than 150 novel candidate sRNAs in the P. aeruginosa strains PAO1 and PA14, which differ in virulence degree. We confirmed by Northern blotting the expression of 52 new sRNAs, substantially increasing the number of known sRNAs expressed by this bacterium. In this context we developed a genetic screen for the identification of genes post-transcriptionally regulated by RNA determinants and applied this system to the search of RNA thermometers (RNATs), i.e. mRNA determinants that couple translation with temperature changes. We identified four putative RNATs and validated two of them in E. coli. Interestingly, the two are located upstream of genes previously implicated in P. aeruginosa pathogenesis, namely dsbA and ptxS. ptxS RNAT was validated also in P. aeruginosa and represents the first RNAT ever described in this bacterium.

Resistance to antimicrobials is a well-known phenomenon leading to difficulties in the treatment of infectious diseases. The genetic determinants of such resistance are in general well understood: plasmids, transposons, insertion sequences and integrons are the most frequently related genetic elements. The word epigenetics refers to changes in the phenotype or in the gene expression caused by mechanisms other than underlying DNA sequence. In some cases these changes can remain for generations. Serratia marcescens is an enterobacterium characterized by its natural (intrinsic) resistance to most antibiotics. It is also a relevant opportunistic pathogen which has been involved in several pathologies such as urinary tract infections, prostheses infections, cellulitis, bacteremia and others. P. aeruginosa is a Gram-negative bacterium considered one of the major nosocomial pathogens worldwide. It causes several infections such as wound and burn infections as well as respiratory tract infections mostly affecting cystic fibrosis patients. An increasing prevalence of infections caused by multidrug-resistant (MDR) isolates has been reported in many countries and is actually a cause of concern. Both, P. aeruginosa and S. marcescens are relevant nosocomial pathogens. Some of the classic antimicrobials used to treat these pathogens are out-of-date and several of the new drugs available have already become targets for bacterial mechanism of resistance. Environmental conditions exert high pressure not only in the selection of genes encoding resistance to antibiotics or integron fixation in bacterial genomes or plasmids and other mobile elements transmission, but also in the expression of these potentialities that leads to resistance. Thus the role of epigenetics remains to be investigated. In addition it is well known that bacteria causing infections are naturally forming part of biofilms instead the planktonic way of life normally assumed to be in laboratory conditions. The aim of this thesis was the study of unconventional mechanisms of antimicrobial resistance contributing to MDR phenotypes in both S.marcescens and P. aeruginosa. Also the exploration of changes in antimicrobial susceptibilities of Serratia marcescens in the last 50 years by comparing isolates collected between 1945 and 1950, and current isolates. ¬The main conclusions obtained from this study are: 1. The resistome of Serratia marcescens did not change significantly during the antibiotic era. 2. Antibiotic withdrawing tends to restore original susceptible phenotypes, irrespective to the molecular mechanism involved in resistance. 3. None of Serratia strains studied presented integrons, any extended spectrum ß-lactamases. 4. Phenotypically determination of susceptibilities of old strains inactive during the last 60 years have confirmed results obtained by metagenomics i.e. the genes of resistance already existed before antibiotics discovery and use. 5. Multiresistant Pseudomonas aeruginosa harbored class 1 integrons containing a cassette encoding aminoglycoside adenylyltransferase (aadB). 6. Multiresistant Pseudomonas aeruginosa overexpressed MexAB-OprM and MexXY efflux machinery. 7. CCCP use should be avoided in experiments performed with P. aeruginosa and probably in other aerobic bacteria. 8. Meropenem induces the formation of aberrant long rods which can survive, accumulate less antibiotic than normal bacteria, and can revert to normal forma when antibiotic pressure disappears. 9. Colistin, the last therapeutic option to fight against Pseudomonas infections in cystic fibrosis patients, is normally active although cases of resistance have arisen recently. 10. Resistance to colistin seems to be mediated by lipopolysaccharide singular properties. 11. Colistin induces injuries in lipid bilayers, which can be studied by means of planar black lipid bilayer techniques. Preliminary results showed the ability of colistin to induce transient channels in the bilayers, with some dependence to voltage. 12. Recovery of susceptibility to imipenem is slower than acquision of resistance, since the selective advantage conferred by imipenem resistance in the presence of the antimicrobial is strong whereas OprD expression is likely evolutionarily advantageous only under certain and unknown environmental conditions.<br>Es van explorar els mecanismes no convencionals de resistència als agents antimicrobians que contribueixen a l’aparició de fenotips multiresistents en els bacteris Gram-negatius Serratia marcescens i Pseudomonas aeruginosa. Es van examinar també els canvis en la susceptibilitat als agents antimicrobians en S. marcescens durant els darrers 50 anys, comparant soques aïllades entre els anys 1945-1950 y soques actuals. ¬Les principals conclusions obtingudes d’aquest estudi són les següents: 1. El resistoma de Serratia marcescens no ha canviat significativament des de l’era pre-antibiòtica fins l’actualitat. 2. La retirada dels antibiòtics tendeix a recuperar els fenotips de susceptibilitat originals, independentment del mecanisme molecular implicat en la resistència. 3. Cap de las soques de Serratia estudiades va presentar integrons ni tampoc ß-lactamases d’espectre estès. 4. La determinació fenotípica de les susceptibilitats de las soques “antigues” de Serratia inactives durant 60 anys ha confirmat els resultats obtinguts mitjançant metagenòmica, és a dir, els gens de resistència als antibiòtics ja existien amb anterioritat al descobriment i ús dels antibiòtics. 5. En les soques clíniques de P. aeruginosa multiresistents es va detectar un integró de classe 1 que contenia el cassette gènic aadB, que codifica l’enzim aminoglicòsid 2’-O- adeniltransferasa, que confereix resistència a gentamicina, tobramicina i kanamicina. 6. Les soques multiresistents de P. aeruginosa van sobreexpressar els sistemes de reflux MexAB-OprM i MexXY-OprM. 7. L’ús de l’inhibidor de bombes de reflux CCCP s’hauria d’evitar als experiments realitzats amb P. aeruginosa i probablement amb altres bacteris de metabolisme aeròbic. 8. El meropenem indueix la formació de llargs bacils aberrants capaços de sobreviure en presència de l’antibiòtic. Aquests bacils acumulen menys antibiòtic que els bacteris normals, i poden revertir a la forma normal quan s’elimina la pressió selectiva. 9. La colistina, la última alternativa terapèutica per lluitar contra P. aeruginosa en pacients amb fibrosis quística, és normalment efectiva, encara que recentment han sorgit casos de resistència a aquest agent antimicrobià. 10. La resistència a colistina sembla estar causada per propietats singulars del lipopolisacàrid. 11. La colistina produeix danys en les membranes lipídiques que poden ser estudiats mitjançant tècniques de Black lipid bilayer. Estudis preliminars van mostrar la capacitat de la colistina para induir canals transitoris en les bicapes lipídiques, amb certa dependència de voltatge. 12. La recuperació de la susceptibilitat a l’imipenem en P. aeruginosa és més lenta que l’adquisició de resistència, donat que l’avantatge selectiva conferida per la resistència a l’imipenem en presència de l’agent antimicrobià és forta, mentre que l’expressió d’ OprD és probablement avantatjosa solament sota certes i desconegudes condicions ambientals.<br>Se exploraron los mecanismos no convencionales de resistencia a agentes antimicrobianos que contribuyen a la aparición de fenotipos multiresistentes en las bacterias Gram-negativas Serratia marcescens y Pseudomonas aeruginosa. Se examinaron también los cambios en la susceptibilidad a los agentes antimicrobianos en S. marcescens en los últimos 50 años, comparando cepas aisladas entre los años 1945-1950 y cepas actuales. ¬Las principales conclusiones obtenidas de este estudio son las siguientes: 1. El resistoma de Serratia marcescens no ha cambiado significativamente desde la era pre-antibiótica hasta la actualidad. 2. La retirada de los antibióticos tiende a recuperar los fenotipos de susceptibilidad originales, independientemente del mecanismo molecular implicado en la resistencia. 3. Ninguna de las cepas de Serratia estudiadas presentó integrones ni tampoco ß-lactamasas de espectro extendido. 4. La determinación fenotípica de las susceptibilidades de las cepas “antiguas” de Serratia inactivas durante 60 años ha confirmado los resultados obtenidos mediante metagenómica, es decir, los genes de resistencia a los antibióticos ya existían con anterioridad al descubrimiento y uso de los antibióticos. 5. En las cepas clínicas de P. aeruginosa multiresistentes se detectó un integrón de clase 1 que contenía el cassette génico aadB, que codifica la enzima aminoglicósido 2’-O- adeniltransferasa, que confiere resistencia a gentamicina, tobramicina y kanamicina. 6. Las cepas multiresistentes de P. aeruginosa sobreexpresaron los sistemas de reflujo MexAB-OprM y MexXY-OprM. 7. El uso del inhibidor de bombas de reflujo CCCP se debería evitar en los experimentos realizados con P. aeruginosa y probablemente con otras bacterias de metabolismo aeróbico. 8. El meropenem induce la formación de largos bacilos aberrantes capaces de sobrevivir en presencia del antibiótico. Estos bacilos acumulan menos antibiótico que las bacterias normales, y pueden revertir a la forma normal cuando se elimina la presión selectiva. 9. La colistina, la última alternativa terapéutica para luchar contra P. aeruginosa en pacientes con fibrosis quística, es normalmente efectiva, aunque recientemente han surgido casos de resistencia a este agente antimicrobiano. 10. La resistencia a colistina parece estar mediada por propiedades singulares del lipopolisacárido. 11. La colistina produce daños en las membranas lipídicas que pueden ser estudiados mediante técnicas de Black lipid bilayer. Estudios preliminares mostraron la capacidad de la colistina para inducir canales transitorios en las bicapas lipídicas, con cierta dependencia de voltaje. 12. La recuperación de la susceptibilidad al imipenem en P. aeruginosa es más lenta que la adquisición de resistencia, dado que la ventaja selectiva conferida por la resistencia al imipenem en presencia del agente antimicrobiano es fuerte, mientras que la expresión de OprD es probablemente ventajosa sólo bajo ciertas y desconocidas condiciones ambientales.

Campylobacter jejuni and Escherichia coli strain F11 are two Gram-negative pathogens with a versatile armament of iron uptake systems to cope with the fluctuating host nutrient environment. Our current understanding of Gram-negative iron uptake systems focuses heavily on a prototypical scheme involving a TonB-dependent outer membrane receptor and an ABC transporter, with little knowledge on systems that do not fall neatly into this paradigm. The primary focus of this thesis is the characterization of three such atypical iron uptake proteins from C. jejuni (ChaN and P19) and pathogenic E. coli (FetP). C. jejuni ChaN is a 30 kDa, iron-regulated lipoprotein hypothesized to be involved in iron uptake. The crystal structure of ChaN reveals that it can bind two cofacial heme groups in a pocket formed by a ChaN dimer. Each heme iron is coordinated by a single tyrosine from one monomer and the propionate groups are hydrogen bonded by a histidine and a lysine from the other monomer. Analytical ultracentrifugation studies demonstrate heme-dependent dimerization in solution. Cell fractionation of C. jejuni shows that ChaN is localized to the outer membrane. Based on these findings, the predicted in vivo role of ChaN in iron uptake is discussed. C. jejuni cFtr1-P19 and E. coli FetMP are homologous iron-regulated systems also proposed to be iron transporters. Through growth studies in both organisms, we show that P19 and FetMP are required for optimal growth under iron-limited conditions. Furthermore, metal binding analysis demonstrates that recombinant P19 and FetP bind both copper and iron. Dimerization of P19 is shown to be metal dependent in vitro and is detected in vivo by cross-linking. Through x-ray crystallography, we have determined the structures of P19 and FetP with various metals bound, thus revealing the locations of the highly conserved copper and iron binding sites. Additionally, the crystal structure of FetP reveals two copper positions in each binding site that is likely functionally important. Through mutagenesis, residues contributing to the alternative copper positions were identified. Together, these studies provide insight into the mechanism of iron transport by the two systems and allow for the development of functional models.

Hexavalent chromium (Cr (VI)), the most toxic form of chromium, is widely used in industrial processes. As a result substantial amounts of Cr (VI) contaminated wastes are produced. The use of microbial cells as bioremediation of heavy metals is a potential alternative to conventional chemical methods. In this work, laboratory- scale experiments were performed to investigate Cr (VI) removal using five environmental Gram-negative bacterial strains, three of which were nosocomial strains. The potential of live and autoclaved bacterial strains was investigated to mitigate Cr (VI) from its initial concentration of 2.54 mg/1. The autoclaved bacteria were used to determine whether Cr (VI) removal was dependent upon metabolism of the cells or a simple chemical reaction. The results showed notable reduction in Cr (VI) concentration (up to 87% and 23% using live and autoclaved bacteria, respectively). Proteus mirabilis and Methylococcus capsulatus (Bath) bacterial strains were selected for further detailed analyses to investigate the enzyme system that is responsible for Cr (VI) reduction. To locate the cell compartment in which Cr (VI) removal took place in P. mirabilis, a standard bacterial cell fractionation method was used. The highest Cr (VI) removal activity resided in the cytoplasm, and there was also some activity in the cell membrane. No chromium VI removal was observed in the cell wall fraction. The removal by M. capsulatus of Cr (VI) in high copper sulfate media was more rapid than in low copper sulfate media. Phenylacetylene, an inhibitor of soluble methane monooxygenase, completely inhibited Cr (VI) removal. The results reveals that pMMO, sMMO or other enzymes that induced by copper were involved in reducing or otherwise removing Cr (VI). The di-heme cytochrome c peroxidase is also a possible candidate enzyme of reducing chromium (VI), since it is known to be present in the periplasm and to play a role in reducing peroxides generated by oxidative metabolism. Inductively coupled plasma mass spectrometry coupled with ion chromatography, for the determination of chromium species in P. mirabilis and M. capsulatus, showed that Cr (VI) was reduced and detoxified to less toxic and less soluble Cr (III). Furthermore, prominent changes in the polysaccharide, fatty acids, phosphate and proteins wereobserved in FTIR spectra of P. mirabilis and M. capsulatus (Bath) with potassium dichromate. These changes were consistent with the adsorption of chromium. BLAST searches using known chromate (VI) reducing enzymes from other bacteria showed a presence of four significant potential chromate reductase genes in the genome sequence of P. mirabilis. During the growth of M. capsulatus (Bath), it was noticed that a contaminant bacterium appeared in some cell cultures. The contaminant bacterium was identified as Bacillus licheniformis (100%) using PCR and 16S rRNA sequencing. The mixed culture that contains M. capsulatus (Bath) plus Bacillus licheniformis was also tested for Cr (VI) mitigation. The results of this work are a step forward in understanding the potential of environmental microorganisms for remediation of hexavalent chromium contamination. The future work may reveal more about the mechanism of Cr (VI) removal by the bacteria studied here as well as how they can be exploited for practical bioremediation.

L’epidemiologia dei plasmidi di resistenza è il principale mezzo per descrivere la diffusione delle resistenze agli antimicrobici. L’identificazione di plasmidi correlati associati a specifici geni di resistenza può aiutare a tracciare la disseminazione di plasmidi epidemici, aprendo nuovi scenari epidemiologici sui meccanismi di diffusione delle resistenze agli antimicrobici. Questo aspetto è particolarmente evidente quando si considerano plasmidi che sono associati alla diffusione delle Beta Lattamasi a Spettro Esteso (ESBL) come gli enzimi CTX-M, SHV, TEM. Lo scopo di questa tesi è stato quello di caratterizzare plasmidi che conferiscono resistenza a farmaci rilevanti per la terapia umana in differenti collezioni di Enterobacteriaceae di origine umana e animale. Verranno discussi diversi esempi di plasmidi epidemici quali: i plasmidi IncHI2 che trasportano i geni blaCTX-M-9 o blaCTX-M-2 provenienti da isolati di origine umana e animale di Escherichia coli e Salmonella dalla Spagna, Belgio e Inghilterra; i plasmidi IncI1 caratterizzati da specifici fattori di virulenza, che trasportano i geni blaTEM-52 e blaCTX-M-1 derivanti da isolati di Salmonella e E. coli di origine umana e animale; i plasmidi IncN che trasportano i geni codificanti la metallo-β-lattamasi VIM-1 da isolati umani di Klebsiella pneumoniae and E. coli dalla Grecia; plasmidi IncA/C2 associati a specifici geni di resistenza come blaCMY-2, blaCMY-4, blaVIM-4 e blaVEB-1 provenienti da diverse Enterobacteriaceae isolate in differenti parti del mondo, ed infine, i plasmidi associati all’enzima SHV-12 isolati in diverse Enterobacteriaceae di origine umana e animale. L’analisi mediante comparazione della struttura plasmidica ha permesso di rilevare la diffusione di plasmidi emergenti e consente di tracciare i percorsi evolutivi e di diffusione attraverso l’acquisizione di una vastità di elementi genetici mobili associati ai geni di resistenza.<br>The epidemiology of resistance plasmids is a major issue for the description of antimicrobial resistance diffusion. The identification of related plasmids associated to specific resistance genes may help to follow the spread of epidemic plasmids, opening new epidemiological scenarios on the mechanism of diffusion of antimicrobial resistance. This aspect is particularly interesting when applied to collections of plasmids that are playing a major role in the diffusions of Extended Spectrum Beta Lactamases (ESBLs) such as CTX-M, SHV, TEM. The aim of this thesis is the molecular characterization of plasmids conferring drug resistances in different collections of Enterobacteriaceae of human and animal origin. Several example of epidemic plasmids will be discussed: the IncHI2 plasmids carrying blaCTX-M-9 or blaCTX-M-2 genes identified from human and animal isolates of Escherichia coli and Salmonella from Spain, Belgium and UK; the IncI1 family of plasmids characterized by specific virulence factors, carrying the blaCMY-2, blaTEM-52 and blaCTX-M-1 genes from Salmonella and E. coli of human and animal origin; the IncN plasmids carrying the gene codifying the metallo-β-lacatamase VIM-1 from human isolates of Klebsiella pneumoniae and E. coli in Greece; the IncA/C2 plasmids carrying specific resistance genes such as blaCMY-2, blaCMY-4, blaVIM-4 and blaVEB-1 from different Enterobacteriaceae isolated worldwide; different plasmid replicons (IncFII, IncA/C1, IncI1) carrying the ESBL SHV-12 from human and animal origin. The comparative analysis of plasmid backbones allowed to ascertain the diffusion of common, emerging plasmids and helped to determine the evolution of these plasmids by acquisition of relevant resistance genes by a panoply of mobile genetic elements and illegitimate recombination events.

Bacteria multi-drug resistance is a challenging problem of contemporary medicine and a new molecular framework for antibiotics is needed. General bacterial porins are recognized as the main pathway for polar antibiotics, but the permeability rules are still under debate. Recent works in literature pointed the electrostatics of the channel to be responsible for its filtering mechanism, and some theoretical investigations are already reported in the literature aimed at characterizing the electrostatics inside water-filled channels. Using Molecular dynamics simulations we revealed the electrostatic filtering mechanism for porins, using water as sensing tool. We further quantify from water polarization density inside the channel the macroscopic internal electric field inside porins. This method allowed us to put forward an ultra-coarse-grained model in which the channel is described by its cross-section area, internal electric field and electrostatic potential along the axis of diffusion. Once these three descriptors are defined, it is possible to estimate the whole free energy along the channel axis of diffusion for a molecule represented by its size, charge and electric dipole moment in a few seconds. This model would allow to virtually screening libraries of molecules searching for hits with enhanced permeability. These results may have important implications for the formulation of a general model for antibiotics translocation, and can be taken into account for rational drug design.<br>Bacteria multi-drug resistance is a challenging problem of contempo- rary medicine and a new molecular framework for antibiotics is needed. General bacterial porins are recognized as the main pathway for po- lar antibiotics, but the permeability rules are still under debate. Recent works in literature pointed the electrostatics of the channel to be respon- sible for its filtering mechanism, and some theoretical investigations are already reported in the literature aimed at characterizing the electrostat- ics inside water-filled channels. Using Molecular dynamics simulations we revealed the electrostatic filtering mechanism for porins, using water as sensing tool. We further quantify from water polarization density inside the channel the macro- scopic internal electric field inside porins. This method allowed us to put forward an ultra-coarse-grained model in which the channel is de- scribed by its cross-section area, internal electric field and electrostatic potential along the axis of diffusion. Once these three descriptors are defined, it is possible to estimate the whole free energy along the chan- nel axis of diffusion for a molecule represented by its size, charge and electric dipole moment in a few seconds. This model would allow to virtually screen libraries of molecules searching for hits with enhanced permeability. These results may have important implications for the formulation of a general model for antibiotics translocation, and can be taken into account for rational drug design.

A novel Gram negative, capsule-forming bacterium was previously isolated in Dr. G. Roland Vela's laboratory. The distribution of this bacterium in soils from various locations was investigated. Soil samples from 188 locations around the world were examined. Isolates of the bacterium were obtained from 50 of these soils, with 48 of the isolates found in soils from the southwestern United States and northern Mexico. This suggests that this region is the natural habitat of the bacterium. The other two isolates were obtained from Madrid, Spain and Taipei, Taiwan. None were found in soils from South America or Australia. A lack of variation in morphology and physiological properties in the isolates suggests that a homogeneous population exists, even from widespread geographical locations.

Investigation of genomic diversity can provide insight into the evolutionary history of bacterial species. However, complete genome sequencing is not yet practical for large strain collections at the beginning of this project. In this project PCR-based methods to investigate the genomic diversity of non-sequenced strains were successfully developed. In \(Escherichia\) \(coli\), the distribution of two Type III secretion system, ETT2 (\(E.\) \(coli\) Type Three Secretion 2) and Flag-2 (\(E.\) \(coli\) Flagellar system 2), were surveyed among a collection of 79 strains. Remnants of both clusters were found to be present in most strains, suggesting that both have a long evolutionary history within \(E.\) \(coli\). The PCR-based methods were also developed for application as part of genome sequencing projects. They were used to explore the co-linear and variable regions between \(Campylobacter\) \(jejuni\) M1 and the genome sequenced strain \(C.\) \(jejuni\) strain 11168. The \(C.\) \(jejuni\) M1 genome was assembled into thirty-four genomic contigs relative to strain 11168, and the size and position of insertions/deletions were characterised. Similar methods were used to facilitate the finishing of the genome of \(Francisella\) \(tularensis\) strain FSC198, using sequence information from strain Schu S4. The completed genome sequence of FSC198 showed it to be almost identical to that of Schu S4. The two genomes differ at only 11 loci, eight SNPs (single nucleotide polymorphisms) and 3 VNTRs (variable number tandem repeats). This surprising finding suggested that the European isolate FSC198 may be derived from the US laboratory strain Schu S4. Two virulence factors, IglA and IglB, from a pathogenicity island of strain FSC198 were further investigated and found to interact at the protein level. These proteins are possibly involved in Type VI secretion, and may represent potential vaccine candidates.

Copper is a trace element utilized by organisms as a cofactor involved in redox chemistry, electron transport, photosynthesis, and oxidation reactions. In excess, copper is toxic; it can generate reactive oxygen species causing cellular damage, or poison other metalloproteins by replacing native metal cofactors. Gram-negative bacteria have developed homeostatic mechanisms to maintain the intracellular copper concentration in the face of changing environmental conditions. For Gram-negative enteric bacteria, like Esherichiacoli and Salmonella enterica serovar typhimurium, copper is encountered in industrial and institutional settings, where the metal is used as a broad-spectrum biocide. For environmental bacteria, such as the marine cyanobacterium Synechococcus sp. WH8102, copper stress occurs because human activity changes the concentration of copper in the ocean. This dissertation contains six chapters, relating four stories of our investigations into the molecular mechanisms of copper homeostasis in Gram-negative bacteria. Chapter I contains literature review and background on the implications of bacterial copper homeostasis. Chapter II reports our work investigating the expression of two E. coli proteins, CusF and CusB, upon copper stress; we show that CusF expresses at a ~10-fold molar excess over CusB. Chapter III describes a collaboration between our lab and Jose Argüello's lab at Worcester Polytechnic Institute, and we show that CusF can acquire Cu(I) from CopA. Our results from Chapters II and III show that CusF functions as a major copper chaperone in the periplasm of E. coli. Chapter IV details our work characterizing a novel protein from marine cyanobacteria, Synw_0921. Although Synw_0921 is believed to be involved in copper homeostasis, we show that it is an iron-sulfur cluster protein. Bioinformatic analysis suggests that Synw_0921 represents a new family of proteins that help marine cyanobacteria adapt to copper changes in their unique environment. Chapter V relates our work on CueR and GolS, two homologous sensor proteins with distinct metal-dependent transcriptional activation; we find that the activity cannot be explained by binding affinity differences. Chapter VI concludes with final thoughts on the intersection of biochemistry and molecular biology in the important process of understanding copper homeostasis.

The most important resistance mechanism to beta-lactam antibiotics is the plasmid-mediated beta-lactamase and the common criterion for the epidemiology of these enzymes is the determination of their biochemical characteristics. Surveys of plasmid-encoded beta-lactamases of Gram-negative bacteria, used to investigate their relative clinical importance, have been poorly performed and rarely conducted outside the developed world. A survey of uropathogenic strains and of salmonellae and shigellae, isolated in south India in 1984, revealed a higher incidence of ampicillin resistance (minimum inhibitory concentration [MIC] > 10mg/1) than had ever been reported before (<i>Enterobacteriacea</i> 82%, salmonellae 90%, shigellae 60%). Only the enterobacterial strains showed any significant resistance to the first generation cephalosporin, cephalosporin, cephaloridine (MIC > 10mg/1). However, 66% of the salmonellae strains were cefuroxime resistant. A small proportion of all species conferred resistance to third generation cephalosporins. In the individual species, there was a very high incidence of ampicillin resistance (<i>E. coli</i> 76% <i>Klebsiella spp</i> 96%) and cephaloridine resistance (<i>E.coli</i> 57% <i>Klebsiella spp</i> 69%). Many of the ampicillin resistant strains harboured either auto-transferable or mobilisable plasmids (40%). Characterisation of the plasmid DNA from the <i>E.coli</i> transconjugants revealed the existence of 37 different plasmids types. The transconjugants from klebsiella, salmonella and shigella possessed fewer plasmids types than those from <i>E. coli</i>. Most plasmids possessed resistance genes to aminoglycosides and to six or more drugs. Beta-lactamase studies revealed that TEM-1 was the most predominant enzyme in all transconjugant strains followed by OXA-1, SHV-1, TEM-2, OXA-2 and the novel enzyme SAR-2. The SAR-2 enzyme was fully characterised and had a higher pI (8.3) than any previously characterised plasmid-mediated beta-lactamase. It had a broad-spectrum activity with the molecular weight of 36000. In addition the unusual observations of <i>E.coli</i> strains producing both the PSE-1 and PSE-2 beta-lactamases and strains hyperproducing the TEM-1 were made and these strains were studied further. The development and mechanisms of resistance to beta-lactam/beta-lactamase inhibitor combinations (ampicillin and clavulanic acid) have been performed with laboratory strains possessing the ampicillin resistance plasmid R1. The results show that challenge with clavulanic acid alone did not affect the expression or integrity of the beta-lactamase whereas challenge with the combination of ampicillin and clavulanic acid caused radical changes with the expression of the beta-lactamase. In some cases there were multiple copies of genes which resulted in hyperproduction of TEM-1 enzyme and this was sufficient to resist the combinations. Unfortunately, these variants also conferred resistance to second and third generation cephalosporins. Evidence of this type of resistance to clavulanic acid is now emerging in clinical practice.

Antibiotic resistance is increasing worldwide, which is threatening the effectiveness of even the most potent and recent antibiotics. The successful treatment of disease is hampered due to the multidrug resistant (MDR) phenotype exhibited by the bacterial pathogens. Therefore, the aims of this thesis were to investigate MDR through several different approaches. Integrons are important contributors to the MDR profile of nosocomial isolates within Australia, therefore the incidence of integrons was assessed in a collection of 72 conjugative clinical plasmids isolated from E. coli, a cohort of 30 urinary tract infection (UTI) isolates and a cohort of four bacteria producing metallo-beta-lactamases (MBLs). Integrons were found in 63% (45/72) of the conjugative plasmids by polymerase chain reaction (PCR). Sequencing of gene cassette arrays revealed that cassettes of the dfr and aadA families were most common. Within the cohort of UTI bacteria, 37% (11/30) were positive for class 1 integrons, and the dfrA17-aadA5 gene cassette array was most common. The four MBL-producers contained the gene cassette blaIMP-4 found within a class 1 integron which was responsible for the MBL phenotype. An assay based on real-time PCR was also developed to measure the recombination activity of the integron integrase (IntI) enzymes. The existing method of IntI measurement, the in vivo conduction assay, was used as a basis for the development of the real-time PCR assay. Five 59-be from the gene cassettes aadB, orfA, sat2, dfrA1 and aacA4 were cloned as recognition sites used in the real-time PCR assay. IntI1 was the most active integrase and showed an activity of 2.31 ?? 10??-1 when recombining the aadB and orfA 59-be. The highest level of class 2 integrase activity was 2.00 ?? 10-1?? during recombination of the sat2 and dfrA1 59-be, while IntI3 showed its highest recombination frequency of 2.29 ?? 10-1?? when the aadB and orfA 59-be were used. Additionally, the real-time PCR assay was used assess the levels of IntI activity over time. Using this method, the level of recombination as time progressed remained stable at a level of 4.10 ?? 10-2????. MDR was also analysed in 37 Acinetobacter baumannii isolates which were collected from four hospitals in Sydney. Minimum inhibitory concentration (MIC) analysis to 25 antibiotics revealed that all isolates showed a reduced susceptibility to between five and 24 antibiotics. PCR was performed to detect the presence of resistance determinants. Class 1 integrons encoding resistance to aminoglycosides, antiseptics and disinfectants were found in 35 % (13/37) of the isolates. Aminoglycoside resistance genes including aphA1 (12/37), strA (1/37) and strB (22/39) were also found. Resistance to beta-lactams was also observed in all isolates, which correlated with the presence of the ampC and blaOXA-51-like genes. The insertion sequence ISAba1 which provides an alternative promoter leading to increased gene expression was found upstream of the ampC gene in 29 isolates; the same isolates also contained the identical insertion sequence upstream of the carbapenemase resistance gene blaOXA-23. These 29 isolates also possessed the tetracycline resistance gene tetB. All but one of these 29 isolates also contained the gene blaTEM-1. Resistance to quinolones and fluoroquinolones was attributed to the presence of a Ser83-Leu83 gyrA mutation present in 36 resistant isolates. Furthermore, a putative dihydrofolate resistance gene, folA, was found in all isolates. Repetitive extragenic palindromic PCR revealed the presence of seven clonal groups. Overall, this study demonstrated the widespread impact and dissemination of MDR within nosocomial settings in Australia. The use of new assays, such as the real-time PCR assay developed in this thesis, is essential to the understanding of dissemination of antibiotic resistance.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2014.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>Chapter 1. Introduction to Iron Homeostasis and Siderophores Iron is an essential nutrient for almost all living organisms. This Chapter presents an overview of iron homeostasis in human and bacteria, as well as the biology and chemistry of siderophores and siderophore conjugates. Siderophores are small-molecule iron chelators synthesized by bacteria for scavenging iron from the environment. Their iron-binding properties, structures, transport machineries and biosynthesis are discussed with a focus on enterobactin and salmochelins, which are the two siderophores studied in this thesis. The notion of conjugating functional molecules to siderophores has been actively studied for decades. The syntheses and applications of siderophore conjugates are summarized, with an emphasis on siderophore-antibiotic conjugates. New antimicrobial strategies targeting bacterial iron acquisition are also described. A summary of project goals and thesis organization is presented at the end of this Chapter. Chapter 2. Siderophore-mediated Cargo Delivery to the Cytoplasm of Escherichia coli and Pseudomonas aeruginosa: Syntheses of Monofunctionalized Enterobactin Scaffolds and Evaluation of Enterobactin-cargo Conjugate Uptake This Chapter describes the design and syntheses of monofunctionalized enterobactin (Ent, L- and Disomers) scaffolds. A family of ten Ent-cargo conjugates is synthesized to probe the extent to which the Gram-negative ferric Ent uptake and processing machinery recognizes, transports, and utilizes derivatized Ent scaffolds. The delivery of Ent-cargo conjugates is evaluated by a series of growth recovery assays. The results demonstrate that the Escherichia coli K12 and Pseudomonas aeruginosa PAO 1 Ent transport machinery identifies and delivers select Ent-cargo conjugates to the cytoplasm, and P. aeruginosa PAO exhibits greater promiscuity than E. coli K12 for the uptake and utilization of the conjugates. This work affords a new native siderophore platform amendable for synthetic modification and cargo attachment. It also indicates that cargo size affects the delivery efficiency, which influences the conjugate design for Ent-mediated delivery strategies described in the following Chapters. Chapter 3. Enterobactin-Mediated P-Lactam Delivery into Gram-negative Bacteria The design, synthesis, and characterization of two enterobactin-antibiotic conjugates, where the [beta]-lactam antibiotics ampicillin (Amp) and amoxicillin (Amx) are linked to a monofunctionalized enterobactin scaffold via a stable polyethyleneglycol linker are reported. Under conditions of iron limitation, these siderophore-modified antibiotics provide greatly enhanced antibacterial activity compared to the parent [beta]-lactams against Escherichia coli strains that include several pathogens. The mechanism of the antimicrobial activity is probed and the results reveal that the improvement is due to Ent-mediated delivery. Strain-specific killing and faster time-kill kinetics are also observed for the conjugates. These studies demonstrate that the native enterobactin platform provides a means to effectively deliver antibacterial cargo across the outer membrane permeability barrier of Gram-negative pathogens that utilize enterobactin for iron acquisition. Chapter 4. Stability Evaluation of Acyloxymethyl/Acyloxyethyl Ester Linker for the Design of Enterobactin-fluoroquinolone Conjugates Fluoroquinolones are widely used antibiotics that target DNA gyrase. Prior studies with siderophore-fluoroquinolone conjugates suggest that a release step is required following cytosolic entry for the conjugates to exhibit antimicrobial activity. We design and synthesize enterobactin-fluoroquinolone conjugates harboring several acyloxymethyl/acyloxyethyl ester-based labile linkers and the stability of these likers and the conjugates are studied. These linkers exhibit relatively short half-lives and afford premature release of the antibiotics under our experimental conditions, which renders them not suitable for siderophore-based antibiotic delivery. This conclusion is also supported by the Ent-independent antimicrobial activity observed for the enterobactin-fluoroquinolone conjugates harboring the labile linkers. Appendix 1. Chemoenzymatic Syntheses of Enterobactin-antibiotic Conjugates and Studies of Antimicrobial Activity In this Appendix, cargo attachment to enterobactin by using chemoenzymatic reactions is described. This approach affords conjugates containing a 10-mer peptide as the linker between enterobactin and the cargo. A series of enterobactin conjugates harboring antimicrobial peptides and fluoroquinolone antibiotics are reported, and their antimicrobial activity against K coli is evaluated. Unfortunately, none of these conjugates afford enhanced activity compared to the unmodified antimicrobial agents, which may result from impropriate cargo selection or linker design. Appendix 2. NMR, HPLC and UV-Vis Characterizations of the Reported Compounds.<br>by Tengfei Zheng.<br>Ph. D.

Proteins fulfil a wide variety of essential functions in the cell. Recombinant protein production in the Gram-negative bacterium Escherichia coli (E. coli) facilitates structural and functional studies of proteins and it has been instrumental in biotechnology for the manufacturing of e.g. many protein-based drugs. However, to obtain sufficient amounts of active recombinant protein is not always a trivial task. The production of proteins that reside in membranes is limited by their complex biogenesis and their hydrophobic nature. Consequently, despite the importance of membrane proteins in health and disease (approx. 70% of today’s drugs act on membrane proteins), structures of only a very small fraction of the existing membrane proteins have yet been solved. Many soluble proteins are also difficult to produce, e.g. those containing disulfide bonds (e.g. antibody fragments and most hormones). Disulfide bond-containing proteins have to be produced in the periplasm of E. coli to be able to fold properly. To reach the periplasm, these proteins have to be ‘secreted’ across the inner membrane, which makes that also their biogenesis is complex. The aim of my PhD studies has been to improve E. coli-based production of recombinant membrane and secretory proteins. I have found that (i) the previously developed Lemo21(DE3) protein production strain can be used to set the expression intensity of a gene encoding a membrane protein such that the protein is optimally produced in the cytoplasmic membrane without causing any notable stress. Also, (ii) membrane protein production using the Lemo21(DE3) strain can be improved and simplified using carefully optimized culturing and induction conditions, demonstrated by the development of the ‘MemStar recipe’. Furthermore, I found that (iii) when using the standard BL21(DE3)/pT7 expression system for the production of membrane and secretory proteins, omitting the inducer IPTG leads to drastically improved yields as compared to when IPTG is added, owing to a lower initial target protein production rate. In the fourth study, I found that (iv) when using the PrhaBAD promoter for expression of the target gene, protein accumulation rates appear to be mostly unaffected by the inducer concentration. Using a strain-engineering approach, PrhaBAD-based protein production rates could be made constant and rhamnose concentration dependent. This dramatically improved production yields of both membrane and secretory proteins, using only very low amounts of inducer. Taken together, in accordance with previous studies, lowering production rates is an efficient strategy to increase production yields of both membrane- and secretory proteins. This is mostly due to alleviating saturation of the machinery involved in the biogenesis of these proteins. Finally, I also conducted a study (v) where, in both E. coli and Salmonella, I orchestrated the production of two membrane proteins (one that mediates the production of antigens on the surface of Gram-negative bacteria and another that makes defined pore-structures in the Gram-negative bacterial cell envelope) for the development of a safe (non-living) vaccine platform.<br><p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>

Urinary catheters are indispensable in healthcare and, with an ageing population, their use will continue to increase. However, they are commonly associated with colonisation and urinary tract infections (UTIs) caused by the attachment of bacteria to the catheter surface. Application of a novel cationic compound as a catheter coating may have a significant impact on the costs associated with treatment of UTIs and reduce the need for catheter replacement, as well as decreasing the number of UTI associated morbidity and mortality. Cationic compounds in particular are known to interact with the negatively charged outer membrane of bacteria, therefore have a broad spectrum of activity. The purpose of this study was to source and evaluate a novel cationic antimicrobial for use as a potential coating to impede biofilm formation on urinary catheters, and to investigate the cellular response to the selected lead compound. This research has demonstrated that the antimicrobial activity of commercially available Byotrol™ was superior to that of polyamines and quaternary ammonium compounds that were screened. Using high-throughput antimicrobial assays, such as the minimum inhibitory concentration and microtitre plate biofilm forming assays, the inhibitory concentrations of Byotrol™ were found to range from 3 µg/mL to 15 µg/mL for planktonic cultures, and 3 µg/mL to 20 µg/mL for the biofilm growth of uropathogenic bacteria. Furthermore, the minimum biofilm eradication concentration assay demonstrated that 200-1000 µg/mL Byotrol™ was able to eradicate an established biofilm. Byotrol™ may also have significant potential as a device coating, as pre-coating data on glass slides and microtitre plates with the compound inhibited bacterial growth on the surface at concentrations of 400 µg/mL for E. coli, and 1000 µg/mL K. pneumoniae. Atomic force microscopy validated the expectation that higher concentrations of Byotrol™ coated a surface more evenly than lower concentrations. Using two-dimensional gel electrophoresis, the metabolic protein tryptophanase was seen to be significantly over-expressed when E. coli K12 was treated with sub-inhibitory concentrations of Byotrol™. A transcriptomic approach using RNA-Seq demonstrated that a majority of the differentially expressed genes were identified in cells that were challenged with 4 times the minimum inhibitory concentration of Byotrol™. Genes associated with protein synthesis and stress response were significantly up-regulated. Interestingly, the global gene regulators AI-2 and indole were significantly up-regulated, which may have an influence on the expression of genes related to motility, biofilm formation and acid-resistance. Genes associated with chemotaxis and motility, acid-resistance and iron transport were significantly down-regulated, particularly in cells challenged with Byotrol™.Byotrol™ displayed antimicrobial activity both in suspension and as a coating. Identification of differentially expressed genes and proteins, when the bacteria were treated and challenged with Byotrol™, has, for the first time, revealed the bacterial cell’s response to this biocide. The findings may enable the development of strategies to prevent or better manage catheter associated urinary tract infection (CAUTI).

Antibiotic resistance, particularly amongst Gram-negative bacteria, has emerged as a major global concern. Working synergistically with the permeability barrier created by the cell envelope, multidrug efflux pumps contribute significantly to the intrinsic resistance of Gramnegative bacteria. Site-directed mutagenesis, informed by computational analyses, has been used on the prototypical tripartite efflux system, AcrAB-ToiC from Escherichia coli. Likely AcrA-ToiC interactions and functional roles of their sub-domains have been assessed using mutants in a combination of in vivo and in vitro approaches. Results indicate that both the tip region and the equatorial domain, halfway up the ToiC channel, are involved in determining the compatibility of AcrA with ToiC. On the other side of the interaction, both the tip region and the helices of the AcrA hairpin are essential for normal function. The hairpin tip is required to maintain the permeability barrier, while the helices are necessary for a stable AcrA-ToiC interaction. Combining these results with available literature, the first dynamic model of tripartite complex assembly is presented here. This model postulates initial bundling of AcrA and ToiC coiled-coil domains to open the ToiC channel followed by transition to a tip-to-tip interaction to drive channel closing and complex disassembly in an energy-dependent manner.

Postantibiotic effect (PAE) describes the suppression of microbial growth occurring after a short exposure to an antimicrobial agent. PAE appears to be a property of the majority of antimicrobial agents and is demonstrated by a wide variety of microorganisms. At present, carbapenems and penems are the only members of the -lactam group of antimicrobial agents that exhibit a significant PAE on Gram-negative bacilli. A standardised method was developed to evaluate the in vitro PAE of three carbapenems; imipenem, meropenem and biapenem on Gram-negative bacteria under reproducible laboratory conditions that partially mimicked those occurring in vivo. The effects on carbapenem PAE of the method of antimicrobial removal, concentration, exposure duration, inoculum size, inoculum growth phase, multiple exposures and pooled human serum were determined. Additionally, the reproducibility, susceptibility prior to and after PAE determination and inter-strain variation of carbapenem PAE were evaluated. The method developed determined PAE by utilising viable counts and demonstrated carbapenem PAE to be reproducible, constant over successive exposures, dependent on genera, concentration, duration of exposure, inoculum size and growth phase. In addition, carbapenem PAE was not significantly effected either by agitation, the antimicrobial removal method or the viable count diluent. At present, the mechanism underlying PAE is undetermined. It is thought to be due to either the prolonged persistence of the antimicrobial at the cellular site of action or the true recovery period from non-lethal damage. Increasing the L-lysine concentration and salinity at recovery decreased and increased the carbapenem and imipenem PAE of Pseudomonas aeruginosa, respectively. In addition, no apparent change was observed in the production of virulence factors by P.aeruginosa in PAE phase. However, alterations in cell morphology were observed throughout PAE phase, and the reappearance of normal cell morphology corresponded to the duration of PAE determined by viable count. Thus, the recovery of the penicillin binding protein target enzymes appears to be the mechanism behind carbapenem PAE in P. aeruginosa.

Parmi les plus grandes menaces de la santé publique dans le monde entier, la résistance aux antibiotiques est à la pointe. Ceci en partie est dû à l'augmentation des infections causées par des bactéries pathogènes résistantes aux antibiotiques ainsi que la diminution du nombre actuel de nouveaux antibiotiques. Dans le souci de remédier à cette situation malheureuse, il y a eu récemment la ré-surfaçage des antibiotiques anciens et abandonnés comme les polymyxines. Colistine, un membre des antibiotiques de polymyxine, est maintenant considéré comme un antibiotique de «dernier recours» pour le traitement des infections bactériennes à Gram-négatives graves en raison de son action puissante contre ces agents pathogènes. Cependant, la résistance à la colistine parmi ces agents pathogènes a émergé dans plusieurs pays et est actuellement en augmentation. En raison de la nouvelle réintroduction relative de cet antibiotique, il ya un manque d'information complètes sur ses propriétés pharmacologiques ainsi que des mécanismes par lequel les bactéries développent une résistance contre celle-ci.Afin de combler ce manque d'information en ce qui concerne le mécanisme de résistance, nous avons donc entrepris ce projet. Tout d'abord, pour procéder à une surveillance épidémiologique des bactéries résistantes à la colistine chez les humains et les animaux domestiques et d'autre part, de décrypter les mécanismes moléculaires de résistance à la colistine parmi les bactéries résistantes isolées<br>Among one of the greatest threats facing public health worldwide, antibiotic resistance is at the forefront. This is partly due to increase in infections caused by antibiotic-resistant pathogenic bacterial as well as the current dwindling number of new antibiotics. In a way to address this unfortunate situation, there have been recent resuscitation of old and abandoned antibiotics such as polymyxins. Colistin, a member of polymyxin antibiotics, is now regarded as a 'last-resort' antibiotic for the treatment of severe Gram-negative bacterial infections owing to its potent action against these pathogens. However, resistance to colistin among these pathogens has emerged in several countries and is currently on increase. Due to the relatively new reintroduction of this antibiotic, there is a lack of comprehensive information on its pharmacological properties as well as mechanisms by which bacteria develop resistance against it.In order to bridge this information gap in relation to the mechanism of resistance, we therefore undertook this project. First, to carry out an epidemiological surveillance of colistin-resistant bacteria in humans and domesticated animals and secondly, to decipher the molecular mechanisms mediating colistin resistance among the isolated resistant bacteria

In this thesis sixty-one clinical <I>Pseudomonas</I> <I>aeruginosa</I> isolates were acquired from hospitals within Japan and fifty-one of these strains were resistant to imipenem and/or meropenem (MIC >4mg/l). Neither IMP-1 nor a novel carbapenemase could be detected in any of these strains; instead synergism between a cephalosporinase and lowered outer membrane permeability was found to be the most prevalent mechanism of imipenem resistance. The carbapenem-hydrolysing metallo-β-lactamase produced by members of the genus <I>Aeromonas</I> have in the past few years demanded attention from a clinical and enzymological point of view. Two imipenem-resistant <I>Aeromonas veronii</I> biovar <I>sobria</I> strains 13 and 99 were isolated from a water source in South India. An imipenem-based detection method applied after isoelectric focusing revealed that a β-lactamase with a pI of 5.84 was responsible for carbapenem hydrolysis in strains 13 and 99 and unlike previously reported <I>Aeromonas </I>metallo-β-lactamases this enzyme could be detected with nitrocephin. Purification of this novel enzyme, nominated AVS-1, further demonstrated the unusual properties of this carbapenemase, most notably its insensitivity to EDTA. A metallo-β-lactamase gene was amplified from <I>A. veronii</I> bv. <I>sobria</I> strains 13 and 99 by PCR. Sequencing of the PCR product revealed that these two strains possess a metallo-β-lactamase gene that is closely related to the metallo-β-lactamase gene <I>imiS</I> previously identified in an isolate of <I>A. veronii</I> bv. <I>sobria</I>. Therefore, minor amino acid substitutions may account for the extended substrate specificity and unusual inhibitor profile of AVS-1. Two non-carbapenem-hydrolysing β-lactamases were also cloned from <I>A. veronii </I>bv. <I>sobria</I> strain 13. One of these β-lactamases a clavulanic acid sensitive β-lactamase was found to be an <I>ampS</I>-like penicillinase. The other cloned β-lactamase could unfortunately not be sequenced.

Das Typ III Sekretionssystem (T3SS) ist ein Proteinkomplex den Gramnegative Bakterien nutzen um in einem Schritt Effektorproteine (Effektoren) aus dem Zytosol über die Doppelmembran zu sekretieren. Für viele Bakterien ist das T3SS ein essenzieller Virulenzfaktor, der es ihnen erlaubt mit ihrem Wirt zu interagieren und diesen zu manipulieren. Charakteristisch für das T3SS ist die strukturelle Komponente, der Nadelkomplex. Dieser ähnelt strukturell einer Spritze, deren Basalkörper die bakteriellen Membranen und das Periplasma durchspannt und einer Nadel, die vom Basalkörper aus dem Bakterium ragt. Basierend auf dem Modell einer Spritze wird angenommen, dass Effektoren entfaltet und anschließend durch Basalkörper und Nadelkanal sekretiert werden. Trotz der kontinuierlichen Forschung an T3SS entbehrt dieses Modell einer experimentellen Grundlage und der Mechanismus ist nicht vollständig erklärt. Ziel der Arbeit war es, eine experimentelle Basis für den Sekretionsmechanismus des T3SS zu schaffen. Um zu verstehen, wie das T3SS Effektoren sekretiert, wurden zunächst Fusionsproteine konstruiert, welche aus einem Effektor und einem stabil gefalteten Knotenprotein bestehen. Aufgrund des Knotens in der Fusion ist davon auszugehen, dass dieser während der Sekretion nicht entfalten kann. Die Effektordomäne wird sekretiert während der Knoten im Kanal verbleibt und diesen verstopft. Nach unseremWissen ist diese Arbeit die erste Visualisierung von Effektorfusionen an isolierten Nadelkomplexen. Die Effektorfusion wird N-terminal voran durch den Kanal sekretiert, wobei der Kanal das Substrat umschließt und gegen Proteasen und chemische Modifikationen abschirmt. Die Ergebnisse dieser Arbeit untermauern eine Grundidee der Funktionsweise des T3SS und liefern eine vielversprechende Strategie für in situ-Strukturanalysen. Dieser Ansatz lässt sich auch auf andere Proteinsekretionssysteme übertragen, bei welchen Substrate vor dem Transport entfaltet werden müssen.<br>The Type III Secretion System (T3SS) is a complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. This work aimed to provide an experimental basis for the model of the T3SS mechanism. In order to elucidate details of the effector secretion mechanism, fusion proteins consisting of an effector and a bulky protein containing a knotted motif were generated. It is assumed that the knot cannot be unfolded during secretion of the chimera. Consequently, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. This is, to our best knowledge, the first time effector fusions have been visualized together with isolated NCs and it demonstrates that effector proteins are secreted directly through the channel with their N-terminus first. The channel encloses the substrate and shields it from a protease and chemical modifications. These results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion.

The Gram-negative, motile bacterium Campylobacter jejuni is a causative agent of food-borne gastroenteritis. Cytolethal distending toxin (CDT) is one of the important virulence factors for C. jejuni pathogenesis. It was not previously known how CDT is released from C. jejuni into the surrounding environment. In our study, CDT proteins were observed in the periplasmic fraction and all CDT subunits from C. jejuni were released from the bacterial cells in association with OMVs. The OMV-associated toxin caused cytolethal distending effects on tissue culture cells. Our results strongly suggest that the release of OMV-associated CDT is a route by which C. jejuni delivers all CDT toxin subunits (CdtA, CdtB, and CdtC) to the surrounding environment, including infected host tissue.The Gram-negative, motile bacterium Vibrio cholerae is primarily known as the causal organism of the severe dehydrating diarrheal disease cholera. OMVs released from non-O1 non-O139 V. cholerae (NOVC) strain V:5/04 induced an inflammatory response in human host cells. The inflammatory potential is mediated by the nucleotide-binding domain, leucine-rich repeat containing family members NOD1 and NOD2. Physiochemical analysis in conjunction with NOD1/2 reporter assays in HEK293T cells confirmed the presence of the NOD1/2 active peptidoglycan (PGN) in OMVs. Deletion of the quorum sensing master regulator HapR specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. These findings suggest that OMVs from a NOVC strain delivered PGN to the host cells, where they elicited an immune response mediated by NOD1 and NOD2.The Gram-negative, non-motile coccobacillus Aggregatibacter actinomycetemcomitans is a natural inhabitant of the oral cavity, but the bacterium can translocate from the oral cavity into the bloodstream and thereby be transported to other regions of the body. A. actinomycetemcomitans is implicated in aggressive forms of periodontitis. The mechanism behind this aggressive periodontitis was not fully known. In addition to several virulence factors, this organism also produces CDT. We have demonstrated that OMVs released by A. actinomycetemcomitans contain several virulence factors, including CDT. We showed that OMVs delivered CDT to the host cells and that CDT was localized inside the nucleus, which led to a cytolethal distending effect on two different cell lines tested: HeLa cells and human gingival fibroblasts (HGF). These results suggest that A. actinomycetemcomitans OMVs could deliver biologically active CDT toxin into the periodontal tissue and may contribute to periodontitis.In our earlier studies, we discovered that an M6 family metalloprotease PrtV was an essential factor for V. cholerae survival from predator grazing. Pure PrtV protein effectively degraded human blood plasma components. In addition, it also showed a dose-dependent cytotoxic effect in the human intestinal HCT8 cell line. V. cholerae produces a large amount of outer membrane vesicles (OMVs) during the normal course of cell growth. OMVs are composed of periplasmic proteins, membrane lipids, lipopolysaccharides and outer membrane proteins. We showed that OMVs can transport several biologically active toxins and enzymes to the surrounding environment and ultimately into the host cells. We have initiated analysis of OMV-associated secretion of virulence factors in V. cholerae. It was observed that PrtV is secreted from V. cholerae wild type strain C6706 into the culture supernatant in association with OMVs and OMV-associated PrtV protein is biologically active and more stable than the free, soluble PrtV protease.

Thesis (M.S. )--University of Tennessee Health Science Center, 2008.<br>Title from title page screen (viewed on July 31, 2008). Research advisor: Edwin L. Thomas, Ph.D. Document formatted into pages (xi, 50 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 44-50).

Bacteria antibiotic resistance to diverse clinically used compounds is becoming a major health risk. One strategy of multidrug resistance is the active extrusion of toxic compounds out of the bacterial cell walls by efflux pumps. These pumps are often regulated at gene transcriptional level. Pseudomonas putida, one of. the most resistant bacteria, is used as a model system to study the gene regulations involved in multidrug resistance. P. putida exhibits resistance to organic molecules, antibiotics and plant antimicrobials by three efflux pumps. One of them contains three membrane proteins TtgABC coded in one operon. The regulation of the efflux pumps genes ttgABC is controlled by a DNA binding repressor protein TtgR that is coded adjacent to the efflux pump operon and transcribed divergently. TtgR forms a homo dimer and consists of a ligand binding domain and a helix tum helix DNA binding domain. TtgR senses and interacts with ligands, leading to its release from DNA and the transcription of the efflux pump genes. The crystal structures of TtgR in complex with chloramphenicol and tetracycline antibiotics, phloretin, quercetin and naringenin plant antimicrobials and the organic solvent butylparaben have .,been determined to high-resolutions. These structures allow us to identify ligand binding pockets of TtgR and explain its diverse ligand binding properties. The structural information are confinned and complement~ by biochemical studies including isothermal titration calorimetry (lTC). Pseudomonas aeruginosa is a main pathogenic invader of the lungs of cystic fibrosis patients and exhibits multidrug resistance by activation of $e efflux pumps. Mex.Z, a homology of TtgR, is shown to be one of the most frequently mutated genes in bacteria isolated from cystic fibrosis patients, highlighting its importance in bacterial adaptation and survival. We have determined the crystal structure of MexZ, allowing us to explain the structural consequences of some of the clinically important mutations.

Autotransporters represent a diverse family of virulence effectors that are secreted from Gram-negative bacteria by the Type V Secretion System. Their initial description coined the term ‘Autotransporter’ to embody the notion that their three-part architecture governs their navigation through the bacterial cell envelope. The Pet cytotoxic autotransporter is secreted by the diarrhoeal pathogen, Enteroaggregative Escherichia coli (EAEC) and was used as a model to study autotransporter biogenesis. Following a global transposon mutagenesis of EAEC, novel accessory factors were identified that are required for Pet biogenesis, including the transcription factors CRP and Fis, periplasmic chaperones and components of the β-barrel assembly machinery (BAM) complex. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation and show that this co-activation mechanism extends to functionally similar autotransporters. Furthermore, we highlight novel components of the BAM complex required for AT secretion. This work builds on previous studies that, in recent years, have challenged the ‘auto’ nature of this secretion process causing a paradigm shift towards a much more complex mechanism of AT secretion than initially suggested.

Mestrado em Microbiologia Molecular<br>Actualmente, é cada vez mais frequente a associação de bactérias oportunistas e comensais resistentes a antibióticos com infecções nosocomiais. Este problema clínico tornou-se preocupante e deve-se ao uso indiscriminado de antibióticos. Perante esta pressão selectiva, as bactérias desenvolvem diferentes mecanismos de resistência a estes compostos. A presença de estruturas capazes de transportar genes de resistência, designadas por integrões, que contribuem para a disseminação destes genes bem como a sua associação com o perfil de resistência de bactérias constitui o objectivo do presente trabalho. Assim, foram recolhidas amostras de superfícies das instalações sanitárias, do serviço de Medicina II, do Hospital Infante D. Pedro, Aveiro. Após o isolamento das bactérias em meio selectivo para Gramnegativas (MacKonkey), todos os isolados foram sujeitos a tipagem molecular por BOX-PCR. O perfil de bandas obtido após electroforese foi analisado com o programa GelCompar II software (Applied Maths, Kortrijk, Belgium), permitindo distinguir diferentes grupos clonais. De cada grupo clonal foi seleccionado um isolado para os estudos posteriores, resultando num total de 45 isolados distintos. A pesquisa de integrões classe 1 iniciou-se por um “screening” para o gene da integrase. Nos 25 isolados positivos para este gene, foi amplificada e caracterizada a respectiva região variável. A sequência nucleotídica dos amplicões foi comparada com outras depositadas na base de dados. Os resultados mostraram a presença de integrões em diferentes espécies (Pseudomonas putida, Klebsiella pneumoniae, Escherichia coli, Pseudomonas mendocina, Proteus mirabillis e Morganella morganii). As regiões variáveis apresentavam diferentes tamanhos e diferentes arranjos de genes. Em geral, predominam gene cassettes que conferem resistência aos aminoglicosídeos, trimetoprime e metalo-β- lactamases. A localização destas estruturas no genoma bacteriano foi efectuada por “southern blot”; o DNA genómico foi digerido com a enzima S1, e sujeito a hibridação com sondas para os genes 16S e da integrase revelando que a maioria dos integrões estão localizados em plasmídeos. Como conclusão geral, verifica-se a prevalência de isolados contendo determinantes genéticos de resistência em superfícies inanimadas do ambiente hospitalar (53.33%), os quais podem constituir um potencial risco para os pacientes, uma vez que se trata de bactérias oportunistas. O facto de estes genes de resistência estarem associados a elementos genéticos móveis, nomeadamente transposões e muitas vezes plasmídeos, facilita a sua disseminação no ambiente hospitalar, principalmente por transferência horizontal de genes.<br>Currently, it is becoming frequent the association of antibiotic resistant opportunistic and commensal bacteria with nosocomial infections. This is a clinical problem of concern and is based on the indiscriminate use of antibiotics. Given the selective pressure within the hospital environment, bacteria develop different resistance mechanisms to these compounds. The presence of structures, referred as integrons, that carry and disseminate these resistance genes among bacteria and their association with the bacteria resistance profile constitutes the aim of the present study. To this end we collected samples from surfaces of sanitary facilities, of the Medicine II service of the Hospital Infante D. Pedro, Aveiro. After bacteria isolation on a selective medium (MacKonkey) for Gram negatives, all the isolates were molecular typed by BOX-PCR. After electrophoresis, the banding pattern was analysed with the GelCompar II software (Applied Maths, Kortrijk, Belgium), which allowed for the selection of different clonal groups. One isolate was selected from each group for further studies. Forty five isolates were selected for the screening of class 1 integrons. In the twenty-five positive isolates respective variable region was amplified and characterized. Amplicons nucleotide sequences were compared with others deposited in databases. The results revealed the presence of integrons in different species (Pseudomonas putida, Klebsiella pneumoniae, Escherichia coli, Pseudomonas mendocina, Proteus mirabillis e Morganella morganii). Different lenghts of variable regions and different genes arrays were found. Generally gene cassettes conferring resistance to aminoglycosides trimethoprim and metallo-β-lactamases were predominante. Southern hybridization of S1 digested genomic DNA with 16 rDNA and integrase genes labeled probes revealed that the majority of the integrons are located in plasmids. To conclude, is important to refer that there is a prevalence of opportunistic bacteria possessing integrons in inanimate surfaces within the hospital environment, which can constitute risk to the debilitated patients. Moreover, these structures are associated with mobile genetic elements, mainly transposons and many times plasmids, which facilitates the dissemination of these antibiotic resistance genes in the hospital environment, mainly by horizontal gene transfer.

KefB and KefC are glutathione-gated potassium channels that play a protective role during the detoxification of electrophiles in Gram negative bacteria. The KefC channel from Klebsiella aerogenes has been characterised and the gene cloned and sequenced. The KefC channel from K. aerogenes is analogous to KefC from Escherichia coli. The electrophile compounds NEM and CDNB are strong activators of potassium loss via the channel, whereas, methylglyoxal is a weak activator of potassium loss. At the amino acid level the putative protein shows a high degree of similarity with KefC from E. coli. While sequencing the kefC gene from K. aerogenes a difference in the genome organisation of K. aerogenes and E. coli was observed, highlighting the presence of an unassigned ORF, yabF, that overlaps the kefC by 8 bp. Clones of the K. aerogenes kefC gene that lack the first 129 bp of yabF exhibit reduced KefC activity. Analysis of the sequence surrounding the kefB gene from E. coli revealed an ORF, yhaH, that encodes a homologue of the putative YabF protein. The amino acid distribution of YabF and YhaH predict soluble proteins with significant similarity to the NAD(P)H dehydrogenase quinone oxidoreductase, DHQV. To investigate the function of YabF, a strain lacking the yabF-kefC region in E. coli was constructed. The strain was transduced into a kefB background and when transformed with a plasmid expressing only KefC channel activity was greatly reduced. The data suggest that the YabF protein is required in trans for the activity of KefC and preliminary evidence from in vitro-galactosidase fusion studies suggest that yabF and kefC genes may form an operon.