Enterobacteriaceae belong to the normal enteric flora in humans and may cause infections. Escherichia coli is the leading urinary tract pathogen with septicaemic potential, whereas Klebsiella pneumoniae causes opportunistic infections and often outbreaks in hospital settings. Beta-lactams are the first choice for treatment of infections caused by Enterobacteriaceae, and might be destroyed by extended-spectrum beta-lactamases, ESBLs. ESBLs hydrolyse all beta-lactams except cephamycin and carbapenems, and constitute a large heterogeneous group of enzymes with different origins. The phenotypic and molecular characteristics of a K. pneumoniae strain causing a major outbreak at Uppsala University Hospital between 2005 and 2008 were described. The strain was multiresistant and produced CTM-M-15, a common ESBL type in Europe. Due to the lack of obvious epidemiological links between patients, a case-control study was performed, which identified risk factors for the acquisition of the outbreak strain in urine cultures. The complex chain of transmission facilitated by patient overcrowding and the interventions applied to curb the outbreak, was revealed in the subsequent study. In the final study, the genetic background of the observed increase in ESBL-producing E. coli isolates during the K. pneumoniae outbreak was explored. The utility of six typing methods in epidemiological investigations of a local outbreak with ESBL-producing E. coli was compared. The increase of ESBL-producing E. coli isolates was not secondary to the K. pneumoniae outbreak. Twentytwo per cent belonged to the epidemic O25b-ST131 clone and only a limited number of infections were caused by nosocomial transmission. ESBL-producing Enterobacteriaceae are a challenge to clinical microbiology laboratories and infection control teams. To investigate their dissemination, typing methods need to be continuously adapted to the current situation. Proper hand disinfection and structural key problems such as over-crowding, under-staffing, lack of single rooms and bathrooms must be adressed to limit transmission.

Acute otitis media (AOM) is a common disease in childhood and is one of the most common causes for outpatient antibiotic treatment. The major aetiological agents of AOM have varied over the decades. Now the three most common pathogens are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. The resistance patterns of these organisms have also varied from the beginning of the antibiotic era to the situation we have today with an increasing incidence of penicillin-resistant S. pneumoniae and a moderate to high frequency of beta-lactamase production in H. influenzae and M. catarrhalis. In Sweden we have continued to use the Scandinavian treatment policy of penicillins as the first-line antibiotic treatment of AOM, which has been implemented with good results in the past. The question is if this policy will continue to have acceptable treatment results. In order to investigate aspects of treatment, protection and structural changes in AOM, an animal model was used. Amoxicillin treatment of AOM caused by H. influenzae was studied. Amoxicillin treatment was shown to shorten the duration of the infection and to reduce the morphological changes normally observed after an untreated AOM. The influence of antibiotic treatment on recurrent AOM was evaluated. Amoxicillin treatment did not lead to less protection against reinfection. Abstaining from antibiotics did not improve the levels of serum IgG antibodies. The IgG levels were significantly higher in treated animals after rechallenge. AOM caused by H. influenzae with a non-beta-lactamase-mediated resistance to beta-lactams was investigated and it was observed that during amoxicillin treatment the chromosomal changes mediating resistance were possibly advantageous for the bacterium. In cultures from children with AOM, there is sometimes growth of several bacteria. The possibility of a sheltering effect of beta-lactamase-producing H. influenzae on a penicillin-sensitive S. pneumoniae in a mixed infection was investigated, and amoxicillin was shown to eradicate the pneumococci from the middle ear despite the presence of beta-lactamase. An increasingly cultured bacterium in nasopharynx and in AOM is M. catarrhalis. It is now beta-lactamase-producing in almost 100% of cases and is thus not eradicated by penicillins. An animal model of AOM caused by beta-lactamase-producing M. catarrhalis was established to study the course of this infection with the possibility of evaluating aspects of virulence between AOM pathogens. The AOM observed was a self-limiting disease. The results obtained in this study in a rat model support the continuing use of penicillins as first-line drugs in the treatment of AOM. Penicillins are not sufficient to treat all causative agents, but the majority of pathogens including the most virulent bacteria are eradicated from the middle ear.

In the first part of my thesis I focus on mapping problems associated with antibiotic therapy and subsequent development of antibiotic resistance. Tracking resistance is based primarily on data collection and evaluation of the results set sensitivity from around the world. Antibiotic resistance is a natural phenomenon that can be observed in the evolution of microbes as one of the mechanisms of adaptation to new conditions in the environment. For this work I have chosen the following research questions. Do the incidence of ESBL strains in the České Budějovice Hospital a.s. increase over time? Are these values comparable to those achieved in another region, namely in Moravian hospitals the University Hospital of Olomouc, Ostrava University Hospital and Regional University Hospital of T. Bata in Zlin? The data collection I made in collaboration with the laboratory technicians and doctors at Hospital?s Bacteriology Laboratory in České Budějovice. Bacteries tested for the detection of ESBL production originated from biological materials, witch came from patients of hospital in České Budějovice. The first objective was to compare the results achieved in the České Budějovice Hospital in the period of 2007 to 2012. If we look at the total number of ESBL strains that have been isolated since 2007, values have upward trend. While in 2007 there were only 64 strains a year later, the number more than doubled. In 2010, the value soared to 281 tribes and in the year 2012, the number was 321 tribes. The incidence of ESBL strains in 2007 increased about five times. In the long term we can say the numbers have increasing tendency and the range of each species in the production of ESBL has significantly changed. In 2007, it was K. pneumoniae strains that dominated the statistics, but over time the strains of E. coli came forefront. Values of 2012 suggest that the presence of ESBL strains of K. pneumoniae is again almost equal to the number of E. coli strains. The second objective was to compare the results of the 2012 with study of the Prevalence of ESBL-positive Enterobacteriaceae in large Moravian hospitals. In the general overview of ESBL producers values in Hospital České Budějovice (5.23%) are comparable to those in Ostrava (4.9%) and in Zlín (4.3%). Number of strains in the Hospital in Olomouc (11.8%) is about twice as high as the numbers in České Budějovice. In this comparison the České Budějovice Hospital is one of the hospitals with a lower incidence of ESBL producers. The České Budějovice Hospital is below the national average, which originate from an elaborate system of care for patients with colonization or infection with ESBL strains, and from therapy control system using antibiotic center. These results may serve to the Hospital in České Budějovice for statistical purposes, and also for proposals for improving patient care. In the discussion, I pointed out the danger of the spread of resistant strains of bacteria in the community and also the associated risks that mentioned bacteria mean for patients injured in mass accidents or disasters. In these cases, number of infections including ESBL producers can penetrate through open wounds into the affected body. Unlike conventional sensitive bacteria those strains are resistant to commonly used antibiotics and thereby endanger the lives of people affected by the accident.

Structure based drug design is a rapidly advancing discipline that examines how protein targets structurally interact with small molecules, or known inhibitors, and then uses this information to lead inhibitor optimization efforts. In the case of novel inhibitors, protein structural information is first obtained via X-ray crystallography, NMR studies, or a combination of both approaches. Then, computational molecular docking is often used to screen, in silico, millions of small molecules and calculate the potential interactions they may have with the target protein’s binding pocket, in hopes of identifying novel low affinity inhibitors. By examining the interactions these small, low affinity, inhibitors have with the binding pocket, optimization efforts can be focused on maximizing interactions with “hot spots” within the pocket, thus leading to larger, high affinity inhibitors. A similar optimization technique can also be applied to known inhibitors. By examining the interactions of a known inhibitor with the binding site, new compounds can be designed to target “hot spots” in the binding pocket using the known inhibitors core structure as a starting point. The affinity of the newly designed compounds can then be compared to the affinity of the original inhibitor, and further rounds of optimization can be carried out. While simple in design, there are many challenges associated with structure based drug design studies, and there is no guarantee novel inhibitors will be found, but ultimately, it is an extremely powerful methodology that results in a much higher hit rate than other, similar, techniques. The work herein describes the use of structure based drug design to target several different proteins involved in bacterial antibiotic resistance, and a protein that has been implicated in the development of Alzheimer’s disease. The goal of the first project was to design a new PBP inhibitor based upon an existing scaffold, and to better understand the binding mechanism and molecular interactions between penicillin binding proteins and their inhibitors. PBPs are a group of proteins that catalyze the last steps of bacterial cell wall formation, and are the targets of the β-lactam antibiotics. Two compounds were designed which conjugated a ferrocene or ruthenocene group to 6-aminopenicillinic acid, and their antibiotic properties were tested against a range of bacterial strains. To get a better understanding of how the 6-APA organometallic compounds interacted with the PBP active site, a CTX-M-14 β-lactamase model system was used for X-ray crystallographic studies. CTX-M-14 was chosen as its active site shares many key catalytic features with PBPs, and it easily, and reproducibly, yields crystals capable of diffracting to sub-atomic (< 1.0 Å) resolution. I determined a 1.18 Å structure of 6-APA-Ru in complex with CTX-M-14 E166A β-lactamase and was able to gain unprecedented details of the interactions of the ruthenocene group with the CTX-M active site. This structure also revealed that the compound bound in the CTX-M active site was actually the decarboxylated and hydrolyzed product, which was the first time a decarboxylated product had been captured in the CTX-M active site. A second, 0.85 Å, structure of CTX-M in complex with 6-APA-Ru was determined and shed light on how the hydrogen bonding network in the CTX-M active site changes in response to the 6-APA-Ru product binding. A final, 1.30 Å, structure captured the carboxylated and hydrolyzed 6-APA-Ru product in complex with CTX-M, which was the first time the carboxylated product had been captured in the CTX-M active with the catalytic Ser70 residue intact. The results show the potential of the ruthenocene group in improving antibiotic potency, and help to better elucidate the changes that occur in the CTX-M active site upon inhibitor binding, while at the same time, telling us what changes could occur in the active site of PBPs. The next project was focused on novel inhibitor discovery against several different PBPs. PBPs have been successfully inhibited by β-lactam antibiotics for decades, but the alarming rise of bacteria resistant to these antibiotics has placed increased urgency on the discovery of novel PBP inhibitors. A fragment based molecular docking approach was employed to virtually screen millions of small compounds for interactions with the targeted active sites, and then high scoring compounds were selected for visual inspection and inhibitory testing. Virtual screening was first done against Staphylococcus aureus monofunctional transglycosylase, a type of PBP. MTG provided a good binding pocket for virtual screening, but proved challenging to purify and crystallize. However, through great effort MTG crystals were eventually obtained. After repeated rounds of virtual screening against MTG, multiple compounds were selected for inhibition testing, and testing is currently ongoing. Virtual screening was also done against Pseudomonas aeruginosa PBP5 and PBP1a. Purification and crystallization of these proteins proved to be easier than MTG, and both yielded diffraction quality crystals. The final project focused on virtual screening against a protein implicated in the development of Alzheimer’s disease, Slingshot Phosphatase 1. The brains of AD patients have been found to contain elevated levels of active Cofilin, and these elevated levels of active Cofilin may lead to the overproduction of amyloid β. Aβ overproduction, and its resulting accumulation, is believed to be one of the pathways that lead to AD symptoms. Cofilin is activated when it is dephosphorylated by SSH1, and inhibiting this activation may decrease the production of Aβ and the development of AD symptoms. There is no known structure of SSH1, so to perform virtual screening a SSH1 homology model was constructed using the homolog SSH2 as a starting point. Virtual screening was then performed using the SSH1 homology model and many compounds were selected for inhibition testing. Initial testing found several compounds that could prevent Cofilin dephosphorylation at levels > 10μM. However, three compounds were found to be exceptionally active, and could prevent Cofilin dephosphorylation at both 1 and 10 μM. One of these three compounds was tested directly against purified SSH1 and found to inhibit its activity, and reduce Aβ production. Crystallization of purified SSH1, and SSH2, was attempted in order to get complex structures with the three best compounds. SSH2 crystals were obtained which diffracted to 1.91 Å, and several initial hits were found for SSH1. Optimization of crystals for both proteins is currently ongoing. The SSH1 inhibitor, along with the two other highly active compounds, provides an excellent starting point for the development of highly potent SSH1 inhibitors.

No description available.

TEM and SHV β-lactamases are the most prevalent β-lactamases among Gram-negative bacteria. The introduction and widespread use of expanded-spectrum antibiotics, particularly third generation cephalosporins, has led to the evolution of bacterial strains expressing extended spectrum β-lactamases (ESBLs). ESBLs emerge by genetic point mutation from non-extended spectrum precursors. It was found that multiple β-lactamase families within single isolates complicate the process of detecting the resistance status of isolate using non-quantitative DNA diagnostic methods. Preliminary phenotypic characterisation of probable β-lactamase enzyme family types present in 100 isolates from the Asia-Pacific and South African locales showed that single isolates frequently contained multiple β-lactamase families. SHV, TEM, AMPC and CTX-M β-lactamase families were detected in these isolates using PCR detection methods. Ninety-eight percent of all isolates tested contained as least one β-lactamase gene, with up to four to β-lactamase gene families found to co-exist in single isolates. Kinetic PCR methods for interrogating the polymorphic sites at blaSHV codons 238 & 240 and blaTEM codons 164, 238, 240 as well as promoter polymorphism were developed. A high proportion of blaSHV 238 and 240 mutant alleles was found to correlate with cefotaxime, ceftazidime and aztreonam resistance levels. In an attempt to understand the molecular basis for the co-existence of multiple blaSHV alleles within single isolates, the blaSHV promoter region was cloned from one ESBL expressing isolate. Experimental results showed that blaSHV can exist downstream of two different promoters within a single isolate. Both promoters have previously been reported, and differ by the presence or absence of IS26, which results in a change in the transcription initiation site. The blaSHV gene copy numbers in cis with the different promoters were measured, and it was found that the copy number of the IS26::blaSHV promoter was positively correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with promoters in individual isolates. However, mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL+ isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL+ phenotype. To further confirm the role of IS26 in resistance acquisition, ESBL negative isolates were subjected to serial passage in vitro evolution experiments and fluctuation assays. Results confirm that the insertion of the IS26 element upstream of blaSHV is positively correlated with the ability to exhibit an ESBL phenotype, when such isolates also contain the critical G238S substitution. It was also found that IS26 can catalyse the duplication and mobilisation of blaSHV within an isolate. Fluctuation experiments have shown that the frequency at which such genomic events occur resulting in ESBL phenotypes is extremely low and requires many generations of selection under sub-lethal conditions. A survey of a geographically diverse set of isolates has shown that IS26-blaSHV was found in all of the bacterial populations surveyed. However, it does not appear to be exclusively associated with SHV-mediated ESBL production.

No description available.

No description available.

Metallohydrolases with dinuclear-zinc active sites perform many important biological hydrolytic reactions on a variety of substrates. In this regard, metallo-β-lactamases (mβ1, class B) represent a unique subset of zine hydrolases that hydrolyze the β-lactam ring in several antibiotics. The antibiotic resistance that results from this hydrolysis is becoming an increased threat for the clinical community. These metalloenzymes can hydrolyze a wide range of β-lactam substrates, such as cephamycins and imipenem that are generally resistant t the serine-containing β-lactamases. Therefore, the clinical application of the entire range of antibiotics is severely compromised in bacteria that produce mβls. Due to the lack of information on the mechanism of mβls, to-date, no clinically known inhibitors is there for mβls. In this present study, we synthesized several mono and dizinc complexes as models for the mβls and investigated the differences in their hydrolytic properties. This study supports the assumption that the second zinc in the dinuclear enzymes does not directly involve in the catalysis, but may orient the substrates for hydrolysis and the basic amino acid residues such as Asp and His may activate the zinc-bound water molecules, fulfilling the role of the second zinc in the mononuclear enzymes. The effect of various side chains on the hydrolysis of some commonly used cephalosporin antibiotics by mβl from B.cereus is described. It is shown that the cephalosporins having heterocyclic thiol side chains are more resistance to mβl-mediated hydrolysis than the antibiotics that do not have such side chains. This is partly due to the inhibition of enzyme activity by the thiol moieties eliminated during the hydrolysis. It is also observed that the heterocyclic side chains in pure form inhibit the lactamase activity of mβl as well as its synthetic mimics. The mode of binding of these heterocyclic side chains to the zinc has been analyzed from the crystal structure of the tetranuclear zinc complexes. The theoretical studies suggest that the eliminated heterocyclic thiols undergo a rapid tautomerism to produce the corresponding thiones. These thiones are found to irreversibly inhibit the LPO-catalyzed iodination reaction. The reaction of various thiones with I2 leads to the formation of thione-iodine complexes similar to that of the most commonly used antithyroid drug methimazole(MMI). These observations suggest that some of the latest generation of antibiotics may show negative effects on thyroid gland upon hydrolysis. Synthetic organophosphorus compounds have been used extensively as pesticides and petroleum additives. These compounds are very toxic to mammals and their widespread use in agriculture leads to serious environmental problems. Therfore, degradation of organophosphorus trimesters and remediation of associated contaminated sites are of worldwide concern. In this regards, the bacterial phsophotriesterase (PTE) enzyme plays an important role in degrading a wide range of organophosphorus esters and the active side of PTE has been shown to be very similar to that of mβl. This identification prompted us to check the hydrolysis of phosphotriesters by the mβl and its mimics. It has been observed that the dinuclear zine(II) complexes that do not allow a strong binding of phosphodiestes would be a better PTE mimics.

A fusion protein composed of antibody fragments and β-lactamase was earlier created by Kojima et al. (2011), with antigen specificities against a bone disease marker and a pesticide. The enzyme was circularly permutated and fused to the variable heavy and light chain antibody fragments, thereby ensuring inactivity until binding of the target antigen triggered enzyme activation. Upon activation, the β-lactamase produced a colorimetric signal, which indicated antigen presence. In this work, a similar strategy was used to create two novel fusion proteins composed of circularly permuted β-lactamase and superfolder green fluorescent protein with anti-benzo[a]pyrene variable antibody fragments. The fusion proteins were designed and expressed in E. coli for the development of a single-step visual immunoassay. It was hypothesized that the cp reporter proteins would be activated once the binding of B[a]P to the variable antibody fragments occurred, and this interaction was expected to produce a detectable colorimetric or fluorescent signal. Although positive results were obtained in one instance, substantial supportive evidence in favour of the hypothesis could not be obtained. / SENTINEL Bioactive Paper Network, Natural Sciences and Engineering Research Council of Canada (NSERC), Canada Research Chairs Program.