Antibiotics, Antimicrobials & Resistance

Adam Whelan completed his PhD in Vaccinology and Immunology of Bo¬vine Tuberculosis at Imperial College London. He has worked as a Research Scientist at UK Government Agencies concerned with infectious diseases of relevance to the veterinary, and more recently, biodefense research com¬munities. He has published more than 63 papers in reputed journals and is currently the Technical Lead on a UK MOD funded antimicrobial therapy programme of work.

Burkholderia mallei is the causative agent of glanders, a disease primarily of solipeds. It is a zoonotic pathogen and is highly infectious by the aerosol route. In humans, it is almost always fatal in the absence of antibiotic therapy and is classified as a Tier 1 bio-threat agent by the US Centers of Disease Control and Prevention. Due to the rarity of human infections, there is limited data on the efficacy of different antibiotic regimes. The purpose of this study was to assess the efficacy of finafloxacin against an inhaled challenge of B. mallei using a Balb/c mouse model of glanders. Finafloxacin is a novel member of the fluoroquinolone class of antibiotics that demonstrates the unique property of having greater activity under acidic conditions. We have previously demonstrated that finafloxacin can protect mice against an inhaled challenge of the related pathogen B. pseudomallei, the causative agent of melioidosis. This protection was superior to that provided by co-trimoxazole, an antibiotic commonly used in the treatment of melioidosis. In this study, mice were infected with an aerosol of B. mallei. Treatment with finafloxacin or co-trimoxazole was initiated 24 hours post-infection and survival and bacterial colonization were determined up to 65 days post-infection. Finafloxacin and co-trimoxazole both offered significant and comparable protection in comparison to the vehicle controls. Neither antibiotic provided complete protection as relapse of infection was observed at days 26 and 23 for finafloxacin and co-trimoxazole, respectively. However, finafloxacin provided improved bacterial control during the early stages of the infection (following the determination of bacterial load in organs at days 1 and 15 post infection) and reduced bacterial dissemination during late-stage infection (day 65). In summary, these data demonstrate the utility of finafloxacin as a promising therapeutic for glanders.

Andrea Szabo has graduated as a Pharmacist and has completed her PhD in Interdisciplinary Medicine at the University of Szeged. She has done post-doctoral studies in the Department of Public Health at the same univer¬sity. She is working as an Assistant Professor of Public Health and Preven¬tive Medicine. She has broad interdisciplinary experience in Neurotoxicology, Nanotoxicology, Nutritional Epidemiology, Epidemiology of Infectious and Non-Infectious Diseases, and Antimicrobial Stewardship. She has partici¬pated in the development of the Hungarian methodological guideline for an¬timicrobial stewardship in the framework of a funded Human Resource De¬velopment program. Her recent research interest is the knowledge, attitude and practice related to antibiotic resistance and infectious diseases among different populations. She is the Principal Investigator of this research. She has published 48 papers in reputed journals.

Antibiotic resistance is one of the major global public health threats. In Hungary, the level of antibiotic resistance is one of the highest among EU countries; however, some health care professionals still neglect this problem. One of the main reasons for development of resistance is non-prudent use of antibiotics (e.g., broad spectrum agents or unjustified indications). 90% of the antibiotics are used in outpatient care in Hungary; therefore, the proper attitude of primary health care professionals (general practitioners-GPs and pharmacists) is extremely essential. The aim of this questionnaire-based study was to reveal the problem perception of antibiotic resistance and the driving forces behind non-prudent use of antibiotics. 105 physicians (mostly GPs) and 192 pharmacists completed the questionnaires; additionally, to reveal the roots of the manner, 161 pregraduate medical students were also involved in the study. 10% of GPs thought that antibiotic resistance is not a problem in Hungary, while pharmacists were totally aware of it. Likewise, worse attitude of physicians was observed in considering antibiotics of critical importance and in giving detailed advice to patients on proper use of antibiotics. One third of doctors thought that culturing the microorganism in question is unnecessary for choosing the appropriate therapy; and one quarter of them agreed with using broad spectrum antibiotics to make the therapy easier. More pharmacists (44% vs. 25% of GPs) admit that they are markedly influenced by the temperament of patients, resulting in dispensing antibiotics without prescription in case of pharmacists and prescribing antibiotics without rational indications in case of physicians. Medical students’ responses were very similar to the licensed physicians’ indicating that the root of the commitment originates from the university studies. Our results underline the role of primary health care professionals in fighting off antibiotic resistance and confirm the instant need for complex antibiotic stewardship program in Hungary.

Jan Kormanec is the Head of Department of Genomics and Biotechnol¬ogy at the Institute of Molecular Biology of Slovak Academy of Sciences (Bratislava, Slovakia), where he has been working since 1984. He graduated from the Comenius University (Bratislava, Slovakia) in 1984 and with a PhD from the Slovak Academy of Sciences in 1991. In 2001, he was awarded the highest scientific title (DrSc) by Comenius University. His main research interests include the role of sigma factors of RNA polymerase in stress-re¬sponse, pathogenicity, and differentiation of bacteria, and investigation of antibiotics production and regulation in Streptomyces. He has published 116 papers in reputed journals and four chapters, which are cited by more than 1700 citations in Web of Science.

Bacteria of the genus Streptomyces are the main producers of bioactive natural products with a broad range of biological activities. A large number of these products belong to polyketides. These structurally diverse natural compounds are synthesized by a repeated decarboxylative condensation from acyl-CoA precursors by a polyketide synthase (PKS). Aromatic polyketides are synthesized by type II PKSs. Although a large repertoire of aromatic polyketides produced by type II PKSs was identified, they all belong to just a few common structural types that include pyranonaphthoquinones, tetracyclines, angucyclines, anthracyclines, tetracenomycines, aureolic acids. We identified the aur1 gene cluster in Streptomyces lavendulae subsp. lavendulae CCM 3239 (formerly Streptomyces aureofaciens CCM 3239), which is responsible for the production of the angucycline-like antibiotic auricin. Interestingly, the aur1 gene cluster is localized on the large (241 kb) linear plasmid pSA3239. Auricin was produced in low levels, which hampered its purification and structural elucidation for many years. Careful investigation revealed that auricin is transiently produced in a very narrow growth phase interval of several hours after entry into stationary phase and afterwards it was degraded to non-active metabolites because of its instability at the high pH reached after the production stage. This unusual pattern of auricin production arises from a strict, but complex regulatory mechanism, involving both feed-forward and feed-back control by auricin intermediates via several transcriptional regulators (gamma-butyrolactone system SagA/SagR, pathway-specific regulators Aur1P, Aur1O, Aur1R, Aur1PR3 and Aur1PR4. Auricin structural analysis revealed that it possesses intriguing structural features, distinguishing it from other known angucyclines. It is modified by D-forosamine and contains a unique aglycone similar to those of spiroketal pyranonaphthoquinones griseusins. In addition to its antibiotic activity, auricin also displayed modest cytotoxicity against several human tumor cell lines.

Renata Novakova has completed her PhD in Molecular Biology from the Slovak Academy of Sciences in 1998 and postdoctoral studies from Max Planck Institute of Molecular Physiology in Dortmund. She has worked as Group Leader in the Department of Genomics and Biotechnology in Institute of Molecular Biology of Slovak Academy of Sciences (Bratislava, Slovakia). Her main research interests include investigation of antibiotics production and regulation in Streptomyces. She has published 26 papers in reputed journals, which are cited by more than 140 citations in Web of Science.

Antibiotics belong to the most successful form of chemotherapy developed in the twentieth century. However, their extensive use inevitably selects for resistant microbes. Therefore, there is a continuing and cyclical need for new efficient antibiotics against these multi-resistant bacteria. Antibiotics have been discovered by screening natural products produced by bacteria and fungi for antibiotic activity and subsequently chemically modifying these structures to incorporate additional desirable pharmacological properties. During the last sixty years, large amount of different types of antibiotics has been discovered and characterized. Although it is estimated that only about 3% of the natural products potential has been discovered yet, it seems obvious that the antibiotics that were easy to discover have already been found, and it is likely that the classical search for new antibiotics will involve a substantial amount of expensive and laborious research. However, the current possibilities of new technologies (molecular biology approaches, gene manipulations, genomics, and synthetic biology) open up exciting possibilities for the discovery of new antibiotics. In this respect, synthetic biology is a very promising approach. This emerging area of interdisciplinary research can be defined as the design and construction of novel artificial biological pathways, organisms or devices, or the redesign of existing natural biological systems for useful purposes. We used this strategy for testing of heterologous production of aromatic polyketides based on the first landomycine biosynthetic genes lanABCFDLE, under the control of two strong promoters, ermEp* and kasOp* and optimised RBS site. Although the production was not higher that in its native form, this approach seems to be promising, since it enables to combine various genes from other aromatic polyketide gene clusters to produce new structural polyketide motifs.

Alex Maughan completed Medical Degree at Bristol University. He complet¬ed his foundation year one training at Hinchingbrooke Hospital and is in the process of completing his second year in Royal Papworth Hospital. He takes active interest in Antimicrobial Stewardship by attending hospital commit¬tee meetings and involving himself in public awareness events.

Background: In March 2017 there was a worldwide shortage of piperacillin-tazobactam due to an explosion in a Chinese pharmaceutical factory. At Hinchingbrooke Hospital (UK), interim guidelines were published with recommended alternative antibiotics. Additionally, piperacillin-tazobactam stocks were removed from wards excluding accident and emergency, intensive care, the cancer centre and the acute medical unit. Therefore, if still deemed necessary by clinicians despite the recommended alternatives, the policy stated that piperacillin-tazobactam required senior microbiological approval. Objectives: To determine the level of compliance with interim guidelines both before and after simple interventions. Methods: We conducted a closed loop audit to investigate the use of piperacillin-tazobactam and compliance with interim guidelines. The three outcomes measured were as follows: 1. correct compliance with interim guidelines; 2. number of patients switched from piperacillin-tazobactam within 24 hours; and 3. number of patients remaining on piperacillin-tazobactam with microbiology approval. Our intervention took place over one month; printed copies of interim guidelines were distributed and clinicians were reminded of the piperacillin-tazobactam shortage at teaching sessions. We measured the practice before and after our intervention. Data was analysed using Chi-square test for the first two outcomes and Fisher’s exact test for the third. Results: 71 patients were included in the initial audit cycle and 104 in the second. There was a significant improvement in appropriate use of interim guidelines pre- vs. post-intervention: 62% vs. 86% (p < 0.05). There was no significant reduction in the number of patients prescribed piperacillin-tazobactam without approval or the number changed from piperacillin-tazobactam within 24-hours; however, the trend showed improvement. Conclusions: Our results suggest educational interventions improve prescribing practice and have a positive impact on antimicrobial stewardship. However, there was no reduction in absolute number of patients prescribed piperacillin-tazobactam without approval, suggesting physicians still choose piperacillin-tazobactam for their sickest patients despite the shortage.

Gosha Wojcik is a doctoral student at Edinburgh Napier University and her main research interest is the growing problem of antibiotic misuse leading to the emergence of antimicrobial-resistant pathogens. Her PhD is focused on the development of a theory-based behaviour change intervention to pro¬mote optimal antibiotic use in acute hospitals – the IMPACT study. Prior to commencing her PhD, in close collaboration with the Health Protection Scotland (HPS) and ICU clinicians, she developed and led a prospective pilot study that compared two surveillance algorithms for ventilator-associated pneumonia. With a grant from HPS, she then helped design a study titled `CDC vs HELICS` comparing new Centre for Disease Control (CDC) and the Hospitals in Europe Link for Infection Control through Surveillance (HELICS) systems in the two Lothian general ICUs, the findings of which have been published in the journal of Intensive Care Medicine.

Introduction: The rapid emergence of antimicrobial resistance (AMR) poses a major threat to public health and patient safety due to associated morbidity, mortality and healthcareassociated infections. With the lack of new antibiotics currently in development, promoting optimal antibiotic use is the only option available to slow the spread of resistance. The aim of the review was to identify, examine and synthesise qualitative studies that explored doctors` perceived barriers and facilitators to appropriate antibiotic use in acute hospitals. Methods: The review was registered on the PROSPERO database. The meta-ethnographic seven-stages approach was applied to provide higher level of analysis and generate conceptually rich insights. Using the SPIDER tool, 20 electronic databases, including grey literature, were systematically searched for studies published between 2007 and 2017. Critical appraisal was conducted by 2 independent reviewers using the CASP tool. EMERGE methodological standards7 were used to guide reporting of the results. Results: Out of 12,254 identified papers, 13 were included in the final synthesis. The seven-steps process of analysis identified five overarching themes: (1) Mastering the art of antibiotic prescribing; (2); Behind the lens of misguided logic; (3) Social navigation of medical culture (4); The dual nature of uncertainty, and (5) Balancing multiple tensions. Discussion: This is the first meta-ethnography on doctor`s experiences of antibiotic prescribing in acute hospitals. Findings demonstrate that social navigation of medical prescribing is an emotionally-fuelled endeavour often performed within the culture of guesswork. The identified themes suggest that `appropriate` prescribing is a complex, context-dependant, fluid and intangible process that may often appear at odds with the evidence and therapeutic guidelines. This process is influenced by multiple factors and tensions, including uncertainty, interpersonal relationships, scarce healthcare resources and the fear of: patient deteriorating, losing professional credibility and increased AMR. The findings provide a conceptual framework that could provide the basis for future antibiotic management interventions.

Erika Kapp is a Pharmacist and has completed her MSc in Pharmaceutical Chemistry at North-West University in 2005. From 2006 to 2011 she worked in the not-for-profit sector with the focus on health systems strengthening and sustainable access to antiretroviral and antimycobacterial treatment in the public sector in South Africa. She currently works as a Lecturer in Phar-maceutical Chemistry at the School of Pharmacy, University of the Western Cape, where she is in the process of completing her PhD. Her research in¬terests center on the synthesis and utilization of polycyclic derivatives as resistance reversal agents in Mycobacterium tuberculosis.

Ever-increasing levels of drug resistance in Mycobacterium tuberculosis necessitate novel approaches in the search for new antimycobacterials. Early identification of likely mechanism of action and probability of successful accumulation of potential antimycobacterials within mycobacterial cells allow for triage of compounds that warrant further investigation. Early knowledge pertaining to novelty of mechanism of action, possible cross resistance to existing antimycobacterials as well as the risk of acquisition of genetic resistance brought on by efflux-mediated sub-therapeutic intracellular compound accumulation, promotes the likelyhood of identification of therapeutically viable antimycobacterials. Antimycobacterial activity as well as synergistic activity in combination with known antimycobacterials was evaluated for a range of polycyclic amine derivatives obtained from the School of Pharmacy compound library. Compounds were selected based on presence of pharmacophoric moieties for antimycobacterial activity, the inclusion of bulky lipophilic carriers to promote intracellular accumulation through increased cell wall penetration or alternatively reduced efflux as well as previously demonstrated bioactivity that may support accumulation of coadministered compounds within mycobacterial cells. The most active compound demonstrated a MIC99 of 9.6 μM against Mycobacterium tuberculosis H37Rv. Genotoxicity and inhibition of the problematic bc1 respiratory complex was excluded as mechanism for all compounds, with the two most active compounds likely inhibiting cell wall synthesis in a fashion similar to ethambutol. Synergism assays with known antimycobacterials are currently underway and preliminary results indicate possible synergistic activity with known antimycobacterials. Further structure activity relationship analysis will inform the development of compounds with increased potency and selectivity while exploring possible resistance reversal activity.

Helene Barreteau has completed her PhD in Biochemistry and Microbiology at the University de Picardie Jules Verne (Amiens, France) and postdoctoral studies from the University Paris-Sud (Orsay, France), CNRS (Grenoble, France) and the Musyum National Natural history museum (Paris, France). She currently works as assistant professor in Biochemistry at the University Paris-Sud and has published 35 papers in reputed journals.

Colicins are plasmid-encoded bacteriocins produced by some Escherichia coli strains in order to kill competitors belonging to the same or related species. All known colicins share a same structural organization in three domains, each of them being devoted to a specific function: translocation through the outer membrane, binding to a specific outer membrane receptor and toxicity, from N to C termini, respectively (Cascalès et al., 2007). Among colicins, colicin M (ColM) is the smallest colicin known to date and the only one known to interfere with peptidoglycan biosynthesis. It targets and cleaves the peptidoglycan lipid II intermediate at the periplasmic face of the inner membrane by exhibiting a phosphodiesterase activity leading to the formation of undecaprenol and 1-pyrophospho-MurNAc-(pentapeptide)- GlcNAc (El Ghachi et al., 2006). These two products cannot be used or recycled for peptidoglycan biosynthesis, leading to cell lysis. During last years, several homologues of ColM produced by various bacteria such as Pseudomonas (Barreteau et al., 2009), Burkholderia (Ghequire and De Mot, 2015) and Pectobacterium (Grinter et al., 2012, 2014) were identified and characterized. These ColM-like proteins exhibit the same mode of action as ColM and displayed cytotoxic activity towards a limited number of bacterial species. Accordingly, no crossed cytotoxic activity has been demonstrated (Barreteau et al., 2009; Chérier et al., 2016), presumably due to the high specificity of reception and translocation machineries. Thus, reaching the lipid II target is clearly the crucial and limiting step to be considered in a perspective of exploitation of these colicin family members as tomorrow’s antibiotics. We recently showed that the pectocin M1 (PcaM1, produced by P. carotovorum) and some of its variants were able to kill E. coli cells once addressed to the periplasm of this species using appropriate pASK vectors (Chérier et al., 2016). In these conditions, PcaM1 and its isolated catalytic domain were effectively able to catalyze the degradation of lipid II, leading to the arrest of peptidoglycan biosynthesis and cell lysis. To the best of our knowledge, this was the first example of a ColM-like protein capable of killing another bacterial species, without any treatment affecting cell wall integrity. This result and the fact that lipid II is an essential and specific component through the whole bacterial world make of ColM and its orthologues very interesting agents to be exploited as new antibacterial agents. We thus propose the creation of a range of chimera colicins by an engineering approach, in order to get potent antibacterial molecules able to fight, either specifically or not, against various pathogenic bacterial species. Our first goal consists in getting chimera proteins with a narrow-spectrum antimicrobial activity. Our presentation will deal with our preliminary and encouraging results.