Yes / On 14/15th March 2016 we held a MRC/DBT funded workshop on the theme of Materials to Combat Antibiotic Resistance. The workshop was part of a continuing series of events that are part of the work of UK-India Centre for Advanced Technologies-Minimising the indiscriminate use of Antibiotics (UKICAT–MA). The following is the collection of presentations and the results of discussions highlighting key themes for future work by this group. Combating antibiotic resistance is perhaps the biggest issue facing the global community in the 21st century and no other area, with the exception perhaps of nuclear conflict, has the capacity to significantly reduce living standards and mortality rates. Key objectives identified by WHO1 in this area among five key aspects, include: Objective 4-to optimize the use of antimicrobial agents Objective 5-new medicines, diagnostic tools, vaccines and other interventions Our aims in this series of workshops are to provide an Indo-UK forum for: discussions of our advances in providing technologies to address these objectives; facilitate the interface between UK and Indian clinicians, materials and biological scientists and to identify key areas for new projects. An important aspect of the work in a global context is that by combining the UK and Indian community and clinical experiences we cover most of the scenarios that the global population might expect to encounter

Yes / Drug resistance diagnostics that rely on the detection of resistance-related mutations could expedite patient care and TB eradication. We perform minimum inhibitory concentration testing for 12 anti-TB drugs together with Illumina whole-genome sequencing on 1452 clinical Mycobacterium tuberculosis (MTB) isolates. We evaluate genome-wide associations between mutations in MTB genes or non-coding regions and resistance, followed by validation in an independent data set of 792 patient isolates. We confirm associations at 13 non-canonical loci, with two involving non-coding regions. Promoter mutations are measured to have smaller average effects on resistance than gene body mutations. We estimate the heritability of the resistance phenotype to 11 anti-TB drugs and identify a lower than expected contribution from known resistance genes. This study highlights the complexity of the genomic mechanisms associated with the MTB resistance phenotype, including the relatively large number of potentially causal loci, and emphasizes the contribution of the non-coding portion of the genome. / Biomedical research grant from the American Lung Association (PI MF, RG-270912-N), a K01 award from the BD2K initiative (PI MF, ES026835), and an NIAID U19 CETR grant (P.I. M.M., AI109755), the Belgian Science Policy (Belspo) (L.R., C.J.M.).

Yes / Antimicrobial resistance (AMR) is a growing global crisis with an increasing number of untreatable or exceedingly difficult-to-treat bacterial infections, due to their growing resistance to existing drugs. It is predicted that AMR will be the leading cause of death by 2050. In addition to ongoing efforts on preventive strategies and infection control, there is ongoing research towards the development of novel vaccines, antimicrobial agents, and optimised diagnostic practices to address AMR. However, developing new therapeutic agents and medicines can be a lengthy process. Therefore, there is a parallel ongoing worldwide effort to develop materials for optimised drug delivery to improve efficacy and minimise AMR. Examples of such materials include functionalisation of surfaces so that they can become self-disinfecting or non-fouling, and the development of nanoparticles with promising antimicrobial properties attributed to their ability to damage numerous essential components of pathogens. A relatively new class of materials, metal-organic frameworks (MOFs), is also being investigated for their ability to act as carriers of antimicrobial agents, because of their ultrahigh porosity and modular structures, which can be engineered to control the delivery mechanism of loaded drugs. Biodegradable polymers have also been found to show promising applications as antimicrobial carriers; and, recently, several studies have been reported on delivery of antimicrobial drugs using composites of MOF and biodegradable polymers. This review article reflects on MOFs and polymer-MOF composites, as carriers and delivery agents of antimicrobial drugs, that have been studied recently, and provides an overview of the state of the art in this highly topical area of research.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Michael D. Apley / The discovery of modern antimicrobials in the early 20th century revolutionized treatment of infectious diseases. Less than 100 years later, antimicrobial resistance has become a global threat to public health. With the rise of antimicrobial resistance, the question that remains to be answered is: Can dosing regimens provide maximal clinical efficacy, yet minimize the development of antimicrobial resistance? A pharmacokinetic / pharmacodynamic approach was utilized to investigate oxytetracycline regimens that would impart efficacy while minimizing the potential for resistance development due to plasmid transfer. An in vitro pharmacodynamic model was used to quantify the response of a Pasteurella multocida isolate to two oxytetracycline dosing regimens. The PK/PD index most closely related to efficacy was the Cmax:MIC. The in vitro pharmacodynamic model was then used to investigate the effects of antimicrobial exposure on plasmid transfer. A mixed population of oxytetracycline-susceptible and resistant bacteria was exposed to two dosing regimens and plasmid transfer was quantified. When oxytetracycline concentrations exceeded the MIC of the recipient, development of resistance was suppressed. The same donor and recipient bacteria were used in an in situ swine model to validate the in vitro findings. Following surgical implantation of porous membrane straws containing the mixed bacterial population, animal subjects in the treatment groups received one of two oxytetracycline treatments. Oxytetracycline concentrations in the plasma and interstitial fluid were quantified. Plasmid transfer within the implant membranes was quantified and correlated to pharmacokinetic measures in the animal. Plasmid transfer rates in the implant membranes did not correlate to the investigated pharmacokinetic parameters. The study methodologies in this dissertation should serve as a foundation for future studies in antimicrobial pharmacokinetic/pharmacodynamic research. The results presented here show that the bacterial response to oxytetracycline can be optimized in a concentration dependent manner and that antimicrobial resistance development through plasmid transfer can be suppressed in vitro when oxytetracycline concentrations exceed the MIC of the recipient bacteria. These results suggest that a proper balance between clinical efficacy and minimizing antimicrobial resistance can be achieved for oxytetracycline through appropriate dosing regimens and drug formulations.

Background: Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA) represent the most worrying Gram-negative and Gram-positive nosocomial pathogens of the present age. They are of increasing concern in the clinical environment due to their multi-drug resistance and the dwindling therapeutic options available. A. baumannii is the most frequently isolated clinical species of the genus, and is able to rapidly acquire resistance. Hypermutators, most frequently deficient in mismatch repair (MMR) via defects in the mutS gene, have been associated with antimicrobial resistance in several bacterial populations. To date, however, the potential role of MMR-deficient mutators in the development of resistance in clinical Acinetobacter spp. has not been investigated. Biocides, most notably chlorhexidine (CHX), are increasingly used in the hospital environment to prevent bacterial spread. This has led to concerns about the development of reduced biocide susceptibility and associated antibiotic resistance in hospital bacterial populations, where there is frequent exposure to both of these factors. The effect of CHX upon defined clinical MRSA isolates is examined here. Methods: The mutS gene of clinical Acinetobacter spp. isolates with varying sensitivities was sequenced and compared to establish whether any variations were present. Mutation studies were performed on isolates by challenging them with ciprofloxacin to determine whether different mutS types correlated with any variation in their ability to develop significant fluoroquinolone resistance. The response of clinical MRSA isolates to a range of CHX concentrations was examined with susceptibility testing methods, and effects were compared with standard strains. Determination of post-exposure minimum inhibitory concentrations (MICs) of a range of antibiotics enabled evaluation of whether exposure to CHX had an effect on susceptibility to antibiotics. Results: Variation was observed in the mutS gene of clinical Acinetobacter spp. isolates, with greater homology observed as resistance increased. A highly conserved and previously unreported amino acid sequence was discovered in resistant isolates. Nonresistant isolates with this ‘R-type’ mutS sequence appeared to have a greater ability to develop significant ciprofloxacin resistance. Clinical MRSA isolates had varying susceptibility to CHX, and there were differences in the susceptibility of standard strains compared to clinical isolates. CHX residues exerted a prolonged minimal inhibitory effect, and several increases in antibiotic MICs following CHX exposure were observed. Conclusions: The correlation of the mutS sequence with mutation ability suggests that defects in the mutS gene may have a role to play in the ability of certain Acinetobacter spp. to rapidly acquire resistance. This could have implications for the treatment of Acinetobacter spp. infections, and may enable quick determination of which clinical isolates have the potential to develop clinically significant resistance. Incomplete eradication due to the prolonged minimal effect of CHX residues may act as a selective pressure in the hospital environment, allowing survival of reduced susceptibility MRSA isolates. Increases in antibiotic MICs following CHX exposure is of grave concern for the future of biocide usage.

Résumé : Le Campylobacter est la première cause de gastro-entérites bactériennes dans les pays industrialisés. La grande majorité des cas sont des infections sporadiques dont la source est rarement identifiée. Le Campylobacter fait partie de la flore intestinale normale d’une large diversité d’animaux et peut également se retrouver dans l’eau. Le but de mon projet de recherche était d’étudier l’épidémiologie clinique et moléculaire des infections à Campylobacter en Estrie afin de déterminer les principales sources d’infections sporadiques et de comparer les génotypes des isolats de Campylobacter selon les différentes niches écologiques. Nous avons déterminé le profil de sensibilité aux antibiotiques d’isolats de différentes sources. Nous avons observé un haut taux de résistance à l’érythromycine et à la tétracycline et un faible taux de résistance à la ciprofloxacine chez les isolats de poulet, pouvant refléter l’utilisation de ces antibiotiques dans cet élevage. Le fait que le taux de résistance à l’érythromycine parmi les isolats humains soit significativement moins élevé que chez les isolats de poulet suggérait l’importance d’autres sources de Campylobacter chez l’humain. Afin de déterminer quelle méthode de typage moléculaire serait la mieux adaptée pour notre devis de recherche, nous avons comparé quatre méthodes (AFLP, MLST, typage du gène fla et EGCP). Seul le MLST a pu attribuer des isolats humains à des niches écologiques particulières comme le poulet, le lait cru et l’eau. Afin d’optimiser la technique de MLST, nous avons développé un système complémentaire basé sur le HRM, qui est beaucoup plus rapide et moins coûteux que le MLST. Nous avons démontré que le HRM a le potentiel de complémenter les méthodes d’analyses basées sur du séquençage pour l’étude des mutations ponctuelles et de faciliter une vaste gamme d’études basées sur des méthodes génotypiques, telle la détection de mutations ponctuelles qui confèrent de la résistance aux antibiotiques. Nous avons entrepris par la suite une étude cas-cas et un vaste projet d’isolement et de caractérisation moléculaire de souches de Campylobacter en Estrie, afin de véritablement cerner les mécanismes de transmission de la bactérie et de comparer les sources d’infections sporadiques chez les cas acquis en régions rurales vs urbaines. Nous avons confirmé que le poulet était responsable de la majorité des cas de campylobactérioses. Cependant, nos résultats suggèrent que la saisonnalité ainsi que le gradient urbain-rural de la campylobactériose sont dus à l’exposition aux souches bovines, particulièrement chez le groupe d’âge des 15-34 ans via l’exposition professionnelle. Par la détermination des sources d’infections, nous avons établi des pistes d’interventions utilisables par les autorités de santé publique, afin de diminuer l’incidence de la campylobactériose au Québec. / Abstract : Campylobacteriosis is the leading notifiable enteric disease in industrialised countries. It colonizes a wide range of animal which in turn spread the disease. The majority of campylobacteriosis cases are sporadic infections for which the source is rarely apparent. The main goal of my research project is to determine contamination sources of Campylobacter in the Eastern Townships, to identify the sources and routes of transmission and to establish the main sources of sporadic infections. We determined antimicrobial susceptibility profiles of Campylobacter isolates in order to predict which bacterial population will be resistant, caused by antimicrobial selective pressure administered to the host. High levels of resistance of chicken isolates to erythromycin and tetracycline, and low levels of resistance to ciprofloxacin reflect the use of the former antibiotics in animal husbandry. The fact that the erythromycin and tetracycline resistance levels were significantly lower among human isolates suggests that other transmission sources are important for human infection. In order to determine which molecular typing method will be the most relevant for our research design, we compared four typing methods (AFLP, MLST,/7a typing and PFGE). Only MLST has the potential to link isolates to a particular ecological niche, such as chicken, raw milk and water. In order to optimize MLST, we developed a complementary system based on HRM. We demonstrated that HRM has the potential to complement the analysis methods based on sequencing for SNP and facilitate a wide range of studies based on genotypic methods. We have subsequently undertaken a major project of isolation and molecular characterization of Campylobacter in the Eastern Townships, in order to truly understand the mechanisms of transmission of the bacteria and determine the source of sporadic cases. We confirmed that chicken was responsible for the majority of cases of campylobacteriosis. However, we have shown that the urban-rural gradient of campylobacteriosis in the Eastern Townships could be explained by exposure to bovine, especially for the 15-34 year old age group through occupational exposure. By the identification of infection sources, we proposed courses of action that could be used by public health authorities to reduce the incidence of campylobacteriosis in Quebec.

Master of Science / Department of Biomedical Sciences / Sanjeev K. Narayanan / Cefovecin is an extended-spectrum long-acting third generation cephalosporin used to treat canine infections. The study objective was to determine the effect of cefovecin on the absolute number and antimicrobial susceptibility of fecal enteric bacteria in healthy dogs. Fourteen Beagles were randomly assigned to a treated (n = 7, 8 mg/kg cefovecin subcutaneously on day 1) or untreated (n = 7) group. LC/MS was used to determine plasma cefovecin concentration on day 14. E. coli, enterococci, and Salmonella were isolated and enumerated from fecal samples collected on days 0, 3, 7, 14, and 28. Antimicrobial resistance was determined using disc diffusion, MIC, and detected using PCR for the bla[subscript CMY-2] gene on select isolates. Mean plasma concentration of cefovecin on day 14 was 9.59 µg/mL in treated dogs; untreated dogs had no measurable plasma cefovecin. The absolute number of E. coli was lower in treated dogs on day 3 (P ≤ 0.0001), and the absolute number of cefovecin-resistant E. coli was higher in treated dogs on days 7 (P = 0.002), 14 (P = 0.004) and 28 (P ≤ 0.0001), compared to untreated dogs. Enterococci increased and were higher in the treatment group on day 7 (P = 0.0226). Isolation of Salmonella was rare. After cefovecin treatment, beta-lactam resistance was more common in fecal E. coli from treated dogs than untreated dogs, while resistance of enterococci was not altered. On day 28, treated dogs were 3.25 times more likely to carry the bla[subscript CMY-2] gene than untreated dogs (95% CI 1.27 – 8.35). The implications of these findings in clinically ill patients require further research.

Introduction: Southeast Asia has among the highest rates of antibiotic resistance worldwide, particularly in Indonesia, where paediatricians prescribed antibiotics to 94% of children, knowing that the infection was viral. Relevance: There is a gap in understanding of the reasons behind the irrational use of antibiotics by healthcare professionals and patients. Aim: This research aims to explore factors that influence the use of antibiotics and knowledge about antibiotic resistance in Jakarta, Indonesia. Methods: In December 2014, the researcher conducted thirteen semi-structured interviews with four stakeholder groups, which are involved in the “Smart Use of Antibiotics” campaign in Jakarta. Qualitative Content Analysis was used to identify the theme “unite our voice to address antibiotic resistance from all angles” as well as the five categories: Education, Media, Policy, Culture and Trust. Results: Each category presented one factor, which was divided into the sub-factors education of patients and professionals; online and offline media; policy and guidelines, drug availability and accessibility and stakeholder involvement; habit and behaviour, doctor-patient relationship, environment / surroundings; and trust in the system, in the healthcare professionals, among professionals and in medicine. Conclusion: All stakeholders need to unite their voices together to achieve a smarter use of antibiotics and increase the knowledge about antibiotic resistance.

Antibiotic resistant bacteria, a growing health crisis, arise due to antibiotic overuse and misuse. Resistant infections endanger the lives of patients and are financially burdensome. Aggregate antimicrobial susceptibility reports, called antibiograms, are critical for tracking antibiotic susceptibility and evaluating the likelihood of the effectiveness of different antibiotics to treat an infection prior to the availability of patient specific susceptibility data. This research leverages the Massachusetts Statewide Antibiogram database, a rich dataset composed of antibiograms for $754$ antibiotic-bacteria pairs collected by the Massachusetts Department of Public Health from $2002$ to $2016$. However, these antibiograms are at least a year old, meaning antibiotics are prescribed based on outdated data which unnecessarily furthers resistance. Our objective is to employ data science techniques on these antibiograms to assist in developing more responsible antibiotic prescription practices. First, we use model selectors with regression-based techniques to forecast the current antimicrobial resistance. Next, we develop an assistant to immediately identify clinically and statistically significant changes in antimicrobial resistance between years once the most recent year of antibiograms are collected. Lastly, we use k-means clustering on resistance trends to detect antibiotic-bacteria pairs with resistance trends for which forecasting will not be effective. These three strategies can be implemented to guide more responsible antibiotic prescription practices and thus reduce unnecessary increases in antibiotic resistance.

Antibiotic resistant bacteria, a growing health crisis, arise due to antibiotic overuse and misuse. Resistant infections endanger the lives of patients and are financially burdensome. Aggregate antimicrobial susceptibility reports, called antibiograms, are critical for tracking antibiotic susceptibility and evaluating the likelihood of the effectiveness of different antibiotics to treat an infection prior to the availability of patient specific susceptibility data. This research leverages the Massachusetts Statewide Antibiogram database, a rich dataset composed of antibiograms for $754$ antibiotic-bacteria pairs collected by the Massachusetts Department of Public Health from $2002$ to $2016$. However, these antibiograms are at least a year old, meaning antibiotics are prescribed based on outdated data which unnecessarily furthers resistance. Our objective is to employ data science techniques on these antibiograms to assist in developing more responsible antibiotic prescription practices. First, we use model selectors with regression-based techniques to forecast the current antimicrobial resistance. Next, we develop an assistant to immediately identify clinically and statistically significant changes in antimicrobial resistance between years once the most recent year of antibiograms are collected. Lastly, we use k-means clustering on resistance trends to detect antibiotic-bacteria pairs with resistance trends for which forecasting will not be effective. These three strategies can be implemented to guide more responsible antibiotic prescription practices and thus reduce unnecessary increases in antibiotic resistance.